Vaccines Ip

INDIAN PHARMACOPOEIA 2007MONOGRAPHS VACCINES AND IMMUNOSERA FOR HUMAN USE General Requirements ..................................................................................................... Monographs ..................................................................................................................... Adsorbed Diphtheria, Tetanus and Hepatitis B (rDNA) Vaccine ........................................ Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component) and Haemophilus Type b Conjugate Vaccine ........................................................................ Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component) and Hepatitis B (rDNA) Vaccine ........................................................................................... Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component), Inactivated Poliomyelitis Vaccine and Haemophilus Type b Conjugate Vaccine ................. Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component) and Inactivated Poliomyelitis Vaccine ................................................................................... Adsorbed Diphtheria, Tetanus, Pertussis and Poliomyelitis (Inactivated) Vaccine .................... Adsorbed Diphtheria, Tetanus, Pertussis, Poliomyelitis (Inactivated) and Haemophilus Type b Conjugate Vaccine ........................................................................ Adsorbed Pertussis Vaccine (Acellular Component) Adsorbed Pertussis Vaccine (Acellular, Co-purified) ......................................................... ......................................................... Bacillus Calmette-Guerin Vaccine (Freeze-Dried) ............................................................... Diphtheria and Tetanus Vaccine (Adsorbed) ........................................................................ Diphtheria and Tetanus Vaccine (Adsorbed) for Adults and Adolescents .............................. Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed) ..................................................... Diphtheria, Tetanus, Pertussis (Whole Cell), Hepatitis B (rDNA) and Haemophilus Type b Conjugate Vaccine (Adsorbed) .................................................... Diphtheria, Tetanus, Pertussis (Whole Cell) Hepatitis B (rDNA) Vaccine (Adsorbed) ................................................................................................... Diphtheria, Tetanus, Pertussis (Whole Cell) and Haemophilus Type b Conjugate Vaccine (Adsorbed) ...................................................................................... Diphtheria Vaccine (Adsorbed) ........................................................................................... Haemophilus Type b Conjugate Vaccine ............................................................................. Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed) ................................. Hepatitis B Vaccine (rDNA) ............................................................................................. Inactivated Hepatitis A Vaccine (Adsorbed) ....................................................................... Inactivated Hepatitis B Vaccine ........................................................................................... 1269 MONOGRAPHS INDIAN PHARMACOPOEIA 2007 Inactivated Influenza Vaccine (Split Virion) ........................................................................... Inactivated Influenza Vaccine (Surface Antigen) .................................................................... Inactivated Influenza Vaccine (Whole Virion) .................................................................... Japanese Encephalitis Vaccine (Human) .............................................................................. Measles and Rubella Vaccine (Live) ..................................................................................... Measles Vaccine (Live) ...................................................................................................... Measles, Mumps and Rubella Vaccine (Live) ....................................................................... Meningococcal Polysaccharide Vaccine .............................................................................. Mumps Vaccine (Live) ...................................................................................................... Pertussis Vaccine ................................................................................................................ Pneumococcal Polysaccharide Vaccine ................................................................................ Poliomyelitis Vaccine (Inactivated) ...................................................................................... Poliomyelitis Vaccine, Live (Oral) ....................................................................................... Rabies Vaccine, Human ...................................................................................................... Rubella Vaccine (Live) ....................................................................................................... Tetanus Vaccine (Adsorbed) ............................................................................................... Tick-borne Encephalitis Vaccine (Inactivated) ..................................................................... Tuberculin Purified Protein Derivative ................................................................................... Typhoid (Strain Ty 21a) Vaccine, Live (Oral) .................................................................... Typhoid Polysaccharide Vaccine ......................................................................................... Typhoid Vaccine ................................................................................................................ Typhoid Vaccine (Freeze Dried) ......................................................................................... Varicella Vaccine (Live) ....................................................................................................... Viper Venom ........................................................................................................................ Yellow Fever Vaccine (Live) ............................................................................................... 1270 IP 2007 VACCINES : GENERAL REQUIREMENTS Vaccines : General Requirements Vaccines are preparations of antigenic substances that are administered for the purpose of inducing in the recipient a specific and active immunity against the infective agent or toxin produced by it. Vaccines may contain living micro-organisms suitably treated to attenuate their virulence but retain their antigenic potency or they may consist of pathogenic organisms which have been killed or inactivated. Some vaccines consist of antigenic fractions or substances produced by the same pathogenic organisms but rendered harmless whilst retaining their antigenic efficiency. Vaccines may be prepared from one species only or from a mixture of two or more species. Vaccines may be prepared by the method described in the individual monographs or by the general methods given below or in any other manner provided the identity of the antigens is maintained and the vaccines are free from microbial contamination and extraneous agents. Suitable adjuvants may be added during the preparation but streptomycin, penicillin or other β-lactam antibiotics may not be added at any stage of manufacture or in the final vaccine. A suitable bactericide may be added to sterile and inactivated vaccines. The final products are distributed aseptically into sterile containers which are then sealed to exclude extraneous micro-organisms. Unless otherwise indicated in the monograph, the final vaccine may be filled into single dose or multiple dose containers but vaccines in multiple dose containers must invariably contain a bactericide. Bacterial Vaccines. Bacterial vaccines are either sterile suspensions of live or killed bacteria or sterile extracts of derivatives of bacteria. They may be simple vaccines prepared from one species or may be mixed vaccines prepared by blending two or more simple vaccines from different species or strains. Bacterial vaccines may be prepared from cultures grown on suitable solid or liquid media. The whole culture or parts of it may be used in preparing the vaccine. The identity, antigenic potency and purity of each bacterial culture must be carefully controlled. Vaccines containing killed organisms may be prepared by killing the organisms by chemical or physical means provided the antigenic potency of the vaccine is preserved. Vaccines containing living bacteria may be prepared from strains which are avirulent for humans but which stimulate the production of antibodies active against pathogenic strains of the same species. The final vaccines must be free from any substance known to cause toxic, allergic or other undesirable immunological reactions in humans. Bacterial vaccines are suspensions of varying degrees of opacity in colourless or slightly coloured liquids or they may 1 be freeze-dried so that the water content is not more than 3.0 per cent w/w unless otherwise stated in the individual monograph. They may be standardized in terms of interopacity units or, where appropriate, by numbers of living or killed bacteria determined by direct cell count or by viable count. Bacterial toxoids. Bacterial toxoids are toxins or material derived therefrom, the toxicity of which has been reduced to a very low level or completely eliminated by chemical or physical means without destroying their immunizing potency. The toxins are obtained from selected strains of specific micro-organisms, grown in media free from ingredients known to cause toxic, allergic or other undesirable immunological reactions in humans. Toxoids produced by the action of formaldehyde are known as formol toxoids. Bacterial toxoids may be liquid or may be prepared by adsorbing on mineral carriers such as aluminium phosphate, aluminium hydroxide or any other suitable adsorbent; the adsorbed product may be separated, washed and suspended in a saline or other appropriate solution isotonic with blood. Bacterial toxoids are clear or slightly opalescent liquids, colourless or slightly yellow. Adsorbed toxoids may be white or greyish white suspensions or pale-yellow liquids with a sediment at the bottom of the container. Freeze-dried preparations are greyish white or yellowish white powders or pellets. Viral and rickettsial vaccines. Viral and rickettsial vaccines are suspensions of viruses or rickettsiae and are prepared from infected tissues or blood obtained from artificially infected animals, from cultures in fertile eggs, or from cell or tissue culture. Viral vaccines may be live or killed and they may be freeze-dried. Live vaccines are usually prepared using attenuated strains of the specific organisms. Killed vaccines may be inactivated by suitable chemical or physical means. Mixed Vaccines. Mixed vaccines are mixtures of two or more vaccines. A suitable antibacterial substance may be added to inactivated or live viral and rickettsial vaccines provided that it has no action against the specific organisms. Production General provisions. Requirements for production including in-process testing are included in individual monographs. Where justified and authorized, certain tests may be omitted where it can be demonstrated, for example by validation studies, that the production process consistently ensures compliance with the test. Unless otherwise justified and authorized, vaccines are produced using a seed-lot system. The methods of preparation are designed to maintain adequate immunogenic properties, to render the preparation harmless and to prevent contamination with extraneous agents. the adsorbents are prepared in special conditions which confer the appropriate physical form and adsorptive properties. control eggs are incubated and tested as prescribed in the monograph. Stability. Approved animal (but not human) serum may be used in the growth medium for cell cultures but the medium used for maintaining cell growth during virus multiplication shall not contain serum. after freeze-drying where applicable. Culture media. Penicillin and streptomycin are neither used at any stage of production nor added to the final product. Final bulk. Adsorbents. Control cells. 2 Control eggs. unless otherwise stated. unless otherwise stated. Processing of cell banks and subsequent cell cultures is done under aseptic conditions in an area where no other cells are being handled. allergic or other undesirable reactions in man. If an antimicrobial preservative is used. validated purification procedures may be applied. be used for production. Suitable additives. master seed lots prepared with media containing penicillin or streptomycin may. For vaccines for parenteral administration. From the stability data . the final lot is prepared by distributing the final bulk under suitable conditions into sterile. For vaccines produced in cell cultures. Vaccines are as far as possible free from ingredients known to cause toxic. unless otherwise justified and authorised. the degree of adsorption is evaluated as part of the consistency testing. For live vaccines produced in eggs. it shall be shown that it does not impair the safety or efficacy of the vaccine and its effectiveness throughout the period of validity shall be demonstrated. or the number of subcultures of a bacterium. A release specification for the degree of adsorption is established in the light of results found for batches used in clinical testing. however. Seed lot. Purification. A test for inactivation is carried out as soon as possible after the inactivation process. Degree of adsorption. Substrates for propagation comply with the relevant requirements of the Pharmacopoeia or in the absence of such requirements with those of the competent authority. from the master seed lot shall not exceed that used for production of the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. the loss of potency in the recommended storage conditions is assessed and excessive loss even within the limits of acceptable potency may indicate that the vaccine is unacceptable. control cells are maintained and tested as prescribed. Where applicable. Inactivated vaccines are produced using a validated inactivation process whose effectiveness and consistency have been demonstrated. non-SPF flocks. in the production of a final lot of vaccine. Antimicrobial preservative. The final bulk is prepared by aseptically blending the ingredients of the vaccine. In order to provide a valid control. these cells must be maintained in conditions that are rigorously identical with those used for the production cell cultures. including use of the same batches of media and media changes. are closed so as to exclude contamination. where justified and authorized. the number of passages of a virus. Cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration although it is preferable to have a medium free from antibiotics during production. the stability of intermediates in given storage conditions shall be evaluated and a period of validity established. allergic or other undesirable reactions in man. calcium phosphate or other suitable adsorbent. During development of an adsorbed vaccine. for example in vaccines produced in eggs from healthy. including stabilizers and adjuvants may be incorporated. A suitable antimicrobial preservative may be included in sterile and inactivated vaccines and is invariably added if these preparations are issued in multidose containers. it shall be demonstrated that the amount present in the final lot is reduced to such a level as to render the product safe. Serum and trypsin used in the preparation of cell suspensions shall be shown to be free from extraneous agents. No micro-organism other than the seed strain shall be present in a seed lot. Maintenance of potency of the final lot throughout the period of validity shall be demonstrated by validation studies. Where applicable. Where there are recognised potential contaminants of a harvest. The purity of the harvest is verified by suitable tests as defined in the monograph. Inactivation. Propagation and harvest. Vaccines may be adsorbed on aluminium hydroxide. if inclusion of such ingredients is necessary. The seed cultures are propagated and harvested under defined conditions. Intermediates. Substrates for propagation.VACCINES : GENERAL REQUIREMENTS IP 2007 Unless otherwise justified and authorized. For vaccines for administration by a non-parenteral route. the final lot is prepared by aseptically distributing the final bulk into sterile tamper-proof containers which. aluminium phosphate. Culture media are as far as possible free from ingredients known to cause toxic. tamper-proof containers. the inactivation process is also validated with respect to the potential contaminants. The strain of bacterium or virus used in a master seed lot is identified by historical records that include information on the origin of the strain and its subsequent manipulation. Final lot. all vaccines comply with the test for abnormal toxicity. the test in mice may be inappropriate. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus. Reference vaccine(s) Provided valid assays can be performed. Not more than 0. (3) the dose and route of administration. (4) the name and proportion of any antibacterial preservative or other auxiliary substances added to the vaccine. (6) the conditions under which it should be stored. For the preparation of . Adsorbed Diphtheria. Method B. that have not previously been treated with any material that will interfere with the test. Labelling. except that for living bacterial vaccines. the vaccine does not comply with the test.20). Unless otherwise stated. Thiomersal (If present) (2.2. would comply with the test for abnormal toxicity for antisera and vaccines. Maximum 0. respectively. for dried vaccines. NOTE — The statements given in this general chapter is intended to be read in conjunction with the monographs on the individual vaccine in this Pharmacopoeia which refer to preparations for human use. Phenol (If present) (2.IP 2007 ADSORBED DIPHTHERIA. if tested. Unless otherwise stated all vaccines comply with tests for sterility. In vaccines containing phenol as preservative. (2) unless otherwise indicated the minimum number of Units per dose or per ml or. (10) any contraindication to the use of the vaccine. Abnormal toxicity (2.3. tetanus formol toxoid. Aluminium (If present) (2. Sterility (2. the number of doses in the container. Liquid vaccines must be stored at a temperature between 2o and 8o and should not be allowed to freeze unless otherwise specified in the individual monograph. that the vaccine should be shaken well before use. the vaccine does not comply with the test. if more than 1 animal dies in the second test.11).3. The production method is validated to demonstrate that the product. reconstituted where necessary. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine. Between 0. HBsAg is a component protein of hepatitis B virus.48). repeat the test once.005 per cent w/v and 0. 3 Production General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. (9) unless otherwise directed. a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate.36). For each component. Freeze-dried preparations must be stored at temperatures below –20o or as specified in the individual monograph.3. hepatitis B surface antigen (HBsAg). comply with the following requirements unless otherwise stated in the individual monograph. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani.25 mg per dose. Tetanus and Hepatitis B (rDNA) Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid. Not more than 1. and with the following test for specific toxicity of the diphtheria and tetanus components: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs.3. (5) the date after which the vaccine is not intended to be used.25 per cent w/v. each weighing between 250 and 350 g. Tests Vaccines.02 g/l. a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. If more than 1 animal dies from nonspecific causes. Tetanus and Hepatitis B (rDNA) Vaccine Diphtheria. monocomponent reference vaccines may be used for the assays on the combined vaccine. At higher temperatures vaccines deteriorate rapidly.2. Storage.1). The content of bacterial endotoxins in the bulk purified diphtheria toxoid and tetanus toxoid is determined to monitor the purification procedure and to limit the amount in the final vaccine. growth of the organism from which the vaccine was prepared is permitted (sterility means abesence of becterial and fungal contaminants except where specified in the individual monograph). they do not necessarily apply to vaccines for use in veterinary practice. the content of bacterial endotoxin is less than the limit approved for the particular vaccine and in any case the contents are such that the final vaccine contains less than 100 IU per single human dose. The label states (1) for liquid vaccines.02 per cent w/v. (7) for dried vaccines. for viral vaccines. the antigen is obtained by recombinant DNA technology. the total number of ml in the container and. the minimum viral titre. the liquid to be used for reconstitution and its volume.9). Free formaldehyde (If present) (2. (8) that the vaccine should be used immediately after reconstitution. TETANUS AND HEPATITIS B (rDNA) VACCINE generated for the vaccine it must be shown that at the end of the period of validity the degree of adsorption will not be less than for batches used in clinical testing. 25 mg per single human dose. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed). Tests and Assay may be released for use. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent.2.20). applicable to certain vaccines. The assay or. Provided the test for antimicrobial preservative and the assays for the diphtheria and tetanus components have been carried out with satisfactory results on the final bulk vaccine. Where applicable.0 per cent and not greater than 115.0 per cent and not greater than 115. Complies with the test for pyrogens.2 g/l. Free formaldehyde (2. The content is not less than 85. Sterility (2. Osmolality (2. The osmolality of the vaccine is within the limits approved for the particular preparation. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification. determine the amount of antimicrobial preservative by a suitable chemical method. The following method. is given as an example. The lower confidence limit (P = 0. Maintain at 37° for about 16 hours and centrifuge until a clear supernatant is obtained. (2) the amount of HBsAg per single human dose.14). Maximum 0. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). separately or together. they may be omitted on the final lot.8). The lower confidence limit (P = 0. provided it has been carried out with satisfactory results on the final bulk vaccine. The clear supernatant liquid reacts with a suitable diphtheria antitoxin.23). Labelling. Maximum 1. Tests Aluminium (2. is given as an example. Hepatitis B component It complies with the assay of Hepatitis B Vaccine.11). Where applicable. Antimicrobial preservative. of suitable quantities of bulk purified diphtheria toxoid. (3) the type of cells used for production of the HBsAg component. Carry out test for sterility using 10 ml of bulk for each sterility medium. it may be omitted on the final lot.2 g/l. giving a precipitate. determine the amount of antimicrobial preservative by a suitable chemical method. Sterility (2. serves also to identify the hepatitis B component of the vaccine. Pyrogens (2. If an in vivo assay is used for the hepatitis B component.95) of the estimated potency is not less than 40 IU per single human dose. The clear supernatant liquid obtained during identification test A reacts with a suitable tetanus antitoxin. Suitable antimicrobial preservatives may be added.9). C. Diphtheria toxoid is identified by a suitable immunochemical method (2. The content is not less than 85.11).4. B. that the vaccine is intended for primary vaccination of children and is not necessarily 4 Identification A. tetanus toxoid and HBsAg onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate.2. Antimicrobial preservative.0 per cent of the intended amount. (4) where applicable.95) of the estimated potency is not less than 30 IU per single human dose.14).3. Complies with the test for sterility. where applicable. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). Tetanus Vaccine (Adsorbed) and Hepatitis B Vaccine (rDNA). strict adherence to the production process used for the batch tested in clinical trials is necessary.2.2. Inject the equivalent of 1 human dose into each rabbit.ADSORBED DIPHTHERIA. the electrophoretic profile.0 per cent of the intended amount. vaccines. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose. TETANUS AND HEPATITIS B (rDNA) VACCINE IP 2007 a representative batch.2. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption. the test for free formaldehyde may be omitted on the final lot.3. applicable to certain . Tetanus toxoid is identified by a suitable immunochemical method (2. giving a precipitate. The following method. The carrier protein. tetanus formol toxoid. consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. separately or together. FINAL BULK VACCINE Different methods of preparation may be used: a final bulk vaccine may be prepared by adsorption. If the vaccine is presented with the haemophilus component in a separate vial. tetanus 5 Adsorbed Diphtheria. of suitable quantities of bulk purified diphtheria toxoid. would comply with the test for abnormal toxicity for antisera and vaccines. a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties produced by expression of a genetically modified form of the corresponding gene. For subsequent routine control. Pertussis (Acellular Component) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid. For the preparation of a representative batch. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. During development studies and wherever revalidation is necessary. (5) the name and the amount of the adsorbent. monocomponent reference vaccines may be used for the assays on the combined vaccine. the content of bacterial endotoxins is less than the limit approved for the particular vaccine. (7) that the vaccine is not to be frozen. Production of the components The production of the components complies with the tests of the monographs on Diphtheria Vaccine (Adsorbed). Production General provisions The production method shall have been shown to yield . T. P. individually purified antigenic components of Bordetella pertussis. if tested. it shall be demonstrated by tests in animals that the vaccine induces a T-cell dependent B-cell immune response to PRP. is capable of inducing a T-cell-dependent B-cell immune response to the polysaccharide. Pertussis (Acellular Component) and Haemophilus Type B Conjugate Vaccine Diphtheria. as part of consistency studies the assays of the diphtheria. The acellular pertussis component may also contain filamentous haemagglutinin. For each component. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. Reference vaccine Provided valid assays can be performed. tetanus toxoid. with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. D. when conjugated to PRP. the assays of these components may be carried out without mixing with the haemophilus component. Adsorbed) and Haemophilus Type b Conjugate Vaccine. (6) that the vaccine must be shaken before use. the contents of which are mixed with the other components immediately before use. polyribosylribitol phosphate (PRP) covalently bound to a carrier protein. a mineral absorbent such as aluminium hydroxide or hydrated aluminium phosphate. pertactin (a 69 kDa outermembrane protein) and other defined components of B. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. The content of bacterial endotoxins in bulk purified diphtheria toxoid. the contents of the diphtheria. tetanus and pertussis components are carried out on a suitable number of batches of vaccine reconstituted for use. if the vaccine is presented with the haemophilus component in a separate container. The production method is validated to demonstrate that the product. pertussis components and PRP conjugate is determined to monitor the purification procedure and to limit the amount in the final vaccine. Tetanus Vaccine (Adsorbed). The product may be presented with the haemophilus type b component in a separate container. Pertussis Vaccine (Acellular Component. Pertussis toxoid is prepared from pertussis toxin by a method that renders the toxin harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The latter two antigens may be copurified. Tetanus. (ACELLULAR COMPONENT) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE suitable for reinforcing doses or for administration to adults. pertussis such as fimbrial-2 and fimbrial-3 antigens. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine. strict adherence to the production process used for the batch tested in clinical trials is necessary.IP 2007 A. Tetanus. tetanus and pertussis antigens are in any case such that the final vial for these components contains less than 100 IU per single human dose. PRP is a linear copolymer composed of repeated units of 3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n]. 2. D.2. The test is invalid if 1 or more control mice die following histamine challenge. one containing the diphtheria. The following method.0 per cent and not greater than 115.A.24). Where applicable.4. irreversibility of pertussis toxoid.0 per cent of the intended amount.11). Maintain at 37° for about 16 h and centrifuge until a clear supernatant is obtained. Suitable antimicrobial preservatives may be added. T. for example by anion-exchange. is within the limits approved for the particular preparation. not more than 5. the vaccine complies with the test if. the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined. tetanus and pertussis components.0 per cent of the total number of mice die following histamine challenge. applicable to certain vaccines. sterility and pyrogens are carried out on the container with the haemophilus component. The osmolality of the vaccine. D. Free PRP.14). (ACELLULAR COMPONENT) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE IP 2007 toxoid. The following method.14). The clear supernatant reacts with a suitable diphtheria antitoxin.2. The pH of the vaccine. applicable to certain vaccines. the tests for PRP content. Inject diluent into the third group of mice. the other the haemophilus component. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent . is given as an example. free formaldehyde. inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0. irreversibility of pertussis toxoid and antimicrobial preservative and the assays have been carried out with satisfactory results on the final bulk vaccine. giving a precipitate. If the haemophilus component is freeze-dried. Provided the free formaldehyde content has been determined on the bulk purified antigens or the final bulk and it has been shown that the content in the final lot will not exceed 0.2. The amount of free PRP is not greater than that approved for the particular product. P.14). water (where applicable). Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. Antimicrobial preservative. Use 3 groups each of not less than 5 histaminesensitive mice. tetanus and pertussis components. the test for free formaldehyde may be omitted on the final lot.2. Diphtheria toxoid is identified by a suitable immunochemical method (2. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification.23).2 g/l. C.5 ml and observe for 24 h. The pertussis components are identified by a suitable immunochemical method (2. Tetanus toxoid is identified by a suitable immunochemical method (2. determine the amount of antimicrobial preservative by a suitable chemical method. giving a precipitate. 6 Identification If the vaccine is presented with the haemophilus component in a separate vial: identification tests A. antimicrobial preservative and sterility are carried out on the container with the diphtheria. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. applicable to certain vaccines. B. they may be omitted on the final lot. The content is not less than 85.4.16). is given as an example. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Carry out test for sterility using 10 ml of bulk for each sterility medium. some tests may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. which may be freeze-dried. or 2 final bulks may be prepared and filled separately. The following method. w/v solution.14) for PRP. size-exclusion or hydrophobic chromatography (2. Tests and Assay may be released for use. Provided the tests for absence of residual pertussis toxin. Sterility (2. is within the range approved for the particular product. reconstituted if necessary.4. Tests If the product is presented with the haemophilus component in a separate container: the tests for absence of residual pertussis toxin. The haemophilus component is identified by a suitable immunochemical method (2. A. Unbound PRP is determined after removal of the conjugate. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2° to 8°. Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37° for 4 weeks. in both of the groups given the vaccine. aluminium. reconstituted where applicable. Osmolality (2. B and C are carried out using the vial containing the diphtheria. The clear s u p e r n a t a n t obtained as described in Identification test A reacts with a suitable tetanus antitoxin. acellular pertussis components and PRP conjugate onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. is given as an example. The clear supernatant obtained as described in Identification test A reacts with a specific antisera to the pertussis components of the vaccine. ultrafiltration or other validated methods. If 1 mouse dies in either or both of the first and second groups. After 5 days. pH (2. tetanus and pertussis components. identification test D is carried out on the vial containing the haemophilus components. a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate.2. The lower confidence limit (P = 0. (8) that the vaccine is not to be frozen (9) where applicable. pertactin (a 69 kDa outer-membrane protein) and other defined components of B. PRP is determined either by assay of ribose (2.43). Maximum 0. produced by expression of a genetically modified form of the corresponding gene. The content is not less than 85. (5) where applicable. The vaccine may also contain filamentous haemagglutinin.3.2 g/l.0 per cent and not greater than 115. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties.11). PRP. by an immunochemical method (2. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose. (2) the names and amounts of the pertussis components per single human dose. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. Pertussis (Acellular Component) and Hepatitis B (rDNA) Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid. The lower confidence limit (P = 0. Pertussis component The vaccine complies with the assay as the stated Adsorbed Pertussis Vaccine (Acellular Component). individually purified antigenic components of Bordetella pertussis.2. (ACELLULAR COMPONENT) AND HEPATITIS B (rDNA) VACCINE The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0. (7) that the vaccine must be shaken before use. the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine shows symptoms of sensitisation.8). (3) the number of micrograms of PRP per single human dose. (6) the name and the amount of the adsorbent. that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults. pertussis such as fimbrial2 and fimbrial-3 antigens. Aluminium (2. Maximum 1.2.1). Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed).20 ). the minimum potency stated on the label is 30 IU per single human dose. Free formaldehyde (2. Tetanus. hepatitis B surface antigen. Hepatitis B surface antigen is a component protein of hepatitis B virus. tetanus formol toxoid.2 per cent w/v of gelatin and challenge with histamine as above. respectively. Minimum 80.0 per cent for the freeze-dried haemophilus component. AND P.1) or phosphorus (2. Pertussis toxoid is prepared from pertussis toxin by a method that renders the latter harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. Unless otherwise justified and authorised.9). The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani.7.7. Where applicable. Maximum 3. 0. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). Complies with the test for pyrogens.IP 2007 A. Pyrogens (2.95) of the estimated potency is not less than 40 IU per single human dose.3. Complies with the test for sterility. Tetanus and Pertussis (Acellular Component) and Hepatitis B (rDNA) Vaccine Diphtheria.95) of the estimated potency is not less than the minimum potency stated on the label.25 mg per single human dose. 7 Labelling. Water (2. Adsorbed Diphtheria.14) or by anion-exchange liquid chromatography with pulsedamperometric detection. that the vaccine contains a pertussis toxinlike protein produced by genetic modification. Inject per kg of the rabbit’s mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier. 0. determine the amount of antimicrobial preservative by a suitable chemical method. the antigen is obtained by recombinant DNA technology. The latter 2 antigens may be copurified. (4) the type and nominal amount of carrier protein per single human dose. Sterility (2.3.0 per cent of the amount of PRP stated on the label.1 mg of PRP for a vaccine with tetanus toxoid as carrier. D. Production General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven . T.025 mg of PRP for a vaccine with OMP as carrier.0 per cent of the intended amount. Antimicrobial preservative. the electrophoretic profile. irreversibility of pertussis toxoid and antimicrobial preservative and the assays for the diphtheria. B. inject into each mouse 2 mg of histamine . monocomponent reference vaccines may be used for the assays on the combined vaccine. Identification A.14).2. applicable to certain vaccines.A. Diphtheria toxoid is identified by a suitable immunochemical method (2. of suitable quantities of bulk purified diphtheria toxoid. Tetanus Vaccine (Adsorbed). Pertussis Vaccine (Acellular Component. For the preparation of a representative batch. AND P. The clear supernatant obtained as described in identification test A reacts with a suitable tetanus antitoxin.0 per cent of the intended amount. The content of bacterial endotoxins in the bulk purified diphtheria toxoid. Tetanus toxoid is identified by a suitable immunochemical method (2. provided it has been carried out with satisfactory results on the final bulk vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine. Osmolality (2. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed).23). tetanus toxoid.14). separately or together. The clear supernatant obtained as described in identification test A reacts with a specific antisera to the pertussis components of the vaccine. serves also to identify the hepatitis B component of the vaccine. D. Tests Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification.0 per cent and not greater than 115. the test for free formaldehyde may be omitted on the final lot. applicable to certain vaccines. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0. they may be omitted on the final lot. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. (ACELLULAR COMPONENT) AND HEPATITIS B (rDNA) VACCINE IP 2007 clinical efficacy and safety in man. The assay or. Antimicrobial preservative. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification. a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. determine the amount of antimicrobial preservative by a suitable chemical method. applicable to certain vaccines. strict adherence to the production process used for the batch tested in clinical trials is necessary. Tests and Assay may be released for use. After 5 days. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. 8 Provided the tests for absence of residual pertussis toxin. where applicable. Where applicable. D. it may be omitted on the final lot. Inject diluent into the third group of mice. Sterility (2. C. The clear supernatant reacts with a suitable diphtheria antitoxin. The following method. T.11). For each component. The pertussis components are identified by a suitable immunochemical method (2. giving a precipitate.2. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. acellular pertussis components and hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate.2.2 g/l.2.14). Maintain at 37° for about 16 h and centrifuge until a clear supernatant is obtained. Use 3 groups each of not less than 5 histaminesensitive mice. tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine. The content is not less than 85. Adsorbed) and Hepatitis B Vaccine (rDNA). Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37° for 4 weeks. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2° to 8°. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption.2. The osmolality of the vaccine is within the limits approved for the particular preparation. The following method. is given as an example. is given as an example. Carry out test for sterility using 10 ml of bulk for each sterility medium. Reference vaccine(s) Provided valid assays can be performed. tetanus toxoid and pertussis components is determined to monitor the purification procedure and to limit the amount in the final vaccine. The following method. giving a precipitate. is given as an example. the content of bacterial endotoxins is less than the limit approved for the particular vaccine. If an in vivo assay is used for the hepatitis B component. polyribosylribitol phosphate (PRP) covalently bound to a carrier protein. Adsorbed Diphtheria.0 per cent of the intended amount. COMPONENT). Inject the equivalent of 1 human dose into each rabbit. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0. Sterility ( 2. the minimum potency stated on the label is 30 IU per single human dose. the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined. Tetanus. the vaccine complies with the test if. a mineral absorbent such as aluminium hydroxide or hydrated aluminium phosphate. Free formaldehyde (2. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent.2.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis (Acellular Component) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid. determine the amount of antimicrobial preservative by a suitable chemical method. individually purified antigenic components of Bordetella pertussis. (ACELL. The lower confidence limit (P = 0. pertactin (a 69 kDa outer- .11).9).0 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine shows symptoms of sensitisation. Complies with the test for sterility. Complies with the test for pyrogens. in both of the groups given the vaccine.20). that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults. (5) where applicable. T.25 mg per single human dose. The content is not less than 85. not more than 5 per cent of the total number of mice die following histamine challenge. that the vaccine contains a pertussis toxin-like protein produced by genetic modification. D. Unless otherwise justified and authorised.IP 2007 A. Maximum 1. Labelling.2 g/l. The test is invalid if 1 or more control mice die following histamine challenge. The acellular pertussis component may also contain filamentous haemagglutinin. (7) that the vaccine must be shaken before use. Tetanus. Pertussis (Acellular Component). The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. Pyrogens (2.95) of the estimated potency is not less than the minimum potency stated on the label. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose. If 1 mouse dies in either or both of the first and second groups. Hepatitis B component The vaccine complies with the assay as stated under Hepatitis B Vaccine (rDNA). (6) the name and the amount of the adsorbent.2.2 per cent w/v of gelatin and challenge with histamine as above. Antimicrobial preservative. tetanus formol toxoid. P. INACTIVATED POLIOMYLITIS VACCINE AND HAEMOPHILUS TYPE B CONJUGATE base intraperitoneally in a volume not exceeding 0. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties produced by expression of a genetically modified form of the corresponding gene. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). (2) the names and amounts of the pertussis components per single human dose.3. Pertussis component 9 The vaccine complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component). Aluminium (2. Maximum 0. Inactivated Poliomyelitis Vaccine and Haemophilus Type b Conjugate Vaccine Diphtheria. The product may be presented with the haemophilus type b component in a separate container.0 per cent and not greater than 115. Where applicable.5 ml and observe for 24 h.8). Pertussis toxoid is prepared from pertussis toxin by a method that renders the toxin harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. the strain is suitable if more than 50. (3) the amount of HBsAg per single human dose. The lower confidence limit (P = 0. the contents of which are mixed with the other components immediately before use. (8) that the vaccine is not to be frozen (9) where applicable.3. (4) the type of cells used for production of the hepatitis B component. when conjugated to PRP. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine. pertussis and poliomyelitis components are carried out on a suitable number of batches of vaccine reconstituted for use. Reference vaccine(s) Provided valid assays can be performed. Antimicrobial preservative. .2.0 per cent of the intended amount. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. the assays of these components may be carried out without mixing with the haemophilus component. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Pertussis Vaccine (Acellular Component. D. Determine on the poliomyelitis components by a suitable immunochemical method (2. the content of bacterial endotoxins is less than the limit approved for the particular vaccine and. the vaccine does not comply with the test. INACTIVATED POLIOMYLITIS VACCINE AND HAEMOPHILUS TYPE B CONJUGATE IP 2007 membrane protein) and other defined components of B. COMPONENT).14) during preparation of the final bulk vaccine. P. if tested. monocomponent reference vaccines may be used for the assays on the combined vaccine. tetanus toxoid. that have not previously been treated with any material that will interfere with the test. with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. Inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs. determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85. would comply with the following test. Production of the components Production General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. toxaemia or tetanus. separately or together. As part of consistency studies the assays of the diphtheria. in any case. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria.0 per cent and not greater than 115. before addition of the adsorbent.A. During development studies and wherever revalidation is necessary. FINAL BULK VACCINE The final bulk of the diphtheria. Suitable antimicrobial preservatives may be added. 2 and 3 or a suitable quantity of a trivalent pool of such monovalent harvests. Tetanus Vaccine (Adsorbed). pertussis components. tetanus. T. repeat the test once. For each component. each weighing between 250 and 350 g. The carrier protein.2. purified. pertussis such as fimbrial-2 and fimbrial-3 antigens. For subsequent routine control. strict adherence to the production process used for the batch tested in clinical trials is necessary. the contents are such that the final vaccine contains less than 100 IU per single human dose. if more than 1 animal dies in the second test. of suitable quantities of bulk purified diphtheria toxoid. tetanus. Where applicable. the amount of bovine serum albumin is such that the content in the final vaccine will not be more than 50 ng per single human dose. monovalent harvests of human polioviruses 1. a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. 10 The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed). Adsorbed). For the preparation of a representative batch. If more than 1 animal dies from nonspecific causes. (ACELL. The latter 2 antigens may be co-purified. The content of bacterial endotoxins in bulk purified diphtheria toxoid. PRP is a linear copolymer composed of repeated units of 3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n]. the vaccine does not comply with the test. inactivated monovalent poliovirus harvests and bulk PRP conjugate is determined to monitor the purification procedure and to limit the amount in the final vaccine. Sterility (2. bulk purified tetanus toxoid and bulk purified acellular pertussis components onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate and admixture of suitable quantities of purified. Poliomyelitis Vaccine (Inactivated) and Haemophilus Type b Conjugate Vaccine. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. is capable of inducing a Tcell-dependent B-cell immune response to the polysaccharide. The production method is validated to demonstrate that the product.11). A stabiliser may be added. it shall be demonstrated by tests in animals that the vaccine induces a T-cell dependent B-cell immune response to PRP. Bovine serum albumin. Carry out test for sterility using 10 ml of bulk for each sterility medium. pertussis and poliomyelitis components is prepared by adsorption. is within the limits approved for the particular preparation. it may be omitted on the final lot. (ACELL. identification test E is carried out on the vial containing the haemophilus component. Inject diluent into the third group of mice. the test for antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine. The clear supernatant obtained during identification test A reacts with a suitable tetanus antitoxin. tetanus. irreversibility of pertussis toxoid. After 5 days. sterility and pyrogens are carried out on the container with the haemophilus component.14). Tests and Assay may be released for use. The test is invalid if 1 or more control mice die following histamine challenge. Diphtheria toxoid is identified by a suitable immunochemical method (2.4.4.2. The vaccine is shown to contain human polioviruses 1. D. The following method. giving a precipitate. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0. A. The amount of free PRP is not greater than that approved for the particular product. INACTIVATED POLIOMYLITIS VACCINE AND HAEMOPHILUS TYPE B CONJUGATE FINAL LOT The final bulk of the haemophilus component is freeze-dried. is given as an example. T. applicable to certain vaccines. Tests The tests for absence of residual pertussis toxin. The pertussis components are identified by suitable . the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined. aluminium. C. B. applicable to certain vaccines. immunochemical methods (2. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. Osmolality (2. 2 and 3 by a suitable immunochemical method (2. Provided that the test for absence of residual pertussis toxin and irreversibility of pertussis toxoid. is given as an example. such as determination of D-antigen by enzyme-linked mmunosorbent assay (ELISA).23).16). the vaccine complies with the test if. for example by anion-exchange. Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. C and D are carried out using the vial containing the diphtheria. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Tetanus toxoid is identified by a suitable immunochemical method (2. the test for free formaldehyde may be omitted on the final lot. E. Some tests for the haemophilus component may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. The clear supernatant reacts with a suitable diphtheria antitoxin.2. water.14) for PRP.2 g/l. The following method. Free PRP Unbound PRP is determined on the haemophilus component after removal of the conjugate. the 11 Identification Identification tests A. tetanus. they may be omitted on the final lot. P. applicable to certain vaccines. ultrafiltration or other validated methods. Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification. size-exclusion or hydrophobic chromatography (2. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine. free formaldehyde. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2° to 8°.14). The haemophilus component is identified by a suitable immunochemical method (2.5 ml and observe for 24 hours. antimicrobial preservative and sterility are carried out on the container with the diphtheria. inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0. Provided that the free formaldehyde content has been determined on the bulk purified antigens and the purified monovalent harvests or the trivalent pool of polioviruses or the final bulk and it has been shown that the content in the final lot will not exceed 0. pertussis and poliomyelitis components. reconstituted where applicable.IP 2007 A. giving a precipitate. Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37° for 4 weeks. The osmolality of the vaccine. The clear supernatant obtained during identification test A reacts with specific antisera to the pertussis components of the vaccine.14). The following method. is given as an example.2. pertussis and poliomyelitis components. Use 3 groups each of not fewer than 5 histaminesensitive mice. D. If 1 mouse dies in either or both of the first and second groups. B. the tests for PRP content. in both of the groups given the vaccine.14). Maintain at 37° for about 16 hours and centrifuge until a clear supernatant is obtained.2. COMPONENT).2 per cent w/v of gelatin and challenge with histamine as above. not more than 5 per cent of the total number of mice die following histamine challenge.2. (6) the type and nominal amount of carrier protein per single human dose.0 per cent of the amount of PRP stated on the label. Complies with the test for pyrogens. tetanus formol toxoid. (5) the number of micrograms of PRP per single human dose.14) using a reference preparation calibrated in units of D-antigen. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. expressed with reference to the amount of D-antigen stated on the label. the lower confidence limit (P = 0.0 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. individually purified antigenic components of Bordetella pertussis.3.2. Complies with the test for sterility. P.95) of the estimated potency is not less than 30 IU per single human dose. determine the Dantigen content for human polioviruses 1. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). (10) that the vaccine is not to be frozen. Aluminium (2.14) or by anion-exchange liquid chromatography with pulsedamperometric detection.0 per cent for the haemophilus component.2. For each type. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent.3. Free formaldehyde (2. Pertussis (Acellular Component) and Inactivated Poliomyelitis Vaccine Diphtheria. Inject per kg of the rabbit’s mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier.7. (ACELLULAR. Poliomyelitis component D-antigen content As a measure of consistency of production.20). COMPONENT) AND INACTIVATED POLIOMYLITIS VACCINE IP 2007 strain is suitable if more than 50. Maximum 3. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). 2 and 3). Minimum 80. 0. Labelling. Pertussis (Acellular Component) and Poliomyelitis (Inactivated) Vaccine is a combined vaccine containing: diphtheria formol toxoid. is within the limits approved for the particular product.1) or phosphorus ( 2. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties produced by expression of a genetically modified form of the corresponding gene. The lower confidence limit (P = 0. Maximum 0. (11) where applicable. (4) the type of cells used for production of the poliomyelitis component. The Unit and the International Unit are equivalent.3. that the vaccine contains a pertussis toxin-like protein produced by genetic modification. the content. (7) where applicable. T. by an immunochemical method ( 2.7. Tetanus. (3) the nominal amount of poliovirus of each type (1. (8) the name and the amount of the adsorbent.43).2. 2 and 3 grown in suitable cell cultures and inactivated by a validated method.1 mg of PRP for a vaccine with tetanus toxoid as carrier.8). Pertussis component It complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component). The content is not less than 85. Water (2. a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. Pyrogens (2.95) of the estimated potency is not less than 40 IU per single human dose.025 mg of PRP for a vaccine with OMP as a carrier. Unless otherwise justified and authorised.2. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and intended for use in the assay of D-antigen. PRP. 0.1). (9) that the vaccine must be shaken before use. Pertussis toxoid is prepared from pertussis toxin by a method that renders the toxin harmless while maintaining .2 g/l. Tetanus. 2 and 3 by a suitable 12 immunochemical method (2.A. Adsorbed Diphtheria. Sterility (2. Antimicrobial preservative.9). suitable strains of human polioviruses 1. determine the amount of antimicrobial preservative by a suitable chemical method.0 per cent and not greater than 115. D. PRP is determined either by assay of ribose ( 2. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose (2) the names and amounts of the pertussis components per single human dose.25 mg per single human dose. Maximum 1.0 per cent of the intended amount.11 ). Where applicable. that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults. In vivo test The vaccine complies with the in vivo assay as stated under Inactivated Poliomyelitis Vaccine. expressed in units of D-antigen per single human dose. pertussis such as fimbrial2 and fimbrial-3 antigens. the contents are such that the final vaccine contains less than 100 IU per single human dose. The vaccine may also contain filamentous haemagglutinin. they may be omitted on the final lot.4. Provided the tests for absence of residual pertussis toxin. Antimicrobial preservative. a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. of suitable quantities of 13 Identification A. pertussis components and purified.11). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. the amount of bovine serum albumin is such that the content in the final vaccine will be not more than 50 ng per single human dose. For the preparation of a representative batch.IP 2007 A. Diphtheria toxoid is identified by a suitable immunochemical . 2 and 3 or a suitable quantity of a trivalent pool of such purified monovalent harvests.2 g/l. (ACELLULAR. P. Sterility (2. Determine on the poliomyelitis components by a suitable immunochemical method (2. pertactin (a 69 kDa outer-membrane protein) and other defined components of B.14) after virus harvest and before addition of the adsorbent in the preparation of the final bulk vaccine. tetanus toxoid. The latter 2 antigens may be copurified. For each component. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Osmolality (2. Where applicable. would comply with the test for abnormal toxicity for antisera and vaccines. the content of bacterial endotoxins is less than the limit approved for the particular vaccine and. COMPONENT) AND INACTIVATED POLIOMYLITIS VACCINE adequate immunogenic properties and avoiding reversion to toxin. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed). in any case.2. strict adherence to the production process used for the batch tested in clinical trials is necessary. Pertussis Vaccine (Acellular Component. Adsorbed) and Poliomyelitis Vaccine (Inactivated). Suitable antimicrobial preservatives may be added.0 per cent of the intended amount.0 per cent and not greater than 115. if tested. The osmolality of the vaccine is within the limits approved for the particular preparation. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification. bulk purified diphtheria toxoid. Carry out test for sterility using 10 ml of bulk for each sterility medium. tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine. Reference vaccine(s) Provided valid assays can be performed. the test for free formaldehyde may be omitted on the final lot. T. Production General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine. The production method is validated to demonstrate that the product. it may be omitted on the final lot. Bovine serum albumin. D. The content of bacterial endotoxins in bulk purified diphtheria toxoid.2. Tests and Assay may be released for use. separately or together.23). irreversibility of pertussis toxoid and antimicrobial preservative and the assays for the diphtheria. Tetanus Vaccine (Adsorbed). The content is not less than 85. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. tetanus toxoid. acellular pertussis components and admixture of suitable quantities of purified monovalent harvests of human polioviruses 1. monocomponent reference vaccines may be used for the assays on the combined vaccine. inactivated monovalent poliovirus harvests is determined to monitor the purification procedure and to limit the amount in the final vaccine. Provided that the determination of D-antigen content has been carried out with satisfactory results during preparation of the final bulk before addition of the adsorbent. Provided the free formaldehyde content has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0. it may be omitted on the final lot. determine the amount of antimicrobial preservative by a suitable chemical method. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.5 ml and observe for 24 hours. is given as an example.14). For each type. the content. The Unit and the International Unit are equivalent.3. expressed with reference to the amount of D-antigen stated on the label. D. applicable to certain vaccines. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and intended for use in the assay of D-antigen. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). Aluminium (2. The clear supernatant obtained as described in Identification test A reacts with a specific antisera to the pertussis components of the vaccine. applicable to certain vaccines. is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. 2 and 3 by a suitable immunochemical method (2.14) such as the determination of D-antigen by enzyme-linked immunosorbent assay (ELISA).0 per cent of the total number of mice die following histamine challenge.0 per cent of the animals are .14) following desorption using a reference preparation calibrated in units of D-antigen. Antimicrobial preservative. The pertussis components are identified by a suitable immunochemical method (2. Sterility (2. applicable to certain vaccines.0 per cent of the intended amount. inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.25 mg per single human dose if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. The lower confidence limit (P = 0. Maximum 0. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2° to 8°.2. the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined. sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine shows symptoms of sensitisation. giving a precipitate. Inject diluent into the third group of mice. The following method. COMPONENT) AND INACTIVATED POLIOMYLITIS VACCINE IP 2007 method (2. The lower confidence limit (P = 0. B.2 g/l. (ACELLULAR. is given as an example. T. Poliomyelitis component D-antigen content As a measure of consistency of production. In vivo test The vaccine complies with the in vivo assay as stated under Inactivated Poliomyelitis Vaccine.95) of the estimated potency is not less than the minimum potency stated on the label. Free formaldehyde (2. is within the limits approved for the particular product. 14 Tests Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. 2 and 3 by a suitable immunochemical method (2. The content is not less than 85.A.2 per cent w/v of gelatin and challenge with histamine as above.20).14). P.11). Tetanus toxoid is identified by a suitable immunochemical method (2. Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37° for 4 weeks. The test is invalid if 1 or more control mice die following histamine challenge. Pertussis component The vaccine complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component).2. D. Complies with the test for sterility. The following method. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). Use 3 groups each of not less than 5 histaminesensitive mice. C. in both of the groups given the vaccine. If 1 mouse dies in either or both of the first and second groups. The clear supernatant reacts with a suitable diphtheria antitoxin. The vaccine is shown to contain human polioviruses 1. determine the amount of antimicrobial preservative by a suitable chemical method. the minimum potency stated on the label is 30 IU per single human dose.2.2. the strain is suitable if more than 50.3. Maintain at 37° for about 16 h and centrifuge until a clear supernatant is obtained. not more than 5. The following method. After 5 days. The clear supernatant obtained as described in Identification test A reacts with a suitable tetanus antitoxin. Maximum 1.95) of the estimated potency is not less than 40 IU per single human dose. determine the Dantigen content for human polioviruses 1.0 per cent and not greater than 115.2. the vaccine complies with the test if. Unless otherwise justified and authorised.14). giving a precipitate.2.9). Where applicable. The label complies with the requirements stated under Vaccine and also states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose (2) the names and amounts of the pertussis components per single human dose. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus. (10) that the vaccine is not to be frozen. Pertussis and Poliomyelitis (Inactivated) Vaccine (Adsorbed) is a combined vaccine containing: diphtheria formol toxoid. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. separately or together. Pertussis Vaccine (Adsorbed) and Poliomyelitis Vaccine (Inactivated). (6) the type and nominal amount of carrier protein per single human dose. repeat the test once. specific causes. each weighing between 250 and 350 g. Pertussis and Poliomyelitis (Inactivated) Vaccine Diphtheria. Tetanus Vaccine (Adsorbed). 2 and 3 or a suitable quantity of a trivalent pool of such purified monovalent harvests. expressed in units of Dantigen per single human dose. (4) the type of cells used for production of the poliomyelitis component. that the vaccine contains a pertussis toxin-like protein produced by genetic modification.9 per cent sterile solution of sodium chloride. The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed). Production of the components Adsorbed Diphtheria. (5) the number of micrograms of PRP per single human dose. if more than 1 animal dies in the second test. an inactivated suspension of Bordetella pertussis. (3) the nominal amount of poliovirus of each type (1.IP 2007 ADSORBED DIPHTHERIA TETANUS PERTUSSIS AND POLIOMYLITIS (INACTIVATED) VACCINE Labelling. Tetanus. (8) the name and the amount of the adsorbent. that have not previously been treated with any material that will interfere with the test. Inject each mouse of the control group with 0. Inject each mouse of the vaccine group intraperitoneally with 0. Tetanus. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine. (7) where applicable. (9) that the vaccine must be shaken before use. (11) where applicable. the vaccine does not comply with the test. If more than 1 animal dies from non- . pertussis and purified monovalent harvests of human polioviruses 1. Specific toxicity Use not less than 5 healthy mice each weighing between 14 and 16 g for the vaccine group and for the saline control. containing a quantity of the vaccine equivalent to not less than half the single human dose. preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. Use mice of the same sex or distribute males and females equally between the groups. For the preparation of a representative batch. that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults. 2 and 3). The production method is validated to demonstrate that the product. Weigh the groups of mice immediately before the injection and 72 hours and 7 days after the injection. would comply with the test for abnormal toxicity for antisera and vaccines. the vaccine does not comply with the test. Suitable antimicrobial preservatives may be added. of suitable quantities of bulk purified diphtheria toxoid and bulk purified tetanus toxoid and admixture of suitable quantities of an inactivated suspension of B. a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. monocomponent reference vaccines may be used for the assays on the combined vaccine. suitable strains of human polioviruses 1. strict adherence to the production process used for the batch tested in clinical trials is necessary. 2 and 3 grown in suitable cell cultures and inactivated by a validated method. and with the following test for specific toxicity of the diphteria and tetanus components : inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs.5 ml. a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. Reference vaccine(s) Provided valid assays can be performed. if tested. tetanus formol toxoid. Allow the animals access to food and water for at least 2 hours before injection and during the test. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure.5 ml of a 0. The vaccine 15 Production General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. Where applicable.2.9). Complies with the test for sterility.ADSORBED DIPHTHERIA TETANUS PERTUSSIS AND POLIOMYLITIS (INACTIVATED) VACCINE IP 2007 complies with the test if: (a) at the end of 72 hours the total mass of the group of vaccinated mice is not less than that preceding the injection. The content is not less than 85. they may be omitted on the final lot. Tetanus toxoid is identified by a suitable immunochemical method (2. Where applicable. The following method. Maximum 1. The osmolality of the vaccine is within the limits approved for the particular preparation. Maximum 0. if the test is carried out in mice. applicable to certain vaccines. The estimated potency is not less than 4 IU per single human 16 Identification A.2.2 g/l. pertussis suspension and the purified monovalent harvests or the trivalent pool of polioviruses or on the final bulk and it has been shown that the content in the final lot will not exceed 0. Identify pertussis vaccine by agglutination of the bacteria from the resuspended precipitate by antisera specific to B. (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 per cent of that per control mouse.23).2.20).14).3.14) such as the determination of D-antigen by enzyme-linked immunosorbent assay (ELISA). Sterility (2. Diphtheria toxoid is identified by a suitable immunochemical method (2. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification. B.2. determine the amount of antimicrobial preservative by a suitable chemical method.14).4.0 per cent of the intended amount. reacts with a suitable diphtheria antitoxin. 2 and 3 by a suitable immunochemical method (2.2 g/l. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed).2. The lower confidence limit (P = 0.14) during preparation of the final bulk vaccine. is given as an example. The content is not less than 85. Sterility (2.2. Pertussis component Carry out the assay as stated under Pertussis Vaccine. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine. Antimicrobial preservative. giving a precipitate. the lower confidence limit (P = 0. The following method. Antimicrobial preservative. it may be omitted on the final lot. Tests Aluminium (2.25 mg per single human dose. Other suitable methods for separating the bacteria from the adsorbent may also be used. The test may be repeated and the results of the tests combined.11). pertussis or by the assay of the pertussis component prescribed under Assay. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). D. Determine on the poliomyelitis components by a suitable immunochemical method (2. determine the amount of antimicrobial preservative by a suitable chemical method. and the assays for the diphtheria. Bovine serum albumin. Provided that the tests for specific toxicity and antimicrobial preservative. Free formaldehyde (2.3.0 per cent and not greater than 115. the inactivated B.0 per cent and not greater than 115. tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine. Maintain at 37° for about 16 hours and centrifuge until a clear supernatant is obtained. giving a precipitate. If the test is carried out in guinea pigs. before addition of the adsorbent. the lower confidence limit (P = 0. The centrifugation residue obtained in identification A may be used. Carry out test for sterility using 10 ml of bulk for each sterility medium. The clear supernatant .0 per cent of the intended amount. the test for free formaldehyde may be omitted on the final lot.95) of the estimated potency is not less than 60 IU per single human dose. The clear s u p e r n a t a n t obtained during identification test A reacts with a suitable tetanus antitoxin. and (c) not more than 5 per cent of the vaccinated mice die during the test. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Tests and Assay may be released for use.11). applicable to certain vaccines. The vaccine is shown to contain human polioviruses 1.95) of the estimated potency is not less than 40 IU per single human dose. Provided that the free formaldehyde content has been determined on the bulk purified antigens.95) of the estimated potency is not less than 30 IU per single human dose. Osmolality (2. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. is given as an example. the amount of bovine serum albumin is such that the content in the final vaccine will be not more than 50 ng per single human dose. C. Reference vaccine(s) Provided valid assays can be performed. The product is presented with the haemophilus component in a separate container. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. Production General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. PRP is a linear copolymer composed of repeated units of 3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n]. Tetanus. expressed in units of D-antigen per single human dose. it shall be demonstrated by tests in animals that the vaccine induces a T-cell dependent B-cell immune response to PRP. polyribosylribitol phosphate (PRP) covalently bound to a carrier protein. a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b.2. Pertussis.IP 2007 A.14) using a reference preparation calibrated in Units of D-antigen. 2 and 3). For subsequent routine control. the vaccine does not comply with the test. For the preparation of a representative batch. suitable strains of human polioviruses 1. monocomponent reference vaccines may be used for the assays on the combined vaccine. tetanus. each weighing between 250 and 350 g. Poliomyelitis (Inactivated) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid. the assays of these components may be carried out without mixing with the haemophilus component. tetanus formol toxoid. a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. if more than 1 animal dies in the second test. For each type. pertussis and poliomyelitis components are carried out on a suitable number of batches of vaccine reconstituted for use. The Unit and the IU are equivalent. T.. that have not previously been treated with any material that will interfere with the test. (5) where applicable. Pertussis. The carrier protein. D. the content. (3) the nominal amount of poliovirus of each type (1. . (7) that the vaccine must be shaken before use. when conjugated to PRP. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose. (4) the type of cells used for production of the poliomyelitis component. As part of consistency studies the assays of the diphtheria. If more than 1 animal dies from nonspecific causes. if tested. (8) that the vaccine is not to be frozen. The production method is validated to demonstrate that the product. that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults. expressed with reference to the amount of D-antigen stated on the label. 17 Adsorbed Diphtheria. During development studies and wherever revalidation is necessary. and with the following test for specific toxicity of the diphtheria and tetanus components: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs. is within the limits approved for the particular product. P. (6) the name and the amount of the adsorbent. is capable of inducing a Tcell-dependent B-cell immune response to the polysaccharide.. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus. Poliomyelitis component D-antigen content As a measure of consistency of production. Tetanus. would comply with the test for abnormal toxicity for antisera and vaccines. the vaccine does not comply with the test.95) of the estimated potency is not less than 2 IU per single human dose. POLIOMYLITIS (INACTIVATED) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE dose and the lower confidence limit (P = 0. an inactivated suspension of Bordetella pertussis. repeat the test once. Labelling. the contents of which are mixed with the other components immediately before use. (2) the minimum number of International Units of pertussis vaccine per single human dose. 2 and 3 grown in suitable cell cultures and inactivated by a suitable method. Poliomyelitis (Inactivated) and Haemophilus Type b Conjugate Vaccine Diphtheria. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and is intended for use in the assay of D-antigen. In vivo test The vaccine complies with the in vivo assay as stated under Poliomyelitis Vaccine (Inactivated).. strict adherence to the production process used for the batch tested in clinical trials is necessary. 2 and 3 by a suitable immunochemical method (2. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine. determine the Dantigen content for human polioviruses 1. 4. reconstituted where applicable. identification test E is carried out on the vial containing the haemophilus component. Suitable antimicrobial preservatives may be added. (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 per cent of that per control mouse. POLIOMYLITIS (INACTIVATED) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE IP 2007 The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. pertussis suspension and the purified monovalent harvests or the trivalent pool of polioviruses or on the final bulk and it has been shown that the content in the final lot will not exceed 0. tetanus.2.11). for example by anion-exchange. B. Provided that the free formaldehyde content has been determined on the bulk purified antigens. the amount of bovine serum albumin is such that the content in the final vaccine will not be more than 50 ng per single human dose. Pertussis Vaccine (Adsorbed). Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification. Tests and Assay may be released for use. A stabiliser may be added. Provided that the tests for specific toxicity and antimicrobial preservative.4. pertussis and poliomyelitis components is prepared by adsorption. size-exclusion or hydrophobic chromatography (2.2.5 ml. and bulk purified tetanus toxoid onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate and admixture of suitable quantities of an inactivated suspension of B. Allow the animals access to food and water for at least 2 hours before injection and during the test. FINAL LOT The final bulk of the haemophilus component is freeze-dried.. The following method.0 per cent of the intended amount.14) during preparation of the final bulk vaccine. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. The amount of free PRP is not greater than that approved for the particular product. tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine. Carry out test for sterility using 10 ml of bulk for each sterility medium. is within the limits approved for the particular preparation.. Use mice of the same sex or distribute males and females equally between the groups. and (c) not more than 5 per cent of the vaccinated mice die during the test. the inactivated B. Production of components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed). Only final bulk that complies with the following requirements may be used in the preparation of the final lot. The osmolality of the vaccine. containing a quantity of the vaccine equivalent to not less than half the single human dose. pertussis and poliomyelitis components. FINAL BULK VACCINE The final bulk of the diphtheria.2. A. tetanus. Tetanus Vaccine (Adsorbed). Where applicable. Osmolality (2.23). D. Identification Identification tests A. before addition 18 of the adsorbent.A. Inject each mouse of the control group with 0. Bovine serum albumin.2 g/l. Sterility (2.9 per cent sterile solution of sodium chloride. Specific toxicity Use not less than 5 healthy mice each weighing between 14 and 16 g.. The vaccine complies with the test if (a) at the end of 72 hours the total mass of the group of vaccinated mice is not less than that preceding the injection. monovalent harvests of human polioviruses 1. T. Antimicrobial preservative. P. Free PRP Unbound PRP is determined on the haemophilus component after removal of the conjugate. they may be omitted on the final lot. Inject each mouse of the vaccine group intraperitoneally with 0. of suitable quantities of bulk purified diphtheria toxoid.16). ultrafiltration or other validated methods. for the vaccine group and for the saline control. applicable to certain . The content is not less than 85. and the assays for the diphtheria.0 per cent and not greater than 115. 2 and 3 or a suitable quantity of a trivalent pool of such monovalent harvests. it may be omitted on the final lot.5 ml of a 0. Diphtheria toxoid is identified by a suitable immunochemical method (2. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine. Weigh the groups of mice immediately before the injection and 72 hours and 7 days after the injection.14). Determine on the poliomyelitis components by a suitable immunochemical method (2. determine the amount of antimicrobial preservative by a suitable chemical method. the test for free formaldehyde may be omitted on the final lot. separately or together. pertussis and of purified. Poliomyelitis Vaccine (Inactivated) and Haemophilus Type b Conjugate Vaccine. preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. The test may be repeated and the results of the tests combined. C and D are carried out using the vial containing the diphtheria. . The Unit and the IU are equivalent. Aluminium (2.14) using a reference preparation calibrated in Units of D-antigen. antimicrobial preservative and sterility are carried out on the container with diphtheria.95) of the estimated potency is not less than 60 IU per single human dose.9).2. the tests for PRP content.25 mg per single human dose. E. 2 and 3 by a suitable immunochemical method (2. POLIOMYLITIS (INACTIVATED) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE vaccines. aluminium.2.3.14) for PRP. water.3. the lower confidence limit (P = 0.025 mg of PRP for a vaccine with OMP as carrier. The vaccine is shown to contain human polioviruses 1.43). D. if the test is carried out in mice.IP 2007 A. Other suitable methods for separating the bacteria from the adsorbent may also be used. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose. If the test is carried out in guinea-pigs. Minimum 80. is given as an example. applicable to certain vaccines. In vivo test The vaccine complies with the in vivo assay as stated under Poliomyelitis Vaccine (Inactivated).1). Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. For each type. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). free formaldehyde.0 per cent of the intended amount. Antimicrobial preservative. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and intended for use in the assay of D-antigen.14). expressed with reference to the amount of D-antigen stated on the label.7. Sterility (2. pertussis and poliomyelitis components. Inject per kg of the rabbit’s mass a quantity of the vaccine equivalent to 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier. Complies with the test for sterility. Poliomyelitis component D-antigen content As a measure of consistency of production. expressed in 19 Tests The tests for specific toxicity. Where applicable. Pertussis component Carry out the assay as stated under Pertussis Vaccine. Complies with the test for pyrogens. the lower confidence limit (P = 0. Pyrogens (2. such as determination of D-antigen by enzymelinked immunosorbent assay (ELISA).14) or by anion-exchange liquid chromatography with pulsedamperometric detection. is given as an example. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). the content.20).2. The centrifugation residue obtained in identification A may be used.2.8).1) or phosphorus (2. determine the Dantigen content for human polioviruses 1.11). PRP is determined either by assay of ribose (2. determine the amount of antimicrobial preservative by a suitable chemical method. 2 and 3 by a suitable immunochemical method (2. Labelling.0 per cent of the amount of PRP stated on the label. The lower confidence limit (P = 0. pertussis or by the assay of the pertussis component prescribed under Assay.. P.95) of the estimated potency is not less than 2 IU per single human dose. 2 and 3). The estimated potency is not less than 4 IU per single human dose and the lower confidence limit (P = 0.. Identify pertussis vaccine by agglutination of the bacteria from the resuspended precipitate by antisera specific to B. Maximum 3. sterility and pyrogens are carried out on the container with the haemophilus component. C. T. D.2. Water (2.2.14). if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent.0 per cent and not greater than 115. 0.1 mg of PRP for a vaccine with tetanus toxoid as carrier.. 0. is within the limits approved for the particular product. The clear s u p e r n a t a n t obtained during identification test A reacts with a suitable tetanus antitoxin. Tetanus toxoid is identified by a suitable immunochemical method (2. The content is not less than 85. The clear supernatant reacts with a suitable diphtheria antitoxin. by an immunochemical method (2.0 per cent for the haemophilus component.7. tetanus. Maintain at 37° for about 16 hours and centrifuge until a clear supernatant is obtained. Free formaldehyde (2. PRP. giving a precipitate. (2) the minimum number of International Units of pertussis vaccine per single human dose.2.3. Some tests for the haemophilus component may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test.95) of the estimated potency is not less than 30 IU per single human dose. B. The haemophilus component is identified by a suitable immunochemical method (2. giving a precipitate. Maximum 0. The following method. (3) the nominal amount of poliovirus of each type (1.2 g/l.95) of the estimated potency is not less than 40 IU per single human dose. Maximum 1. ADSORBED PERTUSSIS VACCINE (ACELLULAR COMPONENT) IP 2007 Units of D-antigen per single human dose. (9) that the vaccine is not to be frozen. Pertussis toxoid The toxoid induces in animals production of antibodies capable of inhibiting all the properties of pertussis toxin. (9) that the vaccine must be shaken before use. No dermonecrotic reaction is demonstrable. in a volume of 0. Adenylate cyclase. particularly on storage or exposure to heat. CHARACTERISATION OF COMPONENTS During development of the vaccine. None of the media used at any stage contains blood or blood products of human origin. Pertussis toxin Chinese hamster ovary (CHO) cell-clustering effect and haemagglutination as in vitro methods. Tracheal cytotoxin. by a method that avoids reversion of the toxoid to toxin. Absence of residual dermonecrotic toxin. after demonstration of consistency. The components of the vaccine are analysed by one or more of the methods shown below in order to determine their identity and specific properties (activity per unit amount of protein) in comparison with reference preparations. Specific properties. detoxified using suitable chemical reagents. lymphocytosispromoting activity. PURIFIED COMPONENTS Production of each component is based on a seed-lot system. Not more than 2 pmol in the equivalent of 1 dose of the final vaccine. The vaccine may also contain filamentous haemagglutinin. determined by a suitable method such as a biological assay or liquid chromatography (2. (4) the type of cells used for production of the poliomyelitis component. Not more than 500 ng in the equivalent of 1 dose of the final vaccine. produced by expression of a genetically modified form of the corresponding gene. Reference vaccine A batch of vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. (6) the type and nominal amount of carrier protein per single human dose. strict adherence to the production process used for the batch tested in clinical trials is necessary. pertussis such as fimbrial2 and fimbrial-3 antigens. pertactin (a 69 kDa outer-membrane protein) and other defined components of B. Filamentous haemagglutinin Haemagglutination and inhibition by specific antibody. where applicable. determined by immunoblot analysis or another suitable method.14). histamine-sensitising activity and insulin secretory activity as in vivo methods. The toxin shows ADPribosyl transferase activity using transducin as the acceptor. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. (8) the name and the amount of the adsorbent. Observe for 48 hours. Production General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. the production process shall be validated to demonstrate that it yields consistently . Other components such as fimbrial-2 and fimbrial-3 antigens are purified either 20 Adsorbed Pertussis Vaccine (Acellular Component) Pertussis Vaccine (Acellular Component. The latter 2 antigens may be copurified. The reference vaccine is preferably stabilised by a method that has been shown to have no significant effect on the assay procedure when the stabilised and non-stabilised batches are compared.4. (5) the number of micrograms of PRP per single human dose. Pertactin. the amount of component or antigenic fraction equivalent to 1 dose of the final vaccine.1 ml. like protein free from toxic properties. that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults. Reactivity with specific antibody. Adsorbed) is a preparation of individually prepared and purified antigenic components of Bordetella pertussis adsorbed on a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. fimbrial-2 and fimbrial-3 antigens. The vaccine contains either pertussis toxoid or a pertussis toxin. The seed cultures from which toxin is prepared are managed to conserve or where necessary restore toxinogenicity by deliberate selection. the tests need not be applied routinely to each batch. Inject intradermally into each of 3 unweaned mice. individual components that comply with the following requirements. (7) where applicable. filamentous haemagglutinin and pertactin are purified and. Media used for the preparation of seed lots and inocula may contain blood or blood products of animal origin. Pertussis toxoid is prepared from pertussis toxin by a method that renders the latter harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. Pertussis toxin and. For the preparation of a representative batch. after appropriate characterisation. Only purified components that comply with the following requirements may be used in the preparation of the final bulk vaccine. determine the amount of antimicrobial preservative by a suitable chemical method.5 ml and observe for 24 hours. the preparation complies with the test.2 per cent w/v of gelatin. the purity of the components is determined by a suitable method such as polyacrylamide gel electrophoresis (PAGE) or liquid chromatography.14). Sterility (2. Carry out the test for sterility using for each medium a quantity of purified component equivalent to not less than 100 doses. Examined by a suitable immunochemical method (2. Use 3 groups each of not fewer than 5 histaminesensitive mice. Inject into each mouse the equivalent of 1 human dose intravenously or twice the human dose intraperitoneally. A validated test based on the clustering effect of the toxin for Chinese hamster ovary (CHO) cells may be used instead of the test on mice. Residual detoxifying agents and other reagents The content of residual detoxifying agents and other reagents is determined and shown to be below approved limits unless 21 validation of the process has demonstrated acceptable clearance. Provided that the tests for absence of residual pertussis toxin and irreversibility of pertussis toxoid.30) or another suitable method. FINAL BULK VACCINE The vaccine is prepared by adsorption of suitable quantities of purified components.2.2. SDS-PAGE or immunoblot analysis with specific monoclonal or polyclonal antibodies may be used to characterise subunits. A suitable antimicrobial preservative may be added. The content of bacterial endotoxins is determined to monitor the purification procedure and to limit the amount in the final vaccine. Use a group of not fewer than 5 histaminesensitive mice each weighing between 18 and 26 g. antimicrobial preservative. Before detoxification. Carry out test for sterility using 10 ml of bulk for each sterility medium. free formaldehyde and the assay have been carried out with satisfactory results on the final bulk vaccine. the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. characterised and shown to be free from toxic substances. Inject intraperitoneally into the first group twice .2 per cent w/v of gelatin and challenge with histamine as above. The limits applied for the individual components are such that the final vaccine contains less than 100 IU per single human dose.2. After 5 days. If no animal dies. The content is not less than 85.IP 2007 ADSORBED PERTUSSIS VACCINE (ACELLULAR COMPONENT) separately or together. Requirements are established for each individual product. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject three-fold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0. The purification procedure is validated to demonstrate appropriate clearance of substances used during culture or purification.0 per cent and not greater than 115. Inject diluent into a second group of control mice.0 per cent of the intended amount.11). Antigen content Determine the antigen content by a suitable immunochemical method (2. onto aluminium hydroxide or hydrated aluminium phosphate. Tests and Assay may be released for use. diluted to not more than 0.2.14) and protein nitrogen by sulphuric acid digestion (2. Sterility (2. Where applicable.11). maintain at 37° for about 16 h and centrifuge until a clear supernatant liquid is obtained.2. inject 2 mg of histamine base intraperitoneally in a volume not exceeding 0. Antimicrobial preservative. Tests Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. separately or together. these tests may be omitted on the final lot. Absence of residual pertussis toxin This test is not necessary for the product obtained by genetic modification. FINAL LOT Only a final lot that is satisfactory with respect to each of the requirements given below under Identification.5 ml with phosphate-buffered saline solution containing 0. Identification Subject the vaccine to a suitable desorption procedure such as the following: dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. The ratio of antigen content to protein nitrogen is within the limits established for the product. the clear supernatant liquid reacts with specific antisera to the components stated on the label. The test on mice shown below uses a three-point model but. Assay The capacity of the vaccine to induce the formation of specific antibodies is compared with the same capacity of a reference preparation examined in parallel. Store the sera at -20° until tested for antibody content. The label states (1) the names and amounts of the components present in the vaccine. The test conditions are designed to obtain a linear response for absorbance with respect to antibody content over the range of measurement used and absorbance values within the range 0. Two-fold dilutions of sera from mice immunised with test or reference vaccines are made on the plates. the vaccine complies with the test if. unreacted sites are blocked by incubating with a solution of bovine serum albumin and then washed. After incubation at 22° to 25° for 1 h. The content is not less than 85. A standardised control serum is also included in the test.0 per cent and not greater than 115. in both of the groups given the vaccine. Labelling. The following test model is given as an example of a method that has been found to be satisfactory. antibodies are determined using suitable immunochemical methods (2. bleed the mice individually under anaesthesia.95) less than that of the reference vaccine. CD1 strain) of the same stock 4 to 8 weeks old. Requirement The capacity to induce antibodies is not significantly (P = 0. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0. inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. Sterility (2.IP 2007 the single human dose of the vaccine stored at 2° to 8°. Maximum 1. determine the amount of antimicrobial preservative by a suitable chemical method. a substrate is added from which the bound enzyme conjugate liberates a chromophore which can be quantified by measurement of absorbance. Inject intraperitoneally or subcutaneously into each mouse 0. The test is invalid if 1 or more control mice die following histamine challenge.0 per cent. If 1 mouse dies in either or both of the first and second groups. After washing.2 g/l.3. Complies with the test for sterility. Aluminium (2.20). 22 Selection and distribution of test animals Use in the test healthy mice (for example. ELISA Microtitre plates (poly(vinyl chloride) or polystyrene as appropriate for the specific antigen) are coated with the purified antigen at a concentration of 100 ng per well.2 per cent w/v of gelatin and challenge with histamine as above. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent.0 per cent of the intended amount. Distribute the animals in 6 groups of a number appropriate to the requirements of the assay.9). that .14) such as enzyme-linked immunosorbent assay (ELISA). Inject diluent into the third group of mice.2. after validation. Where applicable.3. (b) the confidence interval of the potency estimate is greater than 50. After 5 days. (2) where applicable. not more than 5. Use 3 dilutions of the vaccine under examination and 3 dilutions of a reference preparation and attribute each dilution to a group of mice. the strain is suitable if more than 50. Maximum 0. A reference antiserum of assigned potency is used in the test and serves as the basis for calculation of the antibody levels in test sera. Antimicrobial preservative. Collection of serum samples 4 to 5 weeks after vaccination. After washing.2. Free formaldehyde (2. Antibody determination Assay the individual sera for content of specific antibodies to each component using a validated method such as the ELISA test shown below.0.0 per cent to 200.0 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. A suitable solution of anti-mouse IgG enzyme conjugate is added to each well and incubated at 22° to 25° for 1 h.1 to 2. The test is not valid if (a) the value found for the control serum differs by more than 2 standard deviations from the assigned value. Calculation The antibody titres in the sera of mice immunised with reference and test vaccines are calculated and from the values obtained the potency of the test vaccine in relation to the reference vaccine is calculated by the usual statistical methods.5 ml and observe for 24 hours.11). the plates are washed.5 ml of the dilution attributed to its group.25 mg per single human dose. the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined. for routine testing a single-dilution method may be used. Inject intraperitoneally into the second group twice the single human dose of the vaccine incubated at 37° for 4 weeks.0 per cent of the total number of mice die following histamine challenge. filamentous haemagglutinin. The seed cultures are managed to conserve or. the purity of the antigenic fraction is 23 Adsorbed Pertussis Vaccine (Acellular. (3) the name and amount of the adsorbent. Observe for 48 hours. Media used for the preparation of seed batches and inocula may contain blood or blood products of animal origin. The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. the production process shall be validated to demonstrate that it yields consistently an antigenic fraction that complies with the following requirements. The vaccine contains an antigenic fraction purified without separation of the individual components. detoxified using suitable reagents by a method that ensures minimal reversion of toxoid to toxin. by a method that has been shown to have no significant effect on the assay procedure when the stabilised and non-stabilised batches are compared. Not more than 2 pmol in the equivalent of 1 dose of the final vaccine. (5) that the vaccine is not to be frozen. The antigenic fraction is composed of pertussis toxoid. Pertussis toxin Chinese hamster ovary (CHO) cell-clustering effect and haemagglutination as in vitro methods. The purification procedure is validated to demonstrate appropriate clearance of substances used during culture or purification. strict adherence to the production process used for the batch tested in clinical trials is necessary. the tests need not be applied routinely to each batch. after demonstration of consistency. Pertactin. fimbrial-2 and fimbrial-3 antigens. histamine-sensitising activity and insulin secretory activity as in vivo methods. after appropriate characterisation. Co-Purified) Pertussis Vaccine (Acellular. Tracheal cytotoxin. Adsorbed) is a preparation of antigenic components of Bordetella pertussis adsorbed on a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. pertactin (a 69 kDa outermembrane protein) and other defined components of B. (4) that the vaccine must be shaken before use. PURIFIED ANTIGENIC FRACTION Production of the antigenic fraction is based on a seed-lot system. Reactivity with specific antibody.IP 2007 ADSORBED PERTUSSIS VACCINE (ACELLULAR CO-PURIFIED) the vaccine contains a pertussis toxin-like protein produced by genetic modification. The content of bacterial endotoxins is determined to monitor the purification procedure and to limit the amount in the final vaccine. lymphocytosispromoting activity. Before detoxification. The antigenic fraction is treated by a method that transforms pertussis toxin to toxoid. the amount of antigenic fraction equivalent to 1 dose of the final vaccine. rendering it harmless while maintaining adequate immunogenic properties of all the components and avoiding reversion to toxin. It may contain residual pertussis toxin up to a maximum level approved by the competent authority. in a volume of 0.1 ml. For the preparation of a representative batch. Inject intradermally into each of 3 unweaned mice. Production General provisions. Absence of residual dermonecrotic toxin. . A batch of vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. The toxin shows ADP-ribosyl transferase activity using transducin as the acceptor. Reference vaccine. pertussis such as fimbrial-2 and fimbrial-3 antigens. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. The antigenic fraction is purified and. Not more than 500 ng in the equivalent of 1 dose of the final vaccine. The reference vaccine is preferably stabilised. Filamentous haemagglutinin Haemagglutination and inhibition by specific antibody. CHARACTERISATION OF COMPONENTS During development of the vaccine. particularly on or exposure to heat. No dermonecrotic reaction is demonstrable.4. Pertussis toxoid The toxoid induces in animals the production of antibodies capable of inhibiting all the properties of pertussis toxin.14). Co-Purified. None of the media used at any stage contains blood or blood products of human origin. determined by a suitable method such as a biological assay or liquid chromatography (2. Adenylate cyclase. The antigenic fraction is analyzed by one or more of the methods shown below in order to determine the identity and specific properties (activity per unit amount of protein) of its components in comparison with reference preparations. restore toxinogenicity by deliberate selection. where necessary. Specific properties. determined by immunoblot analysis or another suitable method. The limits applied are such that the final vaccine contains not more than 100 IU per single human dose. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0. reversibility of toxoid. the clear supernatant reacts with specific antisera to the components in the vaccine. Carry out the test for sterility using for each medium a quantity of purified antigenic fraction equivalent to not less than 100 doses of the final vaccine. Test for residual pertussis toxin. Using phosphate-buffered saline containing 0.ADSORBED PERTUSSIS VACCINE (ACELLULAR CO-PURIFIED) IP 2007 determined by a suitable method such as polyacrylamide gel electrophoresis (PAGE) (2. Determine the complete quantitative antigen composition of the antigenic fraction by suitable immunochemical methods (2. inject intraperitoneally into each mouse 1 mg of histamine base in a volume not exceeding 0. Provided that the tests for residual pertussis toxin. 24 FINAL BULK VACCINE The vaccine is prepared by adsorption of a suitable quantity of the antigenic fraction onto aluminium hydroxide or hydrated aluminium phosphate. Calculate the weight or volume of a preparation that sensitises 50 per cent of the mice injected using a suitable statistical method such as probit analysis. Where applicable. The ratio of total antigen content to protein nitrogen is within the limits established for the product. these tests may be omitted on the final lot. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Record the number of animals that die within 24 h of histamine challenge. free formaldehyde and the assay have been carried out with satisfactory results on the final bulk vaccine. FINAL LOT Only a final lot that is satisfactory with respect to each of the requirements given below under Identification. The residual activity of pertussis toxin does not exceed that of batches shown to be safe in clinical studies.0 per cent of the mice injected using a suitable statistical method such as probit analysis.11). antimicrobial preservative. prepare a series of dilutions of the vaccine under examination that have been shown to yield a graded response and attribute each dilution to a separate group of mice. The content of residual detoxifying agents and other reagents is determined and shown to be below approved limits unless validation of the process has demonstrated acceptable clearance. Calculate the weight or volume of a preparation that sensitises 50. Only a purified antigenic fraction that complies with the following requirements may be used in the preparation of the final bulk vaccine. Note the number of animals that die within 24 h of histamine challenge.0 per cent of the intended amount.11).14). Carry out test for sterility using 10 ml of bulk for each sterility medium. Using phosphate-buffered saline containing 0.5 ml.5 ml.2. After 5 days. A validated test based on the clustering effect of the toxin for Chinese hamster ovary (CHO) cells may be used instead of the test on mice. Tests Test for residual pertussis toxin.4.14). The content is not less than 85. Sterility (2.14) and protein nitrogen by sulphuric acid digestion or another suitable method. Inject intraperitoneally into each mouse the dilution attributed to its group. Requirements are established for each individual product.2.0 per cent and not greater than 115. SDS-PAGE or immunoblot analysis with specific monoclonal or polyclonal antibodies may be used to characterise subunits.2. A suitable antimicrobial preservative may be added. Use 3 groups of not fewer than 5 histamine-sensitive mice each weighing between 18 and 26 g.4. the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. determine the amount of antimicrobial preservative by a suitable chemical method. Tests and Assay may be released for use. Residual detoxifying agents and other reagents. inject intraperitoneally into each mouse 1 mg of histamine base in a volume not exceeding 0. . Inject diluent into a fourth group of control mice. After 5 days. Antimicrobial preservative.12) or liquid chromatography (2. Inject intraperitoneally into each mouse the dilution attributed to its group. Identification Subject the vaccine to a suitable desorption procedure such as the following: dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution.2 per cent of gelatin. prepare a series of dilutions of the purified antigenic fraction that have been shown to yield a graded response and attribute each dilution to a separate group of mice.2 per cent w/v of gelatin. Use 3 groups of not fewer than 5 histamine-sensitive mice (see under Production) each weighing between 18 and 26 g. Inject diluent into a fourth group of control mice.2. Examine by a suitable immunochemical method (2. Antigen content. Sterility (2. maintain at 37° for about 16 h and centrifuge until a clear supernatant is obtained.2 per cent w/v of gelatin and challenge with histamine as described above. The residual activity of pertussis toxin does not exceed that of batches shown to be safe in clinical studies. that have acceptable protective potency in animals and are safe. If a bioluminescence test or other biochemical method is used instead of viable count. The capacity of the working seed lot to induce sensitivity to tuberculin in guinea-pigs is demonstrated. When a new working seed lot is established. Maximum 1. using 10 guinea pigs. Assay The vaccine complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component).IP 2007 BACILLUS CALMETTE-GUERIN VACCINE (FREEZE-DRIED) Reversibility of toxoid. Maximum 0. carried out using 10 ml for each medium. during the course of production cycle nor shall they be exposed to a known risk of tuberculosis infection. designed that all cultures and vaccines are protected from direct sunlight and from ultraviolet light at all stages of manufacture. of passages from the secondary seed lot must not exceed 8. The production method shall have been shown to yield consistently BCG vaccines that induce adequate sensitivity to tuberculin in man.9).3. nucleic acid amplification and restrictionfragment-length polymorphism). Complies with the test for sterility. Bacillus Calmette-Guerin Vaccine (Freeze-Dried) Freeze-dried BCG Vaccine is a preparation of live bacteria derived from a culture of the bacillus of Calmette and Guérin (Mycobacterium bovis BCG) capacity of which to protect against tuberculosis has been established. and its relative absence of pathogenicity for man and laboratory animals. in particular they shall not work with virulent strains of Mycobacterium tuberculosis. The content is not less than 85. The vaccine is prepared from cultures which are derived from the master seed lot by as few subcultures as possible and in any case not more than 12 subcultures e. If the secondary seed lot is 4 culture passages removed from the primary seed lot. Identification The bacteria in the working seed lot are identified as Mycobacterium bovis BCG using microbiological techniques. Production of the vaccine is based on a seed-lot system. testing and storage. determine the amount of antimicrobial preservative by a suitable chemical method. if tested. Sterility (2. Only a working seed lot that complies with the following requirements may be used for propagation. Vaccine complies with the requirements stated under Vaccines with the following modifications. A suitable batch of vaccine is prepared from the first working seed lot and is reserved for use as the comparison/ in-house reference vaccine. The working seed lot complies with the test for sterility except for the presence of mycobacteria. The degree of reversibility does not exceed that of batches shown to be safe in clinical studies. Antimicrobial preservative. The label states (1) the names and amounts of the antigenic components present in the vaccine.2. would comply with the tests for safety and efficacy.g. (3) the maximum degree of reversion of toxoid to toxin during the period of validity. SEED LOT The strain used to establish the master seed lot is chosen for and maintained to preserve its stability.11). (2) the maximum amount of residual pertussis toxin present in the vaccine. Complies with the test for sterility.2 g/l.3. 25 Production General provisions The production method is validated to demonstrate that the product.0 per cent and not greater than 115. the vaccine is shown to be not significantly different in activity from the comparison vaccine. its capacity to sensitise man and guinea-pigs to tuberculin and to protect animals against tuberculosis.0 per cent of the intended amount. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent.20). Carry out the test for residual pertussis toxin described above using the vaccine incubated at 37° for 4 weeks in parallel with a sample stored at 2° to 8°. (5) that the vaccine must be shaken before use. Free formaldehyde (2.2. a suitable test for delayed hypersensitivity in guinea-pigs is carried out on a batch of vaccine prepared from the new working seed lot. The strain used shall be identified by historical records that include information on its origin and subsequent manipulation. Virulent mycobacteria Examine the working seed lot as prescribed under Tests. BCG vaccine is susceptible to sunlight: the procedures for the preparation of the vaccine shall be so .25 mg per single human dose. BCG vaccine shall be produced by a staff consisting of healthy persons who do not work with other infectious agents.11). Labelling. which may be supplemented by molecular biology techniques (for example. Aluminium (2. the no. Where applicable. Sterility (2. (6) that the vaccine is not to be frozen. the method is validated against the viable count for each stage of the process at which it is used. (4) the name and amount of the adsorbent. a quantity of vaccine equivalent to at least 50 human doses. The vaccine complies with the test if not more than one animal dies during the 42 days following the injection and autopsy does not reveal any sign of tuberculosis. FINAL LOT The final bulk vaccine is distributed into sterile containers and freeze-dried to a moisture content favourable to the stability of the vaccine. The culture is harvested and suspended in a sterile liquid medium that protects the viability of the vaccine as determined by a suitable method of viable count. Only a final lot that complies with the following requirement for count of viable units and with each of the requirements given below under Identification. Virulent mycobacteria. Provided the test for virulent mycobacteria has been carried out with satisfactory results on the final bulk vaccine. kill the guinea-pigs and examine by autopsy for signs of infection with tuberculosis. it may be omitted on the final lot. or indirectly by an opacity method that has been calibrated in relation to the mass of the organisms. If 2 animals die during this period and autopsy does not reveal signs of tuberculosis repeat the test on 6 other guinea-pigs. Test for purity. Alternatively. At the end of this period. Tests and Assay may be released for use. Examine as prescribed under Tests. 26 Except where the filled and closed containers are stored at a temperature of -20° or lower. The ratio of the count of viable units to the total bacterial concentration is not less than that approved for the particular product by National Regulatory Authority.2. The ratio of the count of viable units after freezedrying to that before is not less than that approved for the particular product. Bacterial concentration Determine the total bacterial concentration by a suitable method. if the bacterial concentration is determined before addition of a stabiliser. if the stabiliser interferes with the determination of bacterial concentration on the final bulk vaccine. The culture medium shall contain no substances known to cause toxic or allergic reactions in human beings or to cause the bacteria to become virulent for guinea-pigs. molecular biology techniques (like nucleic acid amplification) may be used. Purity is checked by acid fast staining. FINAL BULK VACCINE The final bulk vaccine is prepared from a single harvest or by pooling a number of single harvests. The vaccine complies with the test if none of the guinea-pigs shows signs of tuberculosis and if not more than one animal dies during the observation period. Carry out the test in parallel on a reference preparation of the same strain. A stabiliser may be added. the containers are closed either under vacuum or under a gas that is not deleterious to the vaccine. Sterility (2. the determination is carried out before addition of the stabiliser. each weighing between 250 and 400 g and having received no treatment likely to interfere with the test. the test may be omitted on the final lot. Observe the animals for at least 42 days. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Tests Virulent mycobacteria If this test is satisfactory at final bulk stage it can be omitted at the final lot. either directly by determining the mass of the microorganisms. .11). the concentration in the final bulk vaccine is established by calculation. Count of viable units Determine the number of viable units per ml of the reconstituted vaccine by viable count on solid medium using a method suitable for the vaccine under examination or by determination of adenosine triphosphate by a bioluminescence reaction. Complies with the test for sterility using 10 ml for each medium except for the presence of mycobacteria. Count of viable units Determine the number of viable units per ml by viable count on solid medium using a method suitable for the vaccine under examination or by determination of adenosine triphosphate by a bioluminescence reaction. Provided the test for excessive dermal reactivity has been carried out with satisfactory results on the working seed lot and on 5 consecutive final lots produced from it. Identification BCG vaccine is identified by microscopic examination of the bacilli in stained smears demonstrating their acid-fast property and by the characteristic appearance of colonies grown on solid medium. Inject subcutaneously or intramuscularly into each of 6 guineapigs. the expiry date is not later than 4 years from the date of harvest.BACILLUS CALMETTE-GUERIN VACCINE (FREEZE-DRIED) IP 2007 PROPAGATION AND HARVEST The bacteria are grown in a suitable medium for not more than 21 days by surface or submerged culture. The total bacterial concentration is within the limits approved for the particular product by National Regulatory Authority. ignoring any minor reactions at the site of injection. Animals that die during the observation period are also examined for signs of tuberculosis. . Labelling. The number is within the range stated on the label. except for the presence of adjuvant. Temperature stability Maintain samples of the freeze-dried vaccine at 37° for 4 weeks.1 ml of the reconstituted vaccine and of 2 tenfold serial dilutions of the vaccine and identical doses of the comparison vaccine. 27 Antigenic purity Not less than 1500 Lf per mg of protein nitrogen for diphtheria toxoid and not less than 1000 Lf/mg of protein nitrogen for tetanus toxoid. Carry out test for sterility using 10 ml of bulk for each sterility medium. Each bulk purified toxoid shall be tested to ensure that reversion to toxicity cannot take place on storage. respectively. The number of viable units in the heated vaccine is not less than 20. (5) the dose for each age group. Inject intradermally into each guinea-pig. Production General provisions Bulk purified diphtheria and tetanus toxoids The bulk purified diphtheria and tetanus toxoids are prepared as described in the monographs on Diphtheria vaccine (adsorbed) and Tetanus vaccine (adsorbed) and comply with the requirements prescribed therein. The specification for individual component used in formulation is referred in the text of individual monograph. To determine whether reversion has occurred. The vaccine complies with the test if the reaction it produces is not markedly different from that produced by the comparison vaccine.2.11).0 per cent of that in unheated vaccine. Absence of toxin and irreversibility of toxoid Inject subcutaneously into each of 5 guinea-pigs at least 500 Lf of the non-incubated bulk purified toxoid in a volume of 1 ml. without adsorbent. Diphtheria and Tetanus Vaccine (Adsorbed) Diphtheria and Tetanus Vaccine (Adsorbed) is a preparation of diphtheria formol toxoid and tetanus formol toxoid adsorbed on mineral carrier. Sterility (2. Determine the number of viable units in the heated vaccine and in unheated vaccine as described below. The bulk purified toxoid shall be diluted in order to obtain the same concentration and chemical environment as that present in the final bulk vaccine. diluted toxoids that have been stored at 37° for six weeks shall be tested. FINAL BULK VACICNE The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto mineral carrier such as hydrated aluminium phosphate. Not more than 3. Determine the number of viable units in the comparison vaccine in parallel. The test employed shall be approved by the National Regulatory Authority and should be sufficiently sensitive to detect very small amounts of toxin. The reconstituted vaccine complies with the test for sterility except for the presence of mycobacteria. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani. the sensitivity of the test shall have been demonstrated to be not less than that of the guinea-pig test. according to a randomised plan. each weighing not less than 250 g and having received no treatment likely to interfere with the test.3. and the test procedures shall be approved by the National Regulatory Authority. Excessive dermal reactivity Use 6 healthy white or pale-coloured guinea-pigs. Animals that die shall be autopsied and examined for symptoms of diphtheria intoxication (red adrenals). The label states (1) the minimum and maximum number of viable units per ml in the reconstituted vaccine. determined by the semi-micro determination of water. The bulk purified toxoid shall pass the test if no guinea-pig shows symptoms of specific intoxication within six weeks of injection and if at least 80 per cent of the animals survive the test period.0 per cent. (3) that the vaccine is to be used immediately after broaching the container.2.IP 2007 DIPHTHERIA AND TETANUS VACCINE (ADSORBED) Sterility (2. (4) the age group for which the vaccine is intended. No toxicity shall be detected. in this case. 0. Alternatively. Assay Determine the number of viable units in the reconstituted vaccine by viable count on solid medium or using a suitable validated biochemical method for the vaccine under examination. using the same buffer solution as for the final vaccine.43). a cell-culture test system may be used. Observe the lesions formed at the site of the injection for 4 weeks. Water (2. (6) follow instructions as mentioned in the product insert/leaflet. (2) that the vaccine must be protected from direct sunlight. Intradermal tests in guinea-pigs and cell-culture tests both are considered to be suitable.11). The guinea-pigs shall not have been used previously for experimental purposes. ).3.24). Inject subcutaneously into each animal 5 times the dose stated on the label. determine the amount of antimicrobial preservative by a suitable chemical method. Dissolve in the vaccine under examination by adding sufficient sodium citrate to give a 10 per cent w/v solution.3. Diphtheria toxoid is identified by a suitable immunochemical method (2. Specific toxicity. 28 Identification A.4. Tests Sterility (2.0 per cent and not greater than 115. they may be omitted on the final lot. Where applicable.2.2.0 to 7. Tetanus toxoid Complies with the test as stated under Tetanus Vaccine (Adsorbed). Assay Diphtheria component Carry out one of the described methods for the assay of Diphtheria Vaccine (Adsorbed). FINAL LOT The final bulk vaccine is filled and stored aseptically into sterile containers. Free formaldehyde (2. Suitable antimicrobial preservatives may be added. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. Provided the tests for specific toxicity. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Carry out test for sterility using 10 ml of bulk for each sterility medium. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification. determine the amount of antimicrobial preservative by a suitable chemical method.1). Antimicrobial preservatives of the phenolic type must not be used.4. unrestricted diet. B.0 per cent of the quantity stated on the label.2.9.2. Use 5 normal.24). If an animal dies or loses weight in the second test. The clear supernatant liquid obtained during test A reacts with a suitable tetanus antitoxin. B. Complies with the test for abnormal toxicity Aluminium (2. b) Lethal challenge method. Complies with the test for sterility.0.2. Weigh all the animals at weekly intervals for 6 weeks. The containers are closed so as to prevent contamination. the vaccine fails the test. Tetanus component Carry out one of the described methods for the assay of Tetanus Vaccine (Adsorbed) Viz a) Antibody induction . The content is not less than 85.2 g/l. Antimicrobial preservative. 6.0 per cent and not greater than 115. Weigh the animals separately and record their weights. Identification A. Maintain at 37° for about 16 hours and centrifuge until a clear supernatant liquid is obtained.20).14). giving a precipitate or visible floccules. healthy guinea-pigs weighing between 250 and 350 g which have been maintained for at least 1 week on a uniform. free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. Maximum 0.0). giving a precipitate or visible floccules.0 per cent of the intended amount. Not more than 1. Where applicable. None of the animals shows any symptoms of diphtheria or tetanus toxaemia or dies from diphtheria within 42 days or loses weight at the end of the test. Abnormal toxicity (2. Assay Diphtheria toxoid Complies with the test as stated under Diphtheria Vaccine (Adsorbed).DIPHTHERIA AND TETANUS VACCINE (ADSORBED) IP 2007 aluminium hydroxide.11). The clear supernatant reacts with a suitable diphtheria antitoxin.25 mg per single human dose when hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent.2 g/l Sterility (2. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Antimicrobial preservative. repeat the test. pH (2.0 to 7.20). The amount is not less than 85. The pH of the vaccine is within the range approved for the product (6. pH (2.14). the resulting mixture is approximately isotonic with blood. d) Validated serological assay in guinea pigs or mice as approved by National Regulatory Authority. Free formaldehyde (2. and have not been previously treated with any material that will interfere with the test. Tests and Assay may be released for use.3. c) Antibody induction method. Maximum 0. Tetanus toxoid is identified by a suitable immuno-chemical method (2. Viz a) Intradermal challenge method. Maintain at 37o for about 16 hours and centrifuge.11). If more than one animal dies from non-specific causes or loses weight. 2.4. if more than 1 animal dies in the second test. (5) that the vaccine is not to be frozen. Certain antimicrobial preservatives. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. B. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification. Diphtheria toxoid is identified by a suitable immunochemical method (2. The amount is not less than 85. FINAL BULK VACCINE The vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide. (4) that the vaccine must be shaken before use. they may be omitted on the final lot. The label states (1) the human dose. If a satisfactory result is not obtained with a vaccine adsorbed on aluminium hydroxide. Tests and Assay may be released for use.2. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. Where applicable.20).2.14). Maintain at 37° for about 16 hours and centrifuge until a clear supernatant is obtained. respectively. Provided the tests for free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine. Specific toxicity Inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs. the vaccine does not comply with the test. c) Validated serological assay in guinea pigs or mice as approved by National Regulatory Authority. Free formaldehyde (2. carry out the test as follows. giving a precipitate. Maximum 0. applicable to certain vaccines. Maintain at 37° for not less than 6 hours and centrifuge.0 per cent and not greater than 115.3. 29 . (3) the name and the amount of the adsorbent and preservative. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate.0 per cent of the intended amount. Tetanus toxoid is identified by a suitable immunochemical method (2. B. Suitable antimicrobial preservatives may be added. Centrifuge 15 ml of the vaccine under examination and suspend the residue in 5 ml of a freshly prepared mixture of 1 volume of a 56 g/l solution of sodium edetate and 49 volumes of a 90 g/l solution of disodium hydrogen phosphate. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. 6. The following method. The following method.0. pH (2. Production General provisions Bulk purified diphtheria and tetanus toxoids The bulk purified diphtheria and tetanus toxoids are prepared as described in the monographs on Diphtheria vaccine (adsorbed) and Tetanus vaccine (adsorbed) and comply with the requirements prescribed therein. If more than 1 animal dies from nonspecific causes. the vaccine does not comply with the test.24 ).14). Sterility (2. applicable to certain Identification A.2 g/l. is given as an example. Identification A. tamper-proof containers. each weighing between 250 and 350 g. determine the amount of antimicrobial preservative by a suitable physicochemical method. giving a precipitate. Diphtheria and Tetanus Vaccine (Adsorbed) for Adults and Adolescents Diphtheria and Tetanus Vaccine (Adsorbed) for Adults and Adolescents is a preparation of diphtheria formol toxoid and tetanus formol toxoid adsorbed on a mineral carrier. FINAL LOT The final bulk vaccine is distributed aseptically into sterile. (2) the minimum Lf units per single human dose or the minimum International Units per single human dose if potency test done by challenge method. Labelling. that have not previously been treated with any material that will interfere with the test. repeat the test once. Carry out the test for sterility using 10 ml for each medium.IP 2007 DIPHTHERIA AND TETANUS VACCINE (ADSORBED) FOR ADULTS AND ADOLESCENTS method. particularly those of the phenolic type. b) Challenge method in guinea pigs/mice.0 to 7.11). Maintain at 37o for about 16 hours and centrifuge. The clear supernatant reacts with a suitable diphtheria antitoxin. adversely affect the antigenic activity and must not be used. Antimicrobial preservative. The containers are closed so as to prevent contamination. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus. The clear supernatant reacts with a suitable diphtheria antitoxin. Tetanus Vaccine (Adsorbed) and Pertussis Vaccine. tetanus formol toxoid adsorbed on mineral carrier and a suspension of killed Bordetella pertussis organisms. The Bordetella pertussis suspension is prepared by growth of suitable strains in an appropriate medium. pertussis are used. The containers are closed so as to prevent contamination. Maximum 1.2. Sterility (2. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto hydrated aluminium phosphate or aluminium hydroxide and admixture of an appropriate quantity of a suspension of inactivated B. (3) the name and the amount of the adsorbent.DIPHTHERIA.20).U. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 I.95) of the estimated potency is not less than 40 IU per single human dose. the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. The B.2 g/l. giving a precipitate. The lower confidence limit (P = 0. Tetanus Vaccine (Adsorbed) and Pertussis Vaccine respectively and comply with the respective requirements.2. Carry out test for sterility using 10 ml of bulk for each sterility medium.4.0 percent and not more than115. The amount is not less than 85. The label states (1) the human dose. . If two or more strains of B. is given as an example. The formal toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani. The lower confidence limit (P = 0. if potency determined by challenge method or the minimum Lf units per single human dose if test done by antibody induction method.0.25 mg per single human dose. pertussis. Where applicable. determine the amount of antimicrobial preservative by a suitable chemical method. (5) that the vaccine is not to be frozen. under controlled conditions.0 per cent and not greater than 115. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.9). Labelling. Antimicrobial preservative. Sterility (2.1). Suitable antimicrobial preservatives may be added to the bulk vaccine.0 to 7. Production General provisions The production method must be validated to demonstrate that the product if tested. Abnormal toxicity (2. Antimicrobial preservatives particularly those of phenolic type which affect the antigenic activity must not be used. pertussis suspension The bulk purified diphtheria and tetanus toxoids and inactivated B.24). Antimicrobial preservative. determine the amount of antimicrobial preservative by a suitable physicochemical method.3. TETANUS AND PERTUSSIS VACCINE (ADSORBED) IP 2007 vaccines. would comply with the tests for safety as described under monographs of Diphtheria Vaccine. Bulk purified diphtheria and tetanus toxoids. Tetanus and Pertussis Vaccine (Adsorbed) Diphtheria. pH (2. per single human dose. 6. (4) that the vaccine must be shaken before use.0 percent of the intended amount. The specification for individual component used in formulation is referred in the text of individual monograph. bulk inactivated B.2.11). 30 Diphtheria.95) of the estimated potency is not less than 2 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). Where applicable. Assay Diphtheria component Carry out the prescribed method for assay of Diphtheria Vaccine by lethal challenge method described in the assay of Diphtheria Vaccine (Adsorbed).0 per cent of the intended amount.3. FINAL LOT The final bulk vaccine is filled and stored aseptically into sterile containers. Maximum 0. The content is not less than 85. respectively. Complies with the test for abnormal toxicity.11). pertussis suspension are prepared as described in the monograph on Diphtheria Vaccine (Adsorbed). The clear supernatant obtained during identification test A reacts with a suitable tetanus antitoxin. (2) the minimum number of International Units of each component per single human dose. Complies with the test for sterility. Free formaldehyde (2. Tetanus and Pertussis Vaccine (Adsorbed) is a preparation of diphtheria formol toxoid. Tests Aluminium (2. giving a precipitate. reserve the precipitate for identification test C. diphtheria toxemia or tetanus.11). Inject each mouse of the control group with 0. preferably containing the same amount of antimicrobial preservative as that injected with the vaccine.2. The clear supernatant reacts with a suitable diphtheria antitoxin. The vaccine complies with the test if: (a) at the end of 72 hours the total mass of the group of vaccinated mice is not less than that preceding the injection. Use mice of the same sex or distribute males and females equally between the groups. Labelling.9 per cent sterile solution of sodium chloride. Dissolve in the vaccine under examination by adding sufficient sodium citrate to give a 10 per cent w/v solution.2.25 ml into each of the five mice weighing between 17 to 22 g and at least one human dose but not more than 1. and (c) not more than 5 per cent of vaccinated mice should die during the test. Provided the tests for specific toxicity of diphtheria. that have not previously been treated with any material that will interfere with the test. giving a precipitate.20).4.2. Weigh the mice groups immediately before the injection and 72 hours and 7 days after the injection. Allow the animals access to food and water for at least 2 hours before injection and during the test.2. 6. Pertussis component Carry out the assay as stated under Pertussis Vaccine. each weighing between 250 and 350 g. If more than one animal dies from non-specific causes. the vaccine does not comply with the test. Not more than 1. The test may be repeated and the results of the tests combined. Antimicrobial preservative (2. Abnormal toxicity (2.IP 2007 DIPHTHERIA.3. The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified. If within 42 days of the injection any of the animals shows signs of or dies from . The label states (1) in case done by challenge method the minimum number of International Units. The pertussis component is identified by agglutination of the bacteria from the resuspended centrifugation residue (see identification test A. Tetanus toxoid is identified by a suitable immuno-chemical method (2. repeat the test once. Aluminium (2. Pertussis component.2. Tests Sterility (2. they may be omitted on the final lot. Diphtheria toxoid is identified by a suitable immunochemical method (2.24). Specific toxicity Diphtheria and tetanus components. The clear supernatant obtained during identification test A reacts with a suitable tetanus antitoxin. The preparation passes the test if none of the animals dies or shows signs of ill health in 7 days following the injection. (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 per cent of that per control mouse. Assay Diphtheria component Carry out one of the methods for the assay as stated under Diphtheria Vaccine (Adsorbed). if units (as applicable for each component) per single human dose.14). tetanus and pertussis components. but not more than 0. antimicrobial preservative and the Assay have been carried out with satisfactory results on the final bulk vaccine. TETANUS AND PERTUSSIS VACCINE (ADSORBED) Only a final lot that is satisfactory with respect to each of the requirements given below under Identification. when hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent.2 g/l.3. Free formaldehyde (2. free formaldehyde.9). other suitable methods for separating the bacteria from the adsorbent may also be used) by antisera specific to B. Maintain at 37° for about 16 hours and centrifuge until a clear supernatant is obtained.25 mg per single human dose. containing a quantity of the vaccine equivalent to not less than half the single human dose. if more than one animal dies in the second test.2 ) Where applicable. If one of the animal dies or shows the signs of ill health.0.0 ml into each of the two guinea pigs weighing between 250 and 350 g. B. the vaccine does not comply with the test. Tests and Assay may be released for use.0 per cent of the intended amount. Tetanus component Carry out one of the methods for the assay as stated under Tetanus Vaccine (Adsorbed). C. Use not less than 10 healthy mice each weighing between 14 and 16 g for the vaccine group and for the saline control. The content is not less than 85. determine the amount of antimicrobial preservative by a suitable chemical method.0 per cent and not more than115. Inject subcutaneously five times the single human dose stated on the label into each of five healthy guinea-pigs. repeat the test. 31 Identification A. Maximum 0. Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose . pH (2. Inject each mouse of the vaccine group intraperitoneally with 0.14).5 ml of a 0. The animals should be weighted every week and observations be made.1).0 to 7. Complies with the test for sterility.5 ml. pertussis or by the assay of the pertussis component. purified tetanus formol toxoid containing not less than 1. would comply with the following test for specific toxicity of the diphtheria and tetanus component: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea pigs. (6) for vaccine contained in single-dose containers where the space is too small to accommodate the full name of the vaccine.16).. If the vaccine is presented with the haemophilus component in a separate vial. Hepatitis B (rDNA) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of diphtheria formol toxoid containing not less than 1. when conjugated to PRP. the contents of which are mixed with the other components immediately before or during use. Pertussis (Whole Cell). P. (5) that the vaccine is not to be frozen. For subsequent routine control.2. in saline solution or other appropriate solution isotonic with blood. each weighing between 250 and 350 g. Mineral adsorbent is a suspension of hydrated aluminium hydroxide.16) per mg of protein nitrogen. polyribosyl ribitol phosphate. if more than 1 animal shows signs of or dies in the second test. the vaccine does not comply with the test.. a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. The antigenic properties of the vaccine are adversely affected by the presence of certain antimicrobial preservatives particularly those of the phenolic type and some of the quaternary ammonium type and must not be used. (4) that the vaccine must be shaken before use. (2. Tetanus. monocomponent reference vaccines may be used for the assays on the combined vaccine. Tetanus. the vaccine does not comply with the test. tetanus. the assays of these components may be carried out without mixing with the haemophilus component. Production General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. . release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. is capable of inducing a T-cell dependent B-cell immune response to the polysaccharide. If more than 1 animal dies from nonspecific causes. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria. (2. pertussis and hepatitis B are carried out on a suitable number of batches of vaccine reconstituted as for use.D. strict adherence to the production 32 Diphtheria. repeat the test once. HEPATITIS B (rDNA) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE (ADSORBED) IP 2007 (2) In case done by antibody induction method the minimum number of International Units per single human dose of pertussis component and minimum number of Lf of diphtheria toxoid and tetanus toxoid. For the haemophilus component. Reference vaccine(s) Provided valid assays can be performed. hepatitis B surface antigen and haemophilus type b conjugated to suitable protein with a mineral adsorbent to which a suspension of killed Bordetella pertussis has been added. (3) the name and the amount of the adsorbent and preservative. respectively. the abbreviation ‘DTP’ may be used in the label and the container provided that the same code is also stated in the label on the package. such tests may include determination of molecular size. The formol toxoids are prepared from the toxin produced by the growth of Corynebacterium diphtheriae and Clostridium tetani. aluminium phosphate or calcium phosphate. toxemia or tetanus. The product may be presented with the haemophilus component in a separate container. The polysaccharide. PRP is a linear copolymer composed of repeated units of 3-β-Dribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n]. The production method is validated to demonstrate that the product. if tested. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine. as part of consistency studies the assays of the diphtheria.2. that have not previously been treated with any material that will interfere with the test. Hepatitis B (rDNA) and Haemophilus Type b Conjugate Vaccine (Adsorbed) Diphtheria. The stability of the final lot and the relevant intermediates is evaluated using one or more indicator tests..500 Lf. Hepatitis B surface antigen is a component protein of hepatitis B virus. The final product contains a suitable antimicrobial preservative. Taking account of the results of the stability testing. determination of free PRP in the conjugate and kinetics of depolymerisation. with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. For the preparation of a representative batch. T.000 Lf. in suitable media. Pertussis (Whole cell). per mg of protein nitrogen. The toxins are converted to toxoids by treatment with formaldehyde solution by methods which avoid reversibility of the toxoids. the antigen is obtained by recombinant DNA technology. The carrier protein. (WHOLE CELL). Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. bulk purified tetanus toxoid. Tetanus Vaccine (Adsorbed). 6. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0. B. Osmolality (2.IP 2007 D. separately or together. 33 Antimicrobial preservative. Whitish turbid liquid in which the mineral carrier tends to settle down slowly on keeping. A stabilizer may be added. bulk purified hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate.0 per cent of the intended content. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification. the resulting mixture is approximately isotonic with blood. bulk purified tetanus toxoid. B. The final bulk is filled separately. Description.23). Suitable antimicrobial preservatives may be added.0 per cent and not greater than 115. Sterility (2.16). Pertussis component. C.2.4. tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine. If 2 or more strains of B. The amount of free PRP is not greater than that approved for the particular product.4.4. HEPATITIS B (rDNA) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE (ADSORBED) process used for the batch tested in clinical trials is necessary.. To a suspension of the residue . it may be omitted on the final lot. Test F may be omitted if tests A.2 g/l. Tetanus toxoid. P. D and E are carried out. size exclusion or hydrophobic chromatography (2. Pertussis Vaccine. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. D and E may be omitted if test F is carried out. Vaccine with the haemophilus component in a separate container The final bulk is prepared by adsorption. C. Maintain at 37o for about 16 hours and centrifuge. Provided the tests for specific toxicity of diphtheria toxoid. Free PRP Unbound PRP is determined after removal of the conjugate. provided it has been carried out with satisfactory results on the final bulk vaccine. the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units.0. Diphtheria toxoid. bulk purified hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. pertussis component and admixture of a suitable quantity of PRP conjugate. If an in vivo assay is used for the hepatitis B component. pertussis are used. The amount is not less than 85. of suitable quantities of bulk purified diphtheria toxoid. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. Where applicable. pH (2. (WHOLE CELL).. the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. B. determine the amount of antimicrobial preservative by a suitable chemical method. The B. they may be omitted on the final lot. pertussis are used. Identification Tests A. Tests and Assay may be released for use. of suitable quantities of bulk purified diphtheria toxoid. the test for free formaldehyde may be omitted on the final lot. pertussis component and admixture of a suitable quantity of PRP conjugate. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. The osmolality of the vaccine is within the limits approved for the particular preparation. Carry out test for sterility using 10 ml of bulk for each sterility medium. T. The B. tetanus toxoid and pertussis component and antimicrobial preservative and the assays for the diphtheria. C. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed). Reserve the residue for test C. A. the resulting mixture is approximately isotonic with blood. The reference vaccine may be stabilized by a method that has been shown to have no effect on the assay procedure. ultrafiltration or other validated methods. admixture of an appropriate quantity of a suspension of inactivated B. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. for example by anion exchange.11).0 to 7. FINAL BULK VACCINE Vaccine with all components in the same container The final bulk is prepared by adsorption. If 2 or more strains of B. Suitable antimicrobial preservatives may be added. Hepatitis B Vaccine (rDNA) and Haemophilus Type b Conjugate Vaccine. separately or together. admixture of an appropriate quantity of a suspension of inactivated B.24).. Free formaldehyde (2.3. The suspension of the residue obtained in test A gives a positive reactions when tested by suitable in-vitro assay.0 per cent of the amount of PRP stated on the label. PRP.025 mg of PRP for vaccine with OMP as carrier. aluminium. Hepatitis B surface antigen. If the haemophilus component is freeze-dried.1). if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. repeat the test. The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified.0 ml into each of two guinea pigs weighing between 250 and 350 g. the minimum number of International Units (as applicable for each component) per single human dose.. Complies with the test for sterility. The preparation passes the test if none of the animals dies or shows signs of ill health in seven days following the injection.2. Tetanus and Pertussis Vaccine (Adsorbed). E.1).. If one of the animals dies or . (b) in case done by antibody induction.11). Not more than 1. Tetanus and Pertussis Vaccine (Adsorbed).1 mg of PRP for a vaccine with tetanus toxoid as carrier protein.D. Storage. (5) haemophilus conjugate component 34 Tests If the product is presented with the haemophilus component in a separate container. HEPATITIS B (rDNA) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE (ADSORBED) IP 2007 obtained in test A in saline solution add a suitable Bordetella pertussis antiserum. T. When stored under the prescribed conditions the vaccine may be expected to retain potency for not less than 2 years from the date on which the potency test for the pertussis component was started. The content is not less than 85. F. Pertussis vaccine Complies with the test as stated under Diphtheria. determine the amount of antimicrobial preservative by a suitable chemical method. PRP is determined either by assay of ribose (2. P. pertussis and hepatitis B components. water (where applicable). tetanus toxoid and pertussis component. Antimicrobial preservative. the tests for PRP content. The vaccine complies with the test for pyrogens. Where applicable.0 per cent of the intended amount. antimicrobial preservative and sterility are carried out on the container with the diphtheria.25 mg per single human dose. This test is carried out for Haemophilus influenzae type b vaccine only if Haemophilus influenzae type b vaccine is presented as separate lyophilized vial. Inject per kg of the rabbit’s mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria toxoid as carrier.0 per cent and not greater than 115.14) or by anion exchange liquid chromatography with pulsed amperometric detection. some tests may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. (a) in case done by challenge method. Maximum 0. (WHOLE CELL). sterility and pyrogens are carried out on the container with the haemophilus component. 0. Abnormal toxicity (2. D. Pyrogens (2.7. the tests for specific toxicity of diphtheria toxoid. free formaldehyde. (3) pertussis component – IU or IOU per single human dose. or phosphorus (2.mg HBsAg per single human dose. Hepatitis B surface antigen (adsorbed) Complies with the test as stated under Hepatitis B Vaccine (Adsorbed). Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose. 0. Tetanus toxoid (adsorbed) Complies with the test as stated under assay of Tetanus Vaccine (Adsorbed). shows signs of ill health. Not less than 80. but not more than 0.2. (4) hepatitis B component .25 ml into each of five mice weighing between 17 and 22 g and at least one human dose but not more than 1. Assay Diphtheria toxoid (adsorbed) Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). The suspension of the residue obtained in the test A gives a positive reaction when tested by a suitable immunochemical method for PRP. Pertussis component Complies with the test as stated under Diphtheria. tetanus. PRP. Sterility (2. The label states (1) the human dose. agglutination indicates presence of pertussis component. Labelling. the minimum Lf units per single human dose.3. Aluminium (2.9). The vaccine confers an active immunity in mice and guineapigs when administered as directed in the test for Assay.8).1).2 g/l. (2) Diphtheria and Tetanus components.20).2.. Specific toxicity Diphtheria and tetanus components Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). by an immunochemical method (2.2.7. pertussis components and hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. tetanus toxoid.0. Pertussis and Hepatitis B (rDNA) Vaccine (Adsorbed) is a combined vaccine composed of diphtheria formol toxoid containing not less than 1. per mg of protein nitrogen and hepatitis B surface antigen with a mineral adsorbent to which a suspension of killed Bordetella pertussis has been added. separately or together. Taking account of the results of the stability testing. Mineral adsorbent is a suspension of hydrated aluminium hydroxide. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. aluminium phosphate or calcium phosphate in saline solution or other appropriate solution isotonic with blood. Sterility (2. Provided the tests for specific toxicity of diphtheria toxoid. The antigenic properties of the vaccine are adversely affected by the presence of certain antimicrobial preservatives particularly those of the phenolic type and some of the quaternary ammonium type and must not be used. pH (2. if tested. If within 42 days of the injection any of the animals shows signs of or dies . The final product contains a suitable antimicrobial preservative. If more than 1 animal dies from nonspecific causes.2. (7) the name and amount of adsorbent and added preservative. (8) that the vaccine must be shaken before use. release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. Where applicable. Carry out test for sterility using 10 ml of bulk for each sterility medium. the vaccine does not comply with the test.16). The toxins are converted to toxoids by treatment with formaldehyde solution by methods. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification. (6) the type and nominal amount of carrier protein per single human dose. determine the amount of antimicrobial preservative by a suitable chemical method.IP 2007 DIPHTHERIA TETANUS PERTUSSIS (WHOLE CELL).000 Lf ( 2. repeat the test once. toxaemia or tetanus. (9) that the vaccine is not to be frozen. Production General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. tetanus toxoid and pertussis component and antimicrobial preservative and the assays for the diphtheria. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption. 35 Diphtheria.24).2. from diphtheria. Pertussis (Whole Cell) and Hepatitis B (rDNA) Vaccine (Adsorbed) Diphtheria. which avoid reversibility of the toxoids.500 Limes flocculationis (Lf) (2. a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. HEPATITIS B (rDNA) VACCINE (ADSORBED) . purified tetanus formol toxoid containing not less than 1. Tetanus Vaccine (Adsorbed). if more than 1 animal shows signs of or dies in the second test. the antigen is obtained by recombinant DNA technology.0 to 7. 6. of suitable quantities of bulk purified diphtheria toxoid.4. Reference vaccine(s) Provided valid assays can be performed. Tetanus. respectively.mg PRP per single human dose.0 per cent of the intended content.11). in suitable media. each weighing between 250 and 350 g. Tetanus. Suitable antimicrobial preservatives may be added. the vaccine does not comply with the test. per mg of protein nitrogen. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed). The stability of the final lot and the relevant intermediates is evaluated using one or more indicator tests. Antimicrobial preservative. monocomponent reference vaccines may be used for the assays on the combined vaccine. The amount is not less than 85. Pertussis Vaccine and Hepatitis B Vaccine (rDNA). would comply with the following test for specific toxicity of the diphtheria and tetanus component: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea pigs. The production method is validated to demonstrate that the product.2. that have not previously been treated with any material that will interfere with the test.0 per cent and not greater than 115. The formol toxoids are prepared from the toxin produced by the growth of Corynebacterium diphtheriae and Clostridium tetani. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine.16). Hepatitis B surface antigen is a component protein of hepatitis B virus. Tests and Assay may be released for use. Storage. (a) in case done by challenge method. Maintain at 37o for about 16 hours and centrifuge. Tetanus and Pertussis Vaccine (Adsorbed). Antimicrobial preservative.3. When stored under the prescribed conditions the vaccine may be expected to retain potency for not less than 2 years from the date on which the potency test for the pertussis component was started. the test for free formaldehyde may be omitted on the final lot. Specific toxicity Diphtheria and tetanus components Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). . If an in vivo assay is used for the hepatitis B component.DIPHTHERIA TETANUS PERTUSSIS (WHOLE CELL). Pertussis vaccine Complies with the test as stated under Diphtheria. agglutination indicates presence of pertussis component.4. Reserve the residue for test C. The content is not less than 85. Complies with the test for sterility. HEPATITIS B (rDNA) VACCINE (ADSORBED) IP 2007 tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine.mg HBsAg per single human dose.IU or IOU per single human dose. (7) that the vaccine is not to be frozen. (6) that the vaccine must be shaken before use. (5) the name and amount of adsorbent and added preservative. determine the amount of antimicrobial preservative by a suitable chemical method.20). The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate.1).2.2 g/l. Aluminium (2. Labelling. The preparation passes the test if none of the animals dies or shows signs of ill health in seven days following the injection. C. 6. Where applicable. repeat the test. Hepatitis B surface antigen. Tetanus toxoid. 36 Identification Tests A. Abnormal toxicity (2. B. Diphtheria toxoid. Sterility (2. Not more than 1. If one of the animals dies or shows signs of ill health. it may be omitted on the final lot. A. C and D may be omitted if test E is carried out. The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified.24). the minimum number of International Units (as applicable for each component) per single human dose and (b) in case done by antibody induction. B C and D are carried out. The label states (1) the human dose (ml). Tests pH (2. Free formaldehyde (2. (4) hepatitis B component . The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. Pertussis component Complies with the test as stated under Diphtheria. Tetanus and Pertussis Vaccine (Adsorbed). The suspension of the residue obtained in test A gives a positive reactions when tested by suitable in-vitro assay. Description.9). Assay Diphtheria toxoid (adsorbed) Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). E.0 per cent of the intended amount. (3) pertussis component . pertussis antiserum.0 per cent and not greater than 115. To a suspension of the residue obtained in test A in saline solution add a suitable B.0. Whitish turbid liquid in which the mineral carrier tends to settle down slowly on keeping. they may be omitted on the final lot.25 ml into each of five mice weighing between 17 and 22 g and at least one human dose but not more than 1.4.3.0 to 7. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0. The vaccine confers an active immunity in mice and guineapigs when administered as directed under Assay. Maximum 0. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Osmolality (2.23). Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Hepatitis B surface antigen (adsorbed) Complies with the test as stated under Hepatitis B Vaccine (Adsorbed). The osmolality of the vaccine is within the limits approved for the particular preparation. D. Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose. provided it has been carried out with satisfactory results on the final bulk vaccine. B.0 ml into each of two guinea pigs weighing between 250 and 350 g. Test E may be omitted if tests A. but not more than 0.2 g/l.25 mg per single human dose. the minimum Lf units per single human dose. Pertussis component.11).2. (2) diphtheria and tetanus components. Tetanus toxoid (adsorbed) Complies with the test as stated under assay for Tetanus Vaccine (Adsorbed). For the haemophilus component. The polysaccharide. The mineral adsorbent is a suspension of hydrated aluminium hydroxide. Pertussis and Haemophilus type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of diphtheria formol toxoid containing not less than 1. (2. The final product contains a suitable antimicrobial preservative. such tests may include determination of molecular size. Taking account of the results of the stability testing.. Reference vaccine(s) Provided valid assays can be performed. Tetanus. Tetanus Vaccine (Adsorbed). and Haemophilus type b conjugated to suitable protein with a mineral adsorbent to which a suspension of killed Bordetella pertussis has been added. tetanus and pertussis are carried out on a suitable number of batches of vaccine reconstituted for use. (Lf). The reference vaccine may be stabilized by a method that has been shown to have no effect on the assay procedure. pertussis component and admixture of a suitable 37 Production General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria. when conjugated to PRP.. If more than 1 animal dies from nonspecific causes. The antigenic properties of the vaccine are adversely affected by the presence of certain antimicrobial preservatives particularly those of the phenolic type and some of the quaternary ammonium type must not be used. per mg of protein nitrogen.2. For the preparation of a representative batch. the vaccine does not comply with the test. strict adherence to the production process used for the batch tested in clinical trials is necessary. (2. purified tetanus formol toxoid containing not less than 1. The product may be presented with the haemophilus component in a separate container. Pertussis (Whole Cell) and Haemophilus Type b Conjugate Vaccine (Adsorbed) Diphtheria.IP 2007 D. the contents of which are mixed with the other components immediately before or during use. The production method is validated to demonstrate that the product. the assays of the diphtheria. admixture of an appropriate quantity of a suspension of inactivated B. with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein. PRP is a linear copolymer composed of repeated units of 3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n]. aluminium phosphate or calcium phosphate. bulk purified tetanus toxoid. of suitable quantities of bulk purified diphtheria toxoid. separately or together.16). (WHOLE CELL) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE (ADSORBED) Diphtheria. Pertussis Vaccine (Whole Cell) and Haemophilus influenzae Type b Conjugate Vaccine. FINAL BULK VACCINE Vaccine with all components in the same container The final bulk is prepared by adsorption. release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. T. that have not previously been treated with any material that will interfere with the test.. toxemia or tetanus.500 Limes flocculationis. The formol toxoids are prepared from the toxin produced by the growth of Corynebacterium diphtheriae and Clostridium tetani.000 Lf. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed).16) per mg of protein nitrogen. respectively in suitable media. If the vaccine is presented with the haemophilus component in a separate vial. P. if tested. repeat the test once. onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. would comply with the following test for specific toxicity of the diphtheria and tetanus component: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea pigs. if more than 1 animal shows signs of or dies in the second test. For subsequent routine control. in saline solution or other appropriate solution isotonic with blood. as part of consistency studies.2. the vaccine does not comply with the test. . monocomponent reference vaccines may be used for the assays on the combined vaccine. the assays of these components may be carried out without mixing with the haemophilus component. The stability of the final lot and the relevant intermediates is evaluated using one or more indicator tests. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine. is capable of inducing a Tcell dependent B-cell immune response to the polysaccharide. determination of free PRP in the conjugate and kinetics of depolymerisation. each weighing between 250 and 350 g. Tetanus. The toxins are converted to toxoids by treatment with formaldehyde solution by methods which avoid reversibility of the toxoids. polyribosyl ribitol phosphate. a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. Suitable antimicrobial preservatives may be added.2 g/l.11).D. 6. Carry out test for sterility using 10 ml of bulk for each sterility medium. the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. separately or together. The osmolality of the vaccine is within the limits approved for the particular preparation. antimicrobial preservative and sterility are carried out on the container with the diphtheria. the resulting mixture is approximately isotonic with blood. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0. Antimicrobial preservative. Tetanus toxoid. B. Tests and Assay may be released for use. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate.. Tests If the product is presented with the haemophilus component in a separate container.. bulk purified tetanus toxoid onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. agglutination indicates presence of pertussis component. the resulting mixture is approximately isotonic with blood. Description. pertussis are used.0 to 7. Maintain at 37o for about 16 hours and centrifuge. If 2 or more strains of B. A stabilizer may be added. FINAL LOT Where the haemophilus component is in a separate container.4. The amount is not less than 85. pertussis are used. . Where applicable. sterility and pyrogens are carried out on the container with the haemophilus component. Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification.2. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent.16). tetanus and pertussis components. The final bulk is filled separately. the final bulk of the haemophilus component is freeze-dried.0.23). PRP. the test for free formaldehyde may be omitted on the final lot. Test E may be omitted if tests A. T. the tests for PRP content.0 percent of the intended content. pertussis concentration of the final bulk vaccine does not exceed that corresponding to opacity of 20 IU per single human dose. A. determine the amount of antimicrobial preservative by a suitable chemical method. The B. D. C. Production Identification Tests A. ultrafiltration or other validated methods. Reserve the residue for test C. for example by anion exchange. water (where applicable). The B. The vaccine confers an active immunity in mice and guinea pigs when administered as directed in the test for Potency. Free PRP. Diphtheria toxoid. Sterility (2. they may be omitted on the 38 final lot. Provided the tests specific toxicity of diphtheria toxoid.. P. If 2 or more strains of B.24). pertussis component and admixture of a suitable quantity of PRP conjugate. tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine. Suitable antimicrobial preservatives may be added. aluminium. Osmolality (2. tetanus toxoid and pertussis component and antimicrobial preservative and the assays for the diphtheria. free formaldehyde. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose.4. of suitable quantities of bulk purified diphtheria toxoid. C and D may be omitted if test E is carried out. Pertussis component. C and D are carried out. Whitish turbid liquid in which the mineral carrier tends to settle down slowly on keeping. B. admixture of an appropriate quantity of a suspension of inactivated B. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. (WHOLE CELL) AND HAEMOPHILUS TYPE CONJUGATE VACCINE (ADSORBED) IP 2007 quantity of PRP conjugate. size exclusion or hydrophobic chromatography (2. Unbound PRP is determined after removal of the conjugate. To a suspension of the residue obtained in test A in saline solution add a suitable Bordetella pertussis antiserum. Vaccine with the haemophilus component in a separate container The final bulk is prepared by adsorption. pH (2.4. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. E. tetanus toxoid and pertussis component. the tests for specific toxicity of diphtheria toxoid. The amount of free PRP is not greater than approved for the particular product. The suspension of the residue obtained in test A gives a positive reaction when tested by a suitable immunochemical method for PRP.0 per cent and not greater than 115. B. A highly toxinogenic strain of Corynebacterium diphtheriae with known origin and history is grown in a suitable liquid medium. Where applicable. (3) pertussis component – IU or IOU per single human dose.9). Tetanus and Pertussis Vaccine (Adsorbed). Abnormal toxicity (2. At the end of cultivation. from which toxoid is prepared.3. If one of the animal dies or shows the signs of ill health. Aluminium (2.2. The content is not less than 85. repeat the test .2. where necessary. Inject per kg of the rabbit’s mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria toxoid as carrier. Pertussis vaccine Complies with the test as stated under Diphtheria. Toxin-containing culture medium is separated aseptically from the bacterial mass . Sterility (2. (7) that the vaccine must be shaken before use.1 mg of PRP for a vaccine with tetanus toxoid as carrier protein. Tetanus toxoid (adsorbed) Complies with the test as stated under assay of Tetanus Vaccine (Adsorbed). Complies with the test for sterility. Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose . The formol toxoid is prepared from the toxin produced by the growth of Corynebacterium diphtheriae.14) or by anion exchange liquid chromatography with pulsed amperometric detection. Maximum 0. some tests may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. (5) the type and nominal amount of carrier protein per single human dose.025 mg of PRP for vaccine with OMP as carrier. Maximum 1.1) or phosphorus (2.7.11).0 per cent and not greater than 115. Labelling. but not more than 0.3. Production General provisions The maximum number of Lf units per single human dose of diphtheria vaccine (adsorbed) is 30.2 g/l.1). Storage. determine the amount of antimicrobial preservative by a suitable chemical method.8). (4) haemophilus conjugate component . the purity of each culture is tested and contaminated cultures are discarded. Not less than 80.25 ml into each of the five mice weighing between 17 to 22 g and at least one human dose but not more than 1.25 mg per single human dose. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Assay Diphtheria toxoid (adsorbed) 39 Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). restored by deliberate reselection. by an immunochemical method (2. PRP is determined either by assay of ribose (2. This test is carried out for Haemophilus influenzae type b vaccine only if Haemophilus influenzae type b vaccine is presented as separate lyophilized vial.0 per cent of the amount of PRP stated on the label.1). 0. (2) Diphtheria and Tetanus components (a) in case done by challenge method the minimum number of international units (as applicable for each component) per single human dose. Free formaldehyde (2. Antimicrobial preservative. Tetanus and Pertussis Vaccine (Adsorbed). The label states (1) the human dose (ml). Pyrogens (2. (b) in case done by antibody induction method.2. Diphtheria Vaccine (Adsorbed) Diphtheria Vaccine (Adsorbed) is a preparation of diphtheria formol toxoid with a mineral adsorbent.20). The vaccine complies with the test for pyrogens.IP 2007 DIPHTHERIA VACCINE (ADSORBED) If the haemophilus component is freeze-dried. the minimum Lf units per single human dose. 0. Pertussis component Complies with the test as stated under Diphtheria. (6) the name and amount of adsorbent and added preservative. When stored under the prescribed conditions the vaccine may be expected to retain potency for not less than 2 years from the date on which the potency test for the pertussis component was started.g PRP per single human dose. Specific toxicity Diphtheria and tetanus components Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Bulk purified toxoid For the production of diphtheria toxin. seed cultures are managed in a defined seed-lot system in which toxinogenicity is conserved and.0 per cent of the intended amount.7.0 ml into each of the two guinea pigs weighing between 250 and 350 g. The preparation passes the test if none of the animals dies or shows signs of ill health in 7 days following the injection . (9) that the vaccine is not to be frozen. The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified.2. PRP. Intradermal tests in guinea-pigs and cell-culture tests both are considered to be suitable. Alternatively. Maintain at 37o for about 16 hours and centrifuge. seal the plates and incubate at 37° for 5 to 6 days. Prepare serial twofold dilutions of the diluted diphtheria toxin and use undiluted test samples (50 µl per well). Identification Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. except for the presence of adjuvant. without adsorbent. Carry out test for sterility using 10 ml of bulk for each sterility medium. The bulk purified toxoid complies with the test if no toxicity neutralisable by antitoxin is found in either sample. The guinea-pigs shall not have been used previously for experimental purposes. particularly on exposure to heat.DIPHTHERIA VACCINE (ADSORBED) IP 2007 as soon as possible. Confirm cytopathic effect by microscopic examination or suitable staining such as MTT dye. Divide the solution into 2 equal parts. To determine whether reversion has occurred. adversely affect the antigenic activity and must not be used. a cell-culture test system may be used. Distribute them in the wells of a sterile tissue culture plate containing a medium suitable for Vero cells. Carry out a test in Vero cells for active diphtheria toxin using 50 µl per well of both samples. The purified toxin is detoxified with formaldehyde by a method that avoids destruction of the immunogenic potency of the toxoid and reversion of the toxoid to toxin. Use freshly trypsinised Vero cells at a suitable concentration. The test employed shall be approved by the National Regulatory Authority and should be sufficiently sensitive to detect very small amounts of toxin. A suitable reference diphtheria toxin will contain either not less than 100 LD50/ml or 67 to 133 lr/100 in 1 Lf and 25. The sample should not contain antimicrobial 40 preservatives and detoxifying agents should be determined to be below the concentration toxic to Vero cells. The test is invalid if 5 × 10-5 Lf per ml of reference diphtheria toxin in 100 Lf per ml toxoid has no cytotoxic effect on Vero cells or if the cytotoxic effect of this amount of toxin is not neutralised in the wells containing diphtheria antitoxin. The toxin content (Lf per ml) is checked to monitor consistency of production. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. for example 100 IU/ml. without adsorbent. particularly those of the phenolic type. using the same buffer solution as for the final vaccine.000 minimal reacting doses for guinea-pig skin in 1 Lf (diphtheria toxin RP is suitable for use as the reference toxin). and the test procedures shall be approved by the National Regulatory Authority. for example 2. Certain antimicrobial preservatives. Maintain 1 part at 5° ± 3° and the other at 37° for 6 weeks. the resulting mixture is approximately isotonic with blood. Only bulk purified toxoid that complies with the following requirements may be used in the preparation of the final bulk vaccine. Single harvests may be pooled to prepare the bulk purified toxoid. No toxicity shall be detected. prepare in parallel dilutions where the toxin is neutralised by a suitable concentration of diphtheria antitoxin.500 Lf per mg of protein nitrogen. Suitable antimicrobial preservatives may be added. Dilute the toxin in 100 Lf/ml diphtheria toxoid to a suitable concentration. indicated by the pH indicator of the medium. in this case. Absence of toxin and irreversibility of toxoid Inject subcutaneously into each of 5 guinea-pigs at least 500 Lf of the non-incubated bulk purified toxoid in a volume of 1 ml. for example 2 × 10-4 Lf per ml. Non-specific toxicity may be eliminated by dialysis. The bulk purified toxoid shall pass the test if no guinea-pig shows symptoms of specific intoxication within six weeks of injection and if at least 80 per cent of the animals survive the test period. Add cell suspension to each well. prepare a solution of bulk purified toxoid at 100 Lf per ml.2. Alternatively. Animals that die shall be autopsied and examined for symptoms of diphtheria intoxication (red adrenals). Cytotoxic effect is judged to be present where there is complete metabolic inhibition of the Vero cells. purification may be carried out after detoxification. Antigenic purity. Each bulk purified toxoid shall be tested to ensure that reversion to toxicity cannot take place on storage.11). The toxin is purified to remove components likely to cause adverse reactions in humans. The bulk purified toxoid shall be diluted in order to obtain the same concentration and chemical environment as those present in the final bulk vaccine. To ascertain that any cytotoxic effect noted is specific to diphtheria toxin. The clear supernatant . Cell culture method Using the same buffer solution as for the final vaccine.000 to 50. diluted toxoids that have been stored at 37° for six weeks shall be tested. Include control wells without toxoid or toxin and with non-toxic toxoid at 100 Lf per ml on each plate to verify normal cell growth.5 × 105 per ml and a reference diphtheria toxin diluted in 100 Lf per ml diphtheria toxoid. Not less than 1. the sensitivity of the test shall have been demonstrated to be not less than that of the guinea-pig test. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide. Sterility (2. The test may be repeated but when more than one test is performed the results of all valid tests must be combined in the estimate of potency. If necessary. Suggested method Test animals. healthy guinea-pigs weighing between 250 and 350 g.2 ml. the dilutions form a series differing by not more than 2.2 ml of the challenge toxin solution and with 0. result in an intradermal score of approximately three when the animals are challenged. None of the animals shows any symptoms of diphtheria or tetanus toxaemia or dies from diphtheria within 42 days or loses weight at the end of the test. Standard preparation The Standard preparation is International standard of Diphtheria toxoid. pH (2.0 to 7. Biological assay of adsorbed diphtheria vaccine (a) Intradermal challenge method The potency of adsorbed diphtheria vaccine is determined by comparing the dose necessary to protect guinea-pigs against the erythrogenic effects of a range of intradermal injections of diphtheria toxin with the dose of the Standard preparation of adsorbed diphtheria toxoid necessary to give the same protection. or another suitable preparation the potency of which has been determined in relation to the International Standard. Use white guinea-pigs. Weigh all the animals at weekly intervals for 6 weeks.000 to 50. Tabulate the intradermal challenge scores for all the animals receiving the same dilution of vaccine and use those data with a suitable transformation. the vaccine fails the test. and have not been previously treated with any material that will interfere with the test.0.0 and 200. dilute the challenge toxin with a suitable diluent to obtain a challenge toxin solution containing about 512x10-4 Lf in 0. which have been maintained for at least 1 week on a uniform.4. Inject subcutaneously into each animal 5 times the dose stated on the label. For this comparison. the mean score obtained at the lowest dose level is more than three.0 per cent of the control guineapigs and the dilution that contains 2x10-5 Lf gives no reaction in at least 80 per cent of the guinea-pigs (if these criteria are not met a different toxin has to be selected).0 ml of each dilution into each guinea-pig in the group to which that dilution is allocated. use groups containing a number of animals sufficient to obtain results that fulfill the requirements for a valid Assay prescribed below. weighing between 250 and 350 g. the Standard preparation of adsorbed diphtheria toxoid and a suitable preparation of diphtheria toxin. 4x10-5. for each. such as (score)2 or arcsin [(score/6)2]. for use as a challenge toxin. Allocate the dilutions. If more than one animal dies from non-specific causes or loses weight. Examine all the injection sites 48 hours after injection of the challenge toxin and record the incidence of specific diphtheria erythema. Determination of potency of the vaccine. when injected subcutaneously in 1. (b) Lethal challenge method Test animals.2 ml of each of the five dilutions thereof in such a way as to minimize interference between adjacent sites. Distribute the guinea-pigs into no fewer than six equal groups. unrestricted diet.0 ml volumes into guinea-pigs. it is not necessary to verify the activity for every assay. 1x10-5 and 5x10-6 Lf of the challenge toxin.0 per cent of the estimated potency. Use healthy. The guineapigs are all of the same sex or the males and females are distributed equally among the groups. to obtain an estimate of the relative potency for each of the test preparations by parallel-line quantitative analysis. Determine by any of the methods of biological assay of Adsorbed Diphtheria Vaccine described. Select a preparation of diphtheria toxin containing 67 to 133 Limes reactionis/100 (Lr/ 100) in Limes flocculationis (Lf) and 25. and inject subcutaneously 1. inject the unvaccinated control guinea-pigs with dilutions containing 8x10-5. If the challenge 41 toxin preparation has been shown to be stable. include five guinea-pigs as unvaccinated controls. If an animal dies or loses weight in the second test. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardized.000 minimal reacting doses for guinea-pig skin in 1 Lf. one to each of the groups of guinea-pigs. (d) the statistical analysis shows no deviation from linearity and parallelism. (c) the fiducial limits of the assay fall between 50. are required. Dilute a portion of this challenge toxin solution to give a series of five 4-fold dilutions. Weigh the animals separately and record their weights. repeat the test. Specific toxicity. The lower fiducial limit of error of the estimated potency is not less than 30 Units per dose. Prepare in saline solution dilutions of the vaccine under examination and of the Standard preparation such that. After 28 days shave both flanks of each guinea-pig and inject each animal intradermally with 0. Record also the number of sites free from such reactions as the intradermal challenge score. (b) if applicable. Preparation of the challenge toxin solutions. Immediately prior to use.IP 2007 reacts with a suitable diphtheria antitoxin and yields a precipitate. white or light-coloured guinea- . Selection of the challenge toxin.5 fold steps and in which the dilutions of intermediate concentration. The test is not valid unless (a) for both the preparation under examination and the Standard preparation. Potency. from the same stock. Use 5 normal. adsorbed. the toxin dilution that contains 4x10-5 Lf gives a positive erythema in at least 80. 6.24). 2x10-5. protect approximately 50 per cent of the animals from the lethal effects of the subcutaneous injection of the quantity of diphtheria toxin prescribed for this test. Tests and Assay may be released for use.2. Only a final lot that is satisfactory with respect to each of the requirements given below under. Immediately prior to use.0 per cent confidence interval should be within 50 to 200 of the estimated potency.0 ml of the challenge toxin solution.0 per cent of the intended amount. Identification. 1. Not earlier than 2 weeks and not later than 3 weeks after the second injection.0 ml of each dilution into each guinea-pig in the groups to which that dilution is allocated.0 ml volumes into guineapigs. The containers are closed so as to prevent contamination. Maximum 1. applicable to certain vaccines. (c) statistical analysis shows parallelism. Maintain at 37° for about 16 hours and centrifuge until a clear supernatant liquid is obtained. The following method. Preparation of the challenge toxin solutions. the dilutions form a series differing by not more than 2. weighing between 250 and 350 g. .0 per cent confidence interval of estimated potency is less than 30 IU per single human dose then the 42 limits of the 95. Tests Aluminium (2. The clear supernatant liquid reacts with a suitable diphtheria antitoxin.25 mg per single human dose. healthy guinea-pigs weighing between 250 and 350 g. Where applicable. remove dead animals and kill the animals showing definite signs of diphtheria.3. FINAL LOT The final bulk vaccine is distributed aseptically into sterile. (b) the survivors among the four groups of guinea-pigs injected with the challenge toxin and its dilutions indicate that the challenge was approximately 100 LD50 and. is given as an example. free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine. (c) Antibody induction method Inject subcutaneously on each of two occasions separated by an interval of not more than 4 weeks.0 ml of each toxin solution into each guinea-pig in the group to which that solution is allocated. If the lower limit of 95. linearity and a significant slope of the doseresponse lines. Distribute them into six groups of sixteen. and four groups of four. Allocate the six dilutions. giving a precipitate. collect the serum from each animal and determine the antitoxin content of the serum of each animal.0 Units per ml with reference to the Diphtheria antitoxin standard. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/ v solution. (d) Any other validated serological assay in guinea pigs or mice as approved by National Regulatory Authority Antimicrobial preservative.0 per cent and not greater than 115. they may be omitted on the final lot. The test may be repeated any number of times but when more than one test is performed the results of all valid tests must be combined in the estimate of potency.4. Sterility (2. when injected subcutaneously in 1.0 ml. one to each of the four groups of four guineapigs and inject subcutaneously 1. After 28 days inject subcutaneously into each animal in the six groups of sixteen. The amount is not less than 85. into each of 10 normal. and inject subcutaneously 1. Challenge toxin. The test is not valid unless (a) for the vaccine under examination and the Standard preparation the 50.11). using appropriate statistical methods. Count the number of surviving animals 5 days later and calculate the potency of the vaccine under examination relative to the potency of the Standard preparation on the basis of the number of animals that survive in each of the six groups of sixteen. Select a preparation of diphtheria toxin containing not less than 100 LD50 in 1. Prepare in saline solution three dilutions of the vaccine under examination and three dilutions of the Standard preparation such that for each. one to each of the six groups of sixteen guinea-pigs. determine the amount of antimicrobial preservative by a suitable chemical method. or normal saline a challenge toxin containing approximately 100 LD50 in 1.0 per cent protective doses lie between the largest and smallest doses of the preparations given to the guinea-pigs. Identification Diphtheria toxoid is identified by a suitable immunochemical method. Dilute portions of this challenge toxin solution to 2LD50. if aluminium hydroxide or hydrated aluminium phosphate is used as the absorbent. one-fiftieth of the stated human dose diluted to 1 ml with saline solution. The guinea-pigs should all be of the same sex or the males and females should be distributed equally between the six groups of sixteen. Allocate the challenge toxin solution and the three dilutions made from it. 1 LD50 and ½ LD50 in the same solution Determination of potency of the vaccine. tamper-proof containers. Carry out test for sterility using 10 ml of bulk for each sterility medium. as described under Diphtheria Antitoxin or any other method approved by National Regulatory Authority.0 ml. The geometric mean of the antitoxin contents shall be not less than 2.DIPHTHERIA VACCINE (ADSORBED) IP 2007 pigs from the same stock. Provided the tests for specific toxicity.5 fold steps and in which the dilutions of intermediate concentration. Examine the guinea pigs twice in a day.9). prepare from the challenge toxin by dilution in phosphate buffered saline pH 7. Labelling. determine the amount of antimicrobial preservative by a suitable chemical method. with a defined molecular size. release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity.0 per cent and not greater than 115.e. 6. would comply with the test for safety and efficacy of Vaccines. in its salt form. Where applicable.0. Cultures derived from the working seed shall have the same characteristics as of the master seed lot. The production of PRP and of the carrier protein is based on defined seed lot systems. In the purified polysaccharide is determined by methods such as thermogravimetry. The lower confidence limit (P = 0. when conjugated to PRP. Limits for currently approved products. immunogenicity test on mice. PRP is separated from the culture liquid and purified by a suitable method. (3) the name and the amount of the adsorbent and preservative. Taking account of the results of the stability testing. Volatile matter.0 to 7. covalently bound to a carrier protein. H. is determined by gel filtration or high performance size-exclusion chromatography (HPSEC) (2.g.IP 2007 HAEMOPHILUS TYPE B CONJUGATE Free formaldehyde (2. Antimicrobial preservative. are shown for information in 43 Production General provisions The production method shall have been shown to yield consistently H. The stability of the final lot and relevant intermediates is evaluated using one or more indicator tests.20). (5) that the vaccine is not to be frozen. The partially purified PRP shall be stored frozen at or below -20°. MALLS) or any other suitable method. Karl Fischer or any other suitable method. The carrier protein. influenzae type b conjugate vaccines of adequate safety and immunogenicity in humans.g. Influenzae Type b Polysaccharide (PRP) H. if tested. influenzae Type b is grown in a liquid medium that does not contain high-molecular-weight polysaccharides.4. Such tests may include determination of molecular size.1). SEED LOT The strain of H. is capable of inducing a T-cell-dependent B-cell immune response to the polysaccharide. An acceptable value is established for the particular product and each batch of PRP must be shown to comply with this limit. derived from a suitable strain of Haemophilus influenzae type b.2. (4) that the vaccine must be shaken before use.2. All chemical analysis shall be based on the dry weight of the polysaccharide. pH (2. 1H or 13C NMR spectroscopy). including water. Haemophilus Type b Conjugate Vaccine Haemophilus Type b Conjugate Vaccine is a liquid or freezedried preparation of a polysaccharide. using the indicated stationary phases. multiple angle laser light scattering i. Identification The PRP is identified by an immunochemical method (2. Sterility (2. The polysaccharide.16). if any ingredient of the medium contains blood-group substances. is a linear copolymer composed of repeated units of 3-β-D-ribofuronosyl-(11)-ribitol-5phosphate [(C10H19O12P)n].95) of the estimated potency is not less than 30 IU per single human dose. Only those pools of PRP that comply with the following requirements may be used in the preparation of the conjugate.11).14) or other suitable method (e. Master seed lot and working seed lot shall be properly characterized and defined. referred to as PRP. The label states (1) the human dose. The production method is validated to demonstrate that the product. either alone or in combination with light scattering and refractive index detectors (e. Maximum 0.0 per cent of the intended amount. The culture may be inactivated. Assay Carry out one of the prescribed methods for the assay of Diphtheria Vaccine (Adsorbed). The percentage of PRP eluted before a given K0 value or within a range of K0 values.24). influenzae type b used in preparing Haemophilus type b conjugate vaccine shall be identified by a record of its history. including the source from which it was obtained and the tests made to determine the characteristics of the strain.2 g/l. The content is not less than 85.4. Molecular size. determination of free PRP in the conjugate and the . the process shall be validated to demonstrate that after the purification step they are no longer detectable.2. Abnormal toxicity (2. Complies with the test for sterility. (2) the minimum Lf units per single human dose or the minimum International Units per single human dose if potency test done by challenge method.3. The strain shall have been shown to be capable of producing Type b polysaccharide. polyribosylribitol phosphate. The sample of culture of single harvests taken before killing shall be tested for contamination by examination of Gram-stained smears and by inoculation on suitable media. Complies with the test for abnormal toxicity. 0 per cent <0.0 per cent diphtheria protein 25µg Polysaccharide (size-reduced) K0 0.0 per cent ≤ K0: 0.0 per cent 0.75 0.-AH) + tetanus toxoid + EDAC Direct coupling of PRP to CRM 197 (cyanoboro-hydride activated) Tetanus toxoid >1500 Lf per mg of protein nitrogen 20 µg CRM 197 diphtheria protein >90.0 per cent <1.3-0.7 Polysaccharide: 10 µg PRP ≥ 50.3-0.55 60.0 per cent. cyanogen bromide activated PRP ADH-activated PRP (PRP-cov. Polysaccharide Type of PRP Nominal amount per dose Type Carrier Protein Purity Nominal amount per dose 18 µg Coupling method Cyanogen bromide activation of PRP Carbodiimidemediated coupling Reductive amination one(step method) or N-hydroxysuccinimide activation Thioether bond Conjugation Procedure Polysaccharide 25 µg (size reduced) K0 60. where applicable OMP <15. Table 2 .0 per cent <0.0 per cent Not applicable PRP to protein ratio Molecular size (K0): Crosslinked agarose for chromatography R Cross-linked agarose for chromatography R1 1.Requirements on bulk conjugate Test Specifications Free Polysaccharide (PRP) Unbound protein Protein Carrier Tetanus toxoid CRM 197 <20. 4-diamide carbonyldi-imidazole Dp EDAC IM Mw = = = = degree of polymerization l-ethyl-3-(3-dimethylaminopropyl) carbodimide imidazolium weight-average molecular weight.0 per cent: 0.5 44 .75 95.2 0.7 50. depending on the coupling method 0.25-1.0 per cent.05-0.0 per cent <0.3 – 0.30-0.0 per cent.0 per cent <1.1 85.Specifications for different components of Haemophilus Type b Conjugate Vaccine.6 Diphtheria toxoid <37.6-0.7 85.25 0.HAEMOPHILUS TYPE B CONJUGATE IP 2007 Table 1 .6-0.30 Polysaccharide 10 µg (size reduced) Dp=15-35 or 10-35 Diphtheria toxoid >1500 Lf per mg of protein nitrogen Activated diphtheria toxoid (D-AH+).0 per cent < 0.6 15 µg Meningococcal group B outer membrane protein (OMP) Outer membrane 125 or protein vesicles 250 µg <8.0 per cent <5. where applicable <25.0 per cent of lipopolysaccharide PRP activation by CDI PRP-IM + BuA2 + BrAc = PRP-BuA2-BrAc + thioactivated OMP Abbreviations: ADH = BrAc = BuA2 = CDl = adipic acid dihydrazide bromoacetyl chloride butane-1.0 per cent or <2. Where applicable. Where validation studies have demonstrated removal of a residual reagent. the carrier protein is purified by a suitable method.49).0 per cent. the molecular-size distribution is also determined after chemical modification of the polysaccharide. HPLC.2.g. Not less than 32. tests are carried out to determine residues of reagents used during inactivation and purification. determined by a suitable method.1).1). Not more than 1. Br etc. calculated with reference to the dried substance by spectroscopy or any other suitable method.7. Limits applied to currently approved products for some of these tests are listed for information in Table 2. Lipopolysaccharide. a minimum of 1 mg of PRP). The conjugate is obtained by the covalent binding of PRP and carrier protein.7. circular dichroism.3 ). PRP. fluorescence spectroscopy. using D-ribose as a standard or any other suitable assay.4. it is usually partly depolymerised either before or during this procedure.14) or by any suitable method.8 per cent to 9. Not more than 8. CN. peptide mapping or mass spectroscopy.1) or by assay of ribose (2. A validated determination of the degree of polymerization or of the weight-average molecular weight and the dispersion of molecular masses may be used instead of the determination of molecular size distribution. For each test and for each particular product. the test on bulk conjugate may be omitted. Sterility (2. Carry out the test for sterility using for each medium 10 ml or the equivalent of one hundred doses. Diphtheria toxoid is produced as stated under Diphtheria Vaccine (Adsorbed) and complies with the requirements prescribed there for bulk purified toxoid. limits of acceptance are established and each batch of conjugate must be shown to comply with these limits. for determining the pyrogenic effect. Residual reagents. calculated with reference to the dried substance.2.1) or by an immunochemical method (2. Carrier protein The carrier protein is chosen in a way so that when the PRP is conjugated it is able to induce a T-cell-dependent immune response. Diphtheria toxoid. Bacterial endotoxins (2. OMP (Meningococcal group B outer membrane protein complex) OMP complex of Neisseria meningitidis complies with the following requirements for lipopolysaccharide and pyrogens. Use sufficient PRP to allow detection of 1 per cent of protein (e. as estimated by Bial reaction for pentose. Phosphorus (2. Nucleic acid (2.).7. Where validation studies have demonstrated removal of a residual reagent (eg. Where applicable. Currently approved carrier proteins and coupling methods are listed for information in Table 1. An acceptable value for each reagent is established for the particular product and each batch of PRP must be shown to comply with this limit.0 per cent.2. the bacterial purity of the culture is verified.2.2. Reactive functional groups or spacers may be introduced into the carrier protein or PRP prior to conjugation.14). amino acid sequencing. The PRP content is determined by assay of phosphorus (2. Tetanus toxoid is produced as stated under Tetanus Vaccine (Adsorbed) and complies with the requirements prescribed there for bulk purified toxoid except that the antigenic purity is not less than 1500 Lf per mg of protein nitrogen. Where applicable. 6.3. The protein obtained contains not less than 90. The carrier protein shall be characterized by a suitable chemical or physicochemical method like SDS-PAGE.1). Not more than 1. for validation or routinely.0 per cent.7. Suitable tests are carried out. Protein (2. to demonstrate that the product is non-toxic.0 per cent of diphtheria CRM 197 protein. Bulk conjugate PRP is chemically modified to enable conjugation. Only a carrier protein that complies with the following requirements may be used in preparation of the conjugate. the culture may be inactivated. when prepared by liquid chromatography (2. 45 Identification The carrier protein is identified by a suitable immunochemical method (2.IP 2007 HAEMOPHILUS TYPE B CONJUGATE Tables 1 and 2. The carrier proteins are produced by culture of suitable microorganisms. Tetanus toxoid.14) or any other suitable method. Inject into each rabbit 0. as appropriate. isoelectric focusing. Only a bulk conjugate that complies with the following requirements may be used in preparation of the final bulk vaccine.11).8). whichever is less. Pyrogens (2. calculated with reference to the dried substance.0 per cent. the conjugate is purified to remove reagents.7. Diphtheria protein CRM 197.0 per cent of lipopolysaccharide. Ribose (2. unreacted but potentially reactogenic functional groups are made unreactive by means of capping agents. . calculated with reference to the dried substance. the test on PRP may be omitted. Not more than 25 IU of endotoxin per microgram of PRP.25 µg of OMP per kg body weight. Not less than 80.4. Free carrier protein.2.0 per cent of the intended amount. ultra filtration or other validated methods. Only a final lot that is satisfactory with respect to each of the requirements given under Identification. Antimicrobial preservative.2.1). Determine the ratio by calculation.16).1 µg of PRP for a vaccine with tetanus toxoid as carrier. The label states (1) the number of micrograms of PRP per human dose. When aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free PRP. No unreacted functional groups are detectable in the bulk conjugate unless process validation has shown that unreacted functional groups detectable at this stage are removed during the subsequent manufacturing process (for example. it may be omitted on the final lot.2. Abnormal toxicity (2.4.2.2.7. Removal of residual reagents such as cyanide.7. Molecular size.5 to 7. Unreacted functional groups. an antimicrobial preservative and a stabilizer may be added to the bulk conjugate before dilution to the final concentration with a suitable diluent. Sterility (2.14) with pulsed amperometric detection. determine the amount of antimicrobial preservative by a suitable chemical method. determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85. ultra-filtration or other validated methods. The protein content is determined by a suitable chemical method.43). Pyrogens (2.4. Sterility (2. Free carrier protein is determined by a suitable method (which may include deriving the content by calculation from the results of other tests). 46 Identification The vaccine is identified by a suitable immunochemical method (2. Carry out test for sterility using 10 ml of bulk for each sterility medium. Aluminium (2. appropriate to the expected size of the conjugate. FINAL LOT The final lots are filled in suitable containers. (2) the type and nominal amount of carrier protein per single human dose. 0.14) for PRP or the assay serves also to identify the vaccine.2. The amount is within the limits approved for the particular product. Unbound PRP is determined after removal of the conjugate. FINAL BULK VACCINE An adjuvant.0 per cent and not greater than 115.0 per cent. is within the range approved for the product (6.4. Molecular-size distribution is determined by gel filtration or size-exclusion chromatography (2. Tests and Assay may be released for use. (3) for vaccine contained in single-dose containers where the space is too small to accommodate the full name of the vaccine the abbreviation ‘Hib’ may be used in the label on the container provided that the same code is also stated in the label on the package.16).8). Residual reagents.11).14) or by any other suitable method like colorimetery or by anion exchange liquid chromatography (2. Unbound PRP is determined after removal of the conjugate for example by size-exclusion or hydrophobic chromatography (2. Where applicable.16). using a gel matrix. Tests Sterility (2.11). Where applicable.5).3.1) or by phosphorus assay (2. Not more than 3. Provided the test for antimicrobial preservative has been carried out on the final bulk vaccine. under stringent aseptic conditions. EDAC (ethyldimethylaminopropylcarbodimide) and phenol is confirmed by suitable tests or by validation of the purification process.0 per cent and not greater than 115. Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed) Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed) is a suspension consisting of a suitable strain of hepatitis A virus. Complies with the test for abnormal toxicity.11).7. 0. Labelling. The content is not less than 85.2. PRP.3.0 per cent of the amount of PRP stated on the label as determined by ribose assay (2. Complies with the test for pyrogens. PRP to protein ratio.9). Inject per kg of the rabbit’s mass a quantity of the vaccine equivalent to 1 µg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier. reconstituted if necessary. pH (2.025 µg of PRP for a vaccine with OMP as carrier. owing to short half-life). whichever is less. Carry out the tests for sterility using for each medium 10 ml or the equivalent of one hundred doses.1).0 per cent of the intended amount. grown in cell cultures and inactivated by a .24).1) or by an immunochemical method (2. Complies with the test for sterility.HEPATITIS A (INACTIVATED) AND HEPATITIS B (rDNA) VACCINE (ADSORBED) IP 2007 Protein (2. for example by size-exclusion or hydrophobic chromatography (2. Only a final bulk vaccine that complies with the following requirements may be used in preparation of the final lot. Water (2. The pH of the vaccine.4. Free PRP. Antimicrobial preservative. Labelling.2.0 per cent and not greater than 115. (2) the type of cells used for production of the vaccine. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated harvests of hepatitis A virus and one or more batches of purified antigen of Hepatitis B (rDNA).25 mg per single human dose if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. in clinical studies in young. Reference preparation The reference preparation is part of a representative batch shown to be at least as immunogenic in animals as a batch that. Where applicable. Less than 2 IU per human dose.0 per cent of the intended amount. if tested. using specific antibodies or by the mouse immunogenicity tests described under assay. Antimicrobial preservative. carried out with satisfactory results on the final bulk vaccine.0 per cent of the intended amount. This preparation is white or almost white translucent liquid in which the mineral carrier tends to settle down slowly on keeping but is free from foreign particles/ floccules. Production General provisions The two components are prepared as described in the monographs on Hepatitis A Vaccine (Inactivated. determine the amount of antimicrobial preservative by a suitable chemical method. Bacterial endotoxins (2.20).11). The label states (1) the amount of hepatitis A virus antigen and hepatitis B surface antigen per container.2. The content is not less than 85. the antigens are adsorbed on a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate.3). For hepatitis A.IP 2007 HEPATITIS B (rDNA) VACCINE validated method. If the assay of the hepatitis A and/or the hepatitis B component is carried out in vivo. then provided it has been 47 Tests Aluminium (2. Antimicrobial preservative. Adsorbed) and Hepatitis B Vaccine (rDNA) and comply with the requirements prescribed therein. Identification The vaccine is shown to contain hepatitis A virus antigen and hepatitis B surface antigen by suitable immunochemical methods (2. after a full-course primary immunization. Maximum 0. corresponding to a level of neutralizing antibody recognized to be protective.14).2. Complies with the test for sterility. Hepatitis B Vaccine (rDNA) Hepatitis B Vaccine (rDNA) is a non-infectious preparation containing the purified major surface antigen of Hepatitis B virus (HBsAg). Sterility (2. they may be omitted on the final lot. healthy adults. The production method is validated to demonstrate that the product. produces not less than 95. For hepatitis B. Sterility (2.0 per cent seroconversion. antibody level not less than 10 mIU/ml against HBsAg is recognized as being protective. an antibody level not less than 20 mIU/ml determined by enzymelinked immunosorbent assay is recognized as being protective. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Hepatitis B component Complies with the assay as stated under Hepatitis B Vaccine (rDNA). and of hepatitis B surface antigen (HBsAg). it may be omitted on the final lot. Carry out test for sterility using 10 ml of bulk for each sterility medium.3. The content is not less than 85. Assay Hepatitis A component Complies with the assay as stated under Inactivated Hepatitis A Vaccine (Adsorbed). Where applicable. would comply with the test for abnormal toxicity for antisera and vaccines.2.0 per cent and not greater than 115. (4) that the vaccine must be shaken before use. (3) the name and amount of the adsorbent used.11).3. Provided that the tests for free formaldehyde (where applicable) and antimicrobial preservative content (where applicable) have been carried out on the final bulk vaccine with satisfactory results.2 g/l. Maximum 1.9). Production General provisions The antigen is manufactured by recombinant DNA technology by culturing genetically engineered yeast cells or other suitable . FINAL LOT Only a final lot that complies with each of the requirements given below under Identification. Tests and Assay may be released for use. determine the amount of antimicrobial preservative by a suitable chemical method. a component protein of hepatitis B virus obtained by recombinant DNA technology. (5) that the vaccine must not be frozen. Free formaldehyde (2. PROPAGATION AND HARVEST Identity. The purified antigen is finally adsorbed on aluminium hydroxide or aluminium phosphate. The total protein is determined by a validated method. If mammalian cells are used. a test for residual caesium is carried out on the purified antigen. The protein fraction of the antigen is characterized in terms of the primary structure (for example.15). The production method must be validated to demonstrate that the product if tested. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. The purity of the antigen is determined by comparison with a reference preparation using liquid chromatography or other suitable methods such as SDS-PAGE with staining. Antigenic purity. Antigen content and identification.11). The purified antigen complies with the test for sterility. The content of proteins. It must also have been shown to induce specific.3. the pre-S2 gene. Reference preparation. by partial amino-acid sequence analysis and by peptide mapping). For hepatitis B. lipids. The content is within the limits approved for the specific product.. PURIFIED ANTIGEN Only a purified antigen that complies with the following requirements may be used in the preparation of the final bulk. protective antibodies in human beings. microbial purity.0 per cent of total protein.0 per cent of the total protein consists of hepatitis B surface antigen. plasmid retention and consistency of yield are determined at suitable production stages. enzyme-linked immunosorbent assay (ELISA). Composition. carried out using 10 ml for each medium. not more than 10 pg of DNA in the quantity of purified antigen equivalent to a single human dose of vaccine. If mammalian cells are used for production. nucleic acids and carbohydrates is determined. The purity of the antigen is determined by comparison with a reference preparation using liquid chromatography or other suitable methods such as SDSPAGE with any suitable staining method.2. tests for extraneous agents and mycoplasmas are performed in accordance with tests for extraneous agents in viral vaccines for human use. . A suitable method is sensitive enough to detect a potential contaminant at a concentration of 1.49).2. FINAL BULK VACCINE An antimicrobial preservative and an adjuvant may be included in the vaccine. lipid and carbohydrate structure of the antigen is established. The content is not less than 85.0 per cent of the intended amount. Host-cell and vector-derived DNA (2. Sterility (2. Caesium. Additional tests on the purified antigen may be required depending on the production method used: for example. Several physico-chemical steps are employed to purify the Hepatitis-B surface antigen (HBsAg). determine the amount of antimicrobial preservative by a suitable chemical method. Antimicrobial preservative. The molecular weight of the major band in a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced conditions corresponds to the value expected from the gene sequence and possible glycosylation. The complete protein. Not less than 95. The antigen/protein ratio is within the limits approved for the specific product. by determination of the aminoacid composition. immunoblot (preferably using a monoclonal antibody directed against a protective epitope) or single radial diffusion. a combination of the S gene and pre-S2 gene products (middle protein) or a combination of S gene. Where applicable.HEPATITIS B (rDNA) VACCINE IP 2007 cell lines which carry the gene that codes for major surface antigen of the Hepatitis-B virus as approved by the competent authority. The buoyant density of the antigen particles is determined by a physico-chemical method. A part of a batch shown to be at least as immunogenic as a batch that was used in clinical studies and approved by National Regulatory Authority and determined by any suitable method. Characterisation of the substance Development studies are carried out to characterize the antigen. The morphological characteristics of the antigen particles are established by electron microscopy. antibody level not less than 10 mIU/ml against HBsAg is recognized as being protective. would comply with the tests for safety and efficacy.0 per cent and not greater than 115. a test for residual animal serum where mammalian cells are used for production or tests for residual chemicals used during extraction and purification. If a caesium salt is used during production. The antigenic epitopes are characterized. and preS1 gene products (large protein). by a suitable immunochemical method such as radioimmunoassay (RIA). The content is within the limits approved for the specific product. The method used for production of the vaccine must have been shown to yield a product consistently complying with the requirements for immunogenicity and safety. The quantity and specificity of HBsAg is determined in comparison with the International standard for HBsAg subtype ad or an in-house reference. for example gradient centrifugation. The vaccine may contain the product of the S gene (major protein). 48 Total protein (2. Enzyme Linked Immunosorbent Assay (ELISA) using monoclonal antibodies specific for protection inducing epitopes of HBsAg have been shown to be suitable. Where applicable. From the distribution of reaction levels measured on all the sera in the unvaccinated (control group).0 and 125. where applicable. Inject the equivalent of one human dose into each rabbit. Provided that the tests for free formaldehyde.0 per cent and not greater than 115. The data obtained is analyzed by a parallel-line model and may be suitably transformed for statistical evaluation. Maximum 1. Determine the potency either in animals (Method A) or by a validated in vitro procedure (Method B) described below: Method A (Biological) The potency of the vaccine under examination is determined in animals by comparing in given conditions its capacity to induce specific anti-HBsAg antibodies in mice or guinea-pigs with the same capacity as with the reference standard.9). One group of control animals remains unvaccinated but is injected intraperitoneally with the same volume of the diluent alone. Anaesthetize and bleed the animals 28 to 42 days later. The acceptance criteria are approved for a given reference preparation by the National Regulatory Authority in the light of the validation data.3. Complies with the test for pyrogens. (c) the fiducial limits of the estimated relative potency fall between 33. Adequate number of dilutions and replicates of the vaccine under examination and the reference standard are employed in the assay. the test. Assay the individual sera for specific HBsAg antibody concentration by a suitable immunochemical method such as ELISA or RIA.0 and 300. they may be omitted on the final lot. Use animals of the same sex in the test. where applicable.11). is applied to the data. Calculate the result of the assay by standard statistical methods (5. The test is not valid unless (a) the statistical analysis shows no deviation from linearity or parallelism. Labelling. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbant. weighing between 15 and 20 g (about 5 weeks old).2. Any response in the vaccinated animals that exceeds this level is by definition seroconversion.IP 2007 HEPATITIS B (rDNA) VACCINE Sterility (2. Tests and Assay may be released for use. Sterility (2. The percentage of animals showing seroconversion in each group is transformed (for example.11).7). antimicrobial preservative content and the assay in animals. (b) the fiducial limits of the estimated relative potency fall between 80. using the log dose response curve. the maximum reaction level that can be expected to occur in an unvaccinated animal for that particular assay is determined.0 per cent of the estimated potency.0 per cent of the estimated potency. Healthy guinea pigs weighing between 300 and 350g (about 7 weeks old) that have not been previously treated with any material that will interfere with the test will also be suitable for . determine the amount of antimicrobial preservative by a suitable chemical method.25 mg per single human dose. also serves to identify the vaccine.0. (b) the statistical analysis shows no deviation from linearity or parallelism. The test is not valid unless (a) for both the test and reference vaccine. The vaccine complies with the assay of Hepatitis-B Vaccine (rDNA) described below. Antimicrobial preservative. A validated test for bacterial endotoxins may be used instead of the test for pyrogens. Commercially available kits for measuring HBsAg in vitro may be used provided they are validated to produce equally precise and accurate results. the ED50 lies between the smallest and the largest doses given to animals . The upper fiducial limit (P = 0. The content is not less than 85. Inject similar groups of animals with the reference preparation of HepatitisB vaccine (r DNA). Carry out test for sterility using 10 ml of bulk for each sterility medium. Method B (In vitro) The potency of the vaccine under examination is determined by an in vitro method that has been validated against the biological test. Complies with the test for sterility. Tests Aluminium (2. have been carried out on the final bulk vaccine with satisfactory results.2. Assay. The label states (a) the amount of HBsAg per dose. FINAL LOT Only a final lot that complies with each of the requirements given below under Identification. Pyrogens (2. (b) the type of cells used for production of the vaccine.0 per cent of the intended amount. Potency.2.95) of the estimated relative potency is not less than 1. probit transformation) and a parallel line model. of either sex distributed randomly into several groups of mice. (c) the 49 Identification The assay or. the electrophoretic profile. keeping the individual sera separate. Inject intraperitoneally not less than three doses of suitable dilutions of the vaccine under examination diluted with adjuvant used in the vaccine into groups of a suitable strain of mice.8). The potency of the preparation under examination relative to the reference preparation is thus established. INACTIVATED HEPATITIS A VACCINE (ADSORBED) IP 2007 name and amount of the adjuvant; (d) that the vaccine must be shaken before use; (e) that the vaccine must not be frozen. Virus concentration. The virus concentration of each master and working seed lot is determined to monitor consistency of production. Extraneous agents (2.7.3). The master and working seed lots comply with the requirements for seed lots for virus vaccines. In addition, if primary monkey cells have been used for isolation of the strain, measures are taken to ensure that the strain is not contaminated with simian viruses such as simian immunodeficiency virus and filoviruses. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are being handled. Animal serum (but not human serum) may be used in the cell culture media. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from extraneous agents. The cell culture media may contain a pH indicator, such as phenol red and approved antibiotics at the lowest effective concentration. Not less than 500 ml of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells). Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. Only a single harvest that complies with the following requirements may be used in the preparation of the vaccine. When the determination of the ratio of virus concentration to antigen content has been carried out on a suitable number of single harvests to demonstrate consistency, it may subsequently be omitted as a routine test. Inactivated Hepatitis A Vaccine (Adsorbed) Hepatitis A Vaccine (Inactivated, Adsorbed) is a liquid preparation of a suitable strain of hepatitis A virus grown in cell cultures, inactivated by a validated method and adsorbed on a mineral carrier. The vaccine is an opalescent suspension. The vaccine complies with the monograph on Vaccines. Production Production of the vaccine is based on a virus seed-lot system and a cell-bank system. The production method shall have been shown to yield consistently vaccines that comply with the requirements for immunogenicity, safety and stability. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Unless otherwise justified and authorised, the virus in the final vaccine shall not have undergone more passages from the master seed lot than were used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. Reference preparation. A part of a batch shown to be at least as immunogenic as a batch that, in clinical studies in young healthy adults, produced not less than 95.0 per cent seroconversion, corresponding to a level of neutralising antibody accepted to be protective, after a full-course of primary immunisation is used as a reference preparation. An antibody level of 20 mIU /ml determined by enzyme-linked immunosorbent assay is recognised as being protective. Substrate for virus propagation The virus is propagated in a human diploid cell line or in a continuous cell line approved by the competent authority. SEED LOT The strain of hepatitis A virus used to prepare the master seed lot shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Only a seed lot that complies with the following requirements may be used for virus propagation. Description. A clear, colourless or light coloured liquid. Identification The test for antigen content also serves to identify the single harvest. Sterility (2.2.11). The single harvest complies with the test for sterility, carried out using 10 ml for each medium. Mycoplasmas (2.7.4). The single harvest complies with the test for mycoplasmas carried out using 1ml for each medium. Control cells. The control cells of the production cell culture comply with a test for identity and the requirements for extraneous agents. Antigen content. Determine the hepatitis A antigen content by a suitable immunochemical method (2.2.14) to monitor production consistency; the content is within the limits approved for the particular product. Ratio of virus concentration to antigen content. The consistency of the ratio of the concentration of infectious virus, as determined by a suitable cell culture method, to antigen content is established by validation on a suitable number of single harvests. 50 Identification Each master and working seed lot is identified as hepatitis A virus using specific antibodies. IP 2007 INACTIVATED HEPATITIS A VACCINE (ADSORBED) PURIFICATION AND PURIFIED HARVEST The harvest, which may be a pool of several single harvests, is purified by validated methods. If continuous cell lines are used for production, the purification process shall have been shown to reduce consistently the level of host-cell DNA. Only a purified harvest that complies with the following requirements may be used in the preparation of the inactivated harvest. Virus concentration. The concentration of infective virus in the purified harvest is determined by a suitable cell culture method to monitor production consistency and as a starting point for monitoring the inactivation curve. Antigen:total protein ratio. Determine the hepatitis A virus antigen content by a suitable immunochemical method (2.2.14). Determine the total protein by a validated method. The ratio of hepatitis A virus antigen content to total protein content is within the limits approved for the particular product. Bovine serum albumin. Not more than 50 ng in the equivalent of a single human dose, determined by a suitable immunochemical method (2.2.14). Where appropriate in view of the manufacturing process, other suitable protein markers may be used to demonstrate effective purification. Residual host-cell DNA (2.2.15). If a continuous cell line is used for virus propagation, the content of residual host-cell DNA, determined using a suitable method is not greater than 100 pg in the equivalent of a single human dose. Residual chemicals. If chemical substances are used during the purification process, tests for these substances are carried out on the purified harvest (or on the inactivated harvest), unless validation of the process has demonstrated total clearance. The concentration must not exceed the limits approved for the particular product. INACTIVATION AND INACTIVATED HARVEST Several purified harvests may be pooled before inactivation. In order to avoid interference with the inactivation process, virus aggregation must be prevented or aggregates must be removed immediately before and/or during the inactivation process. The virus suspension is inactivated by a validated method; the method shall have been shown to be consistently capable of inactivating hepatitis A virus without destroying the antigenic and immunogenic activity; as part of the validation studies, an inactivation curve is plotted representing residual live virus concentration measured on at least three occasions (for example, on days 0, 1 and 2 of the inactivation process). If formaldehyde is used for inactivation, the presence of excess free formaldehyde is verified at the end of the inactivation process. Only an inactivated harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. 51 Inactivation. Carry out an amplification test for residual infectious hepatitis A virus by inoculating a quantity of the inactivated harvest equivalent to 5 per cent of the batch or if the harvest contains the equivalent of 30,000 doses or more, not less than 1,500 doses of vaccine into cell cultures of the same type as those used for production of the vaccine and incubating the cells for at least 28 days. Make two passages and at the end of incubation carry out a test of suitable sensitivity for residual infectious virus. No evidence of hepatitis A virus multiplication is found in the samples taken at the end of the inactivation process. Use infective virus inocula concurrently as positive controls to demonstrate cellular susceptibility and absence of interference. Sterility (2.2.11). The inactivated viral harvest complies with the test for sterility, carried out using 10 ml for each medium. Bacterial endotoxins (2.2.3). Not more than 2 IU of endotoxin in the equivalent of a single human dose. Antigen content. Determine the hepatitis A virus antigen content by a suitable immunochemical method (2.2.14). Residual chemicals. As stated under Purification and Purified Harvest. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated harvests. Approved adjuvants, stabilisers and antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers. The containers are then closed so as to avoid contamination. Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde (where applicable) and antimicrobial preservative content (where applicable) and the assay have been carried out on the final bulk vaccine with satisfactory results, these tests may be omitted on the final lot. If the assay is carried out using mice or other animals, then provided it has been carried with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. INACTIVATED HEPATITIS B VACCINE IP 2007 Identification The vaccine is shown to contain hepatitis A virus antigen by a suitable immunochemical method using specific antibodies or by the mouse immunogenicity test described under Assay. antibodies against hepatitis A virus by a suitable immunochemical method (2.2.14). Calculations. Carry out the calculations by the usual statistical methods (5.7) for an assay with a quantal response. From the distribution of reaction levels measured on all the sera in the unvaccinated group, determine the maximum reaction level that can be expected to occur in an unvaccinated animal for that particular assay. Any response in vaccinated animals that exceeds this level is by definition a seroconversion. Make a suitable transformation of the percentage of animals showing seroconversion in each group (for example, a probit transformation) and analyse the data according to a parallelline log dose-response model. Determine the potency of the test preparation relative to the reference preparation. Validity conditions. The test is not valid unless (a) for both the test and the reference vaccine, the ED50 lies between the smallest and the largest doses given to the animals; (b) the statistical analysis shows no significant deviation from linearity or parallelism; (c) the fiducial limits of the estimated relative potency fall between 33.0 and 300.0 per cent of the estimated potency. Potency. The upper fiducial limit (P = 0.95) of the estimated relative potency is not less than 1.0. In vitro assay Carry out an immunochemical determination of antigen content (2.2.14) with acceptance criteria validated against the in vivo test. The acceptance criteria are approved for a given reference preparation by the National Regulatory Authority in the light of the validation data. Labelling. The label states (1) the biological origin of the cells and; (2) the adjuvant used for the preparation of the vaccine. Tests Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent. Free formaldehyde (2.3.20). When formaldehyde has been used for inactivation, the vaccine complies with the test prescribed in Vaccines. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Assay The vaccine complies with the assay of Hepatitis A vaccine. The assay is carried out either in vivo, by comparing in given condition the capacity to induce specific antibodies in mice with the same capacity of a reference preparation or in vitro by an immunochemical determination of antigen content (2.2.14). In vivo assay The test on mice shown below is given as an example of a method that has been found suitable for a given vaccine; other validated methods may also be used. Selection and distribution of the test animals. Healthy mice from the same stock, about 5 weeks old and from a strain shown to be suitable should be used in the test. Use animals of the same sex. Distribute the animals in at least seven equal groups of a number suitable for the requirements of the assay. Determination of potency of the vaccine under examination. Using a 0.9 per cent w/v solution of sodium chloride containing the aluminium adjuvant used for the vaccine, prepare at least three dilutions of the vaccine under examination and matching dilutions of the reference preparation. Allocate the dilutions one to each of the groups of animals and inject subcutaneously not more than 0.5 ml of each dilution into each animal in the group to which that dilution is allocated. Maintain a group of unvaccinated controls, injected subcutaneously with the same volume of diluent. After 28 to 32 days, anaesthetise and bleed all animals, keeping the individual sera separate. Assay the individual sera for specific 52 Inactivated Hepatitis B Vaccine Inactivated Hepatitis B Vaccine is a non-infectious inactivated liquid preparation derived from the surface antigen of Hepatitis B virus (HbsAg). This preparation is white or almost white translucent liquid in which the mineral carrier tends to settle down slowly on keeping but is free from foreign particles / floccules. Production The antigen is harvested and purified from the plasma of human carriers of Hepatitis B virus. The surface antigen contains all the three antigen species (S, Pre-S1, Pre-S2). The individual donor plasma is shown by sensitive tests to be seronegative for HIV-I and HIV-2 and for HCV. The plasma pool is tested for freedom from adventitious viruses and blood borne The purity of the antigen is determined by comparison with a reference preparation using liquid chromatography or other suitable methods such as SDS-PAGE with staining. The complete protein. determine the amount of antimicrobial preservative by a suitable chemical or physico-chemical method. microbial purity. Characterisation of the substance Development studies are carried out to characterize the antigen. Sterility (2. the vaccine . Antimicrobial preservative. nucleic acids and carbohydrates is determined. The content is within the limits approved for the specific product. Total protein (2. The purified antigen complies with the test for sterility carried out using 10 ml for each medium.0 per cent seroconversion. The quantity and specificity of HBsAg is determined in comparison with the International standard for HBsAg subtype ad or an in-house reference. The total protein is determined by a validated method. A part of a batch shown to be at least as immunogenic as a batch that produced in clinical studies in young healthy adults not less than 95. plasmid retention and consistency of yield are determined at suitable production stages. where applicable. safety and stability. The molecular weight of the major band in a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced conditions corresponds to the value expected from the gene sequence and possible glycosylation.2. Antigenic purity. antimicrobial preservative content and the assay in animals. where applicable. The content of proteins. by determination of the aminoacid composition.3. the electrophoretic profile. also serves to identify the vaccine.3. Composition. enzyme-linked immunosorbent assay (ELISA). The purified antigen is further inactivated by a validated method.IP 2007 INACTIVATED HEPATITIS B VACCINE transmissible pathogens by appropriate methods. corresponding to a level of neutralizing antibody accepted to be protective (HbsAg antibody titre not less than 10 mIU/ml after a full course of primary immunization determined by enzyme-linked immunosorbent assay (ELISA) is used as a reference preparation. lipid and carbohydrate structure of the antigen is established. The buoyant density of the antigen particles is determined by a physico-chemical method (2. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. immunoblot (preferably using a monoclonal antibody directed against a protective epitope) or single radial diffusion. Tests and Assay may be released for use. PURIFIED ANTIGEN Only a purified antigen that complies with the following requirements may be used in the preparation of the final bulk. The preparation is also tested for the residual HBV DNA using a sensitive test approved by the competent authority and the level is shown to be less than 1 pg HBV DNA per 50 doses. for example gradient centrifugation.0 per cent of that stated on the label. Carry out test for sterility using 10 ml of bulk for each sterility medium. they may be omitted on the final lot. The amount is not less than the 85. to render the hepatitis B virus harmless. Sterility (2. if tested. The method used for production of the vaccine must have been shown to yield a product consistently complying with the requirements of immunogenicity. would comply with the tests for safety and efficacy.11).11). FINAL LOT Only a final lot that complies with each of the requirements given below under Identification. FINAL BULK VACCINE An antimicrobial preservative and an adjuvant may be included in the vaccine. When hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent.29). lipids. The antigenic epitopes are characterized. The morphological characteristics of the antigen particles are established by electron microscopy.0 per cent and not greater than 115. A suitable method is sensitive enough to detect a potential contaminant at a concentration of 1. 53 The antigen/protein ratio is within the limits approved for the specific product.9).49).2. PROPAGATION AND HARVEST Identity. Reference preparation. have been carried out on the final bulk vaccine with satisfactory results. Antigen content and identification. by partial amino-acid sequence analysis and by peptide mapping). Where applicable. Additional tests on the purified antigen may be required depending on the production method used.0 per cent of total protein.0 per cent of the total protein consists of hepatitis B surface antigen. usually with formalin or any other inactivating agent. Tests Aluminium (2. Identification The assay or. Not less than 95.4. The production method must also be validated to demonstrate that the product. The protein fraction of the antigen is characterized in terms of the primary structure (for example. by a suitable immunochemical method such as radio immunoassay (RIA). Provided that the tests for free formaldehyde. After incubation at a controlled temperature. or a mixture of strains of the two types grown individually in eggs derived from specific pathogen free flock or cell cultures. Antimicrobial preservative. recommends new strains corresponding to prevailing epidemiological evidence. MONOVALENT POOLED HARVEST To limit the possibility of contamination.0 per cent of the intended amount.7. The content is not less than 85. Carry out the test for sterility using 10 ml for each medium. Labelling. The label states (1) the amount of HBsAg per dose. PROPAGATION AND HARVEST An antimicrobial agent may be added to the inoculum. unless clinical evidence supports the use of a different amount. Determine the potency by method A (Biological) as described under Hepatitis-B vaccine (rDNA). A validated test for bacterial endotoxins (2. the process should cause minimum alteration of the haemagglutinin and neuraminidase antigens. Inject the equivalent of one human dose into each rabbit. (2) the name and amount of inactivating agent. Complies with the test for pyrogens. Carry out the test for mycoplasmas using 10 ml. The inactivation process shall have been shown to be capable of inactivating the influenza virus without destroying its antigenicity. type A or B. inactivated and treated so that the integrity of the virus particles has been disrupted without diminishing the antigenic properties of the haemagglutinin and neuraminidase antigens.2.3) may be used instead of the test for pyrogens. An antimicrobial agent may be added at the time of harvest. Only a working virus seed lot that complies with the following requirements may be used in the preparation of the monovalent pooled harvest.4).0 per cent and not greater than 115. The concentration shall not exceed a specified upper limit. Test for inactivating agent. At no stage in the production. Assay The upper fiducial limit (P = 0. The concentration of any inactivating agent remaining in the final vaccine shall be determined by methods approved by the competent authority. Mycoplasmas (2. Substrate for virus propagation Influenza virus seed to be used in the production of vaccine is propagated in fertilised eggs from chicken flocks free from specified pathogens or in suitable cell cultures. The inactivation process shall also have been shown to be capable of inactivating avian leucosis viruses and mycoplasmas.11). The final vaccine represents one passage from the working seed lot. The virus is inactivated by a method that has been demonstrated on three consecutive batches to be consistently effective for the manufacturer. (4) that the vaccine must be shaken before use. For production. Production The production method is validated to demonstrate that the product. Sterility (2. Complies with the test for sterility.11). The origin and passage history of virus strains shall be approved by the National Regulatory Authority. (3) the name and amount of the adjuvant. SEED LOT The production of vaccine is based on a seed-lot system. such as chickembryo fibroblasts or chick kidney cells obtained from chicken flocks free from specified pathogens. Sterility (2. If the monovalent pooled harvest is stored after inactivation. The stated amount of haemagglutinin antigen for each strain present in the vaccine is 15 µg per dose.95) of the estimated relative potency is not less than 1. determine the amount of antimicrobial preservative by a suitable chemical method. the virus of each strain is grown in the allantoic cavity of eggs derived from specific pathogen free flocks.2.INACTIVATED INFLUENZA VACCINE (SPLIT VIRION) IP 2007 complies with the test prescribed in the monograph on Vaccines. The haemagglutinin and neuraminidase antigens of each seed lot are identified as originating from the correct strain of influenza virus by suitable methods. Pyrogens (2. Inactivated) is a sterile. it 54 Inactivated Influenza Vaccine (Split Virion) Influenza Vaccine (Split Virion. (5) that the vaccine must not be frozen. aqueous suspension of a strain or strains of influenza virus.2.2. if tested. Choice of vaccine strain The World Health Organisation reviews the world epidemiological situation annually and if necessary . inactivation is initiated as soon as possible after preparation. Working seed lots represent not more than fifteen passages from the approved reassorted virus or the approved virus isolate.8). penicillin or streptomycin is used.0. would comply with the test for safety and efficacy. the allantoic fluids are harvested and combined to form a monovalent pooled harvest. Where applicable. 2 ml of the pooled fluid into a further ten fertilised eggs and incubate at 33° to 37° for 3 days. Carry out as described below under Tests.14) on the first three monovalent pooled harvests from each working seed lot. Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. Neuraminidase antigen. The content is not less than 85. determine the amount of antimicrobial preservative by a suitable chemical method. Identification The assay serves to confirm the antigenic specificity of the vaccine. the concentration does not exceed 0. Tests are carried out on the monovalent pooled harvest for the chemicals used for disruption. for example. Provided that the test for viral inactivation has been performed with satisfactory results on each monovalent pooled harvest and that the tests for free formaldehyde. FINAL LOT The final bulk vaccine is distributed aseptically into sterile. the physical form of the haemagglutinin particles prevents quantitative determination by immunodiffusion after inactivation of the virus.3. by comparison with a haemagglutinin antigen reference preparation or with an antigen preparation calibrated against it. For some vaccines. Viral inactivation.1 per cent v/v at any time during inactivation. the concentration does not exceed 0. Total protein (2. Haemagglutinin antigen. a validation test is carried out to show that the monovalent bulk consists predominantly of disrupted virus particles. carry out for that fluid a further passage in eggs and test for haemagglutination.2.49).11). determine the amount of antimicrobial preservative by a suitable chemical method.2.11). they may be omitted on the final lot.IP 2007 INACTIVATED INFLUENZA VACCINE (SPLIT VIRION) is held at a temperature of 5±3°. Where applicable. The content is not less than 85. Not more than 1 µg of ovalbumin per human dose.5 ml of the allantoic fluid from each surviving embryo and pool the fluids. Carry out the test for sterility using 10 ml for each sterility medium. The production process is validated to demonstrate suitable conservation of haemagglutinin antigen and a suitable tracer is used for formulation.2. 55 Sterility (2. Harvest 0. For these vaccines. If formaldehyde solution is used. Chemicals used for disruption. Antimicrobial preservative. Complies with the test for sterility.0 per cent and not greater than 115. Before or after the inactivation procedure. The vaccine is a slightly opalescent liquid. not more than 100 µg of protein per virus strain per human dose and not more than a total of 300 µg of protein per human dose. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Harvest about 0.0 per cent and not greater than 115. Sterility (2.1). The presence and type of neuraminidase antigen are confirmed by suitable enzymatic or immunological methods (2. . no haemagglutination occurs. The test is not valid unless at least eight of the ten embryoes survive.2. if betapropiolactone is used. tamper-proof containers.14). the limits being approved by the National Regulatory Authority. a determination of haemagglutinin antigen is made on the monovalent pooled harvest before inactivation.0 per cent of the intended amount. The containers are closed so as to prevent contamination.0 per cent of the intended amount.2 g/l of formaldehyde at any time during inactivation. ovalbumin and total protein have been performed with satisfactory results on the final bulk vaccine. Complies with the test for abnormal toxicity. Inoculate 0. Sterility (2. Ovalbumin.1 ml of the allantoic fluid from each surviving embryo and examine each individual harvest for live virus by a haemagglutination test. Tests Viral inactivation. Determine the content of haemagglutinin antigen by an immunodiffusion test (2.2 ml of the vaccine into the allantoic cavity of each of ten fertilised eggs and incubate at 33° to 37° for 3 days. Inoculate 0. FINAL BULK VACCINE Appropriate quantities of the monovalent pooled harvests are blended to make the final bulk vaccine. The test is not valid unless at least eight of the ten embryos survive. Not more than six times the total haemagglutinin content of the vaccine as determined in the assay. protein content. Where applicable. Description. Only a final lot that is satisfactory with respect to each of the requirements given below under Tests and Assay may be released for use.2. For each new strain.11). Antimicrobial preservative.2. determined by a suitable technique using a suitable reference preparation of ovalbumin. Carry out the test at 20° to 25°. If haemagglutination is found for any of the fluids. but in any case. Abnormal toxicity (2. the monovalent pooled harvest is concentrated and purified by high-speed centrifugation or other suitable method and the virus particles are disrupted into component subunits by the use of approved procedures. Carry out the test for sterility using 10 ml for each medium. PROPAGATION AND HARVEST An antimicrobial agent may be added to the inoculum.0 per cent of the estimated content. The inactivation process shall have been shown to be capable of inactivating the influenza virus without destroying its antigenicity. quantitative determination of haemagglutinin antigen with respect to available reference preparations is not possible.0 per cent to 125.0 per cent of the amount stated on the label for each strain. For production. the concentration does not exceed 56 Production The production method is validated to demonstrate that the product. The lower confidence limit (P = 0. The label complies with the requirements stated under Vaccines and also states (a) that the vaccine has been prepared on eggs. inactivated and treated so that the preparation consists predominantly of haemagglutinin and neuraminidase antigens.2 g/l of formaldehyde at any time during inactivation. Working seed lots represent not more than fifteen passages from the approved reassorted virus or the approved virus isolate. The inactivation process shall also have been shown to be capable of inactivating avian leucosis viruses and mycoplasmas.7.14). Assay Determine the content of haemagglutinin antigen by an immunodiffusion test (2. The final vaccine represents one passage from the working seed lot. An antimicrobial agent may be added at the time of harvest. Carry out the test for mycoplasmas using 10 ml. Not more than 100 IU of endotoxin per human dose.INACTIVATED INFLUENZA VACCINE (SURFACE ANTIGEN) IP 2007 Bacterial endotoxins (2. the process should cause minimum alteration of the haemagglutinin and neuraminidase antigens. The stated amount of haemagglutinin antigen for each strain present in the vaccine is 15 µg per dose. type A or B. (d) the haemagglutinin content in µg per virus strain per dose. Carry out the test at 20 to 25°. (e) the season during which the vaccine is intended to protect. MONOVALENT POOLED HARVEST To limit the possibility of contamination.2. without diminishing the antigenic properties of these antigens. For some vaccines. If formaldehyde solution is used. Only a working virus seed lot that complies with the following requirements may be used in the preparation of the monovalent pooled harvest. inactivation is initiated as soon as possible after preparation. the concentration does not exceed 0. if betapropiolactone is used. Substrate for virus propagation Influenza virus seed to be used in the production of vaccine is propagated in fertilised eggs from chicken flocks free from specified pathogens or in suitable cell cultures. Sterility (2. If the monovalent pooled harvest is stored after inactivation. by comparison with an appropriate haemagglutinin antigen reference preparation. At no stage in the production penicillin or streptomycin is used. such as chickembryo fibroblasts or chick kidney cells obtained from chicken flocks free from specified pathogens. The haemagglutinin and neuraminidase antigens of each seed lot are identified as originating from the correct strain of influenza virus by suitable methods. Carry out the test for sterility using 10 ml for each medium. the virus of each strain is grown in the allantoic cavity of eggs derived from SPF flocks.95) of the assay is not greater than 80. Inactivated) is a sterile. An immunological identification of the haemagglutinin antigen and a semi-quantitative determination are carried out instead by suitable methods. Inactivated Influenza Vaccine (Surface Antigen) Influenza Vaccine (Surface Antigen. The origin and passage history of virus strains shall be . SEED LOT The production of vaccine is based on a seed-lot system. it is held at a temperature of 5±3°. Choice of vaccine strain The World Health Organisation reviews the world epidemiological situation annually and if necessary recommends new strains corresponding to prevailing epidemiological evidence. After incubation at a controlled temperature. Labelling. Mycoplasmas (2. the allantoic fluids are harvested and combined to form a monovalent pooled harvest. unless clinical evidence supports the use of a different amount. aqueous suspension of a strain or strains of influenza virus. or a mixture of strains of the two types grown individually in eggs derived from specific pathogen free flocks or cell cultures.4).95) of the estimate of haemagglutinin antigen content is not less than 80. would comply with the test for safety and efficacy. The confidence interval (P = 0.2. (c) the method of inactivation.2.3). if tested.11). approved by the competent authority. The virus is inactivated by a method that has been demonstrated on three consecutive batches to be consistently effective for the manufacturer. (b) the strain or strains of influenza virus used to prepare the vaccine. Where applicable.0 per cent of the intended amount.2. Neuraminidase antigen. ovalbumin and total protein have been performed with satisfactory results on the final bulk vaccine.2.49). The containers are closed so as to prevent contamination. Sterility (2.1). Determine the content of haemagglutinin antigen by an immunodiffusion test (2. Where applicable.2 ml of the pooled fluid into a further ten fertilised eggs and incubate at 33° to 37° for 3 days. Inoculate 0.3).5 ml of the allantoic fluid from each surviving embryo and pool the fluids. Viral inactivation. Complies with the test for abnormal toxicity. Abnormal toxicity (2. Identification The assay serves to confirm the antigenic specificity of the vaccine. The test is not valid unless at least eight of the ten embryos survive.3. Harvest 0. Carry out the test for sterility using 10 ml for each medium. Tests Viral inactivation. Ovalbumin. 57 Only a final lot that is satisfactory with respect to each of the requirements given below under Identification. determined by a suitable technique using a suitable reference preparation of ovalbumin. determine the amount of antimicrobial preservative by a suitable chemical method. Antimicrobial preservative.20). Complies with the test for free formaldehyde as stated under General Requirements for Vaccines for Human Use.2. Virus particles are disrupted into component subunits by approved procedures and further purified so that the monovalent bulk consists mainly of haemagglutinin and neuraminidase antigens. Sterility (2. The test is not valid unless at least eight of the ten embryos survive. Bacterial endotoxins (2. using 10 ml for each medium. Complies with the test for sterility.11). Carry out the test described below under Tests. The content is not less than 85. Haemagglutinin antigen. Before or after the inactivation process. Tests and Assay may be released for use. Not more than 40 µg of protein other than haemagglutinin per virus strain per human dose and not more than a total of 120 µg of protein other than haemagglutinin per human dose. Total protein (2. Inoculate 0. Purity.3.0 per cent and not greater than 115. If haemagglutination is found for any of the fluids.2.11). Free formaldehyde (2. carry out for that fluid a further passage in eggs and test for haemagglutination.2.2 ml of the vaccine into the allantoic cavity of each of ten fertilised eggs and incubate at 33° to 37° for 3 days.1 ml of the allantoic fluid from each surviving embryo and examine each individual harvest for live virus by a haemagglutination test. Carry out the test at 20° to 25°.2. Assay Determine the content haemagglutinin antigen by an . Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. no haemagglutination occurs. FINAL LOT The final bulk vaccine is distributed aseptically into sterile. Provided that the test for viral inactivation has been performed with satisfactory results on each monovalent pooled harvest and that the tests for free formaldehyde.1 per cent v/v at any time during inactivation. Not more than 1 µg of ovalbumin per human dose. the limits being approved by the competent authority. The vaccine is a clear liquid. Carry out the test for sterility.14) on the first three monovalent pooled harvests from each working seed lot. The presence and type of neuraminidase antigen are confirmed by suitable enzymatic or immunological methods (2. Chemicals used for disruption and purification. Harvest about 0.0 per cent and not greater than 115. they may be omitted on the final lot. The content is not less than 85. tamper-proof containers.11). Sterility (2. Not more than 100 IU of endotoxin per human dose. Mainly haemagglutinin and neuraminidase antigens shall be present. by comparison with a haemagglutinin antigen reference preparation or with an antigen preparation calibrated against it. The purity of the monovalent pooled harvest is examined by polyacrylamide gel electrophoresis or by other approved techniques.IP 2007 INACTIVATED INFLUENZA VACCINE (SURFACE ANTIGEN) 0. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. the monovalent pooled harvest is concentrated and purified by high-speed centrifugation or other suitable method. Tests are carried out on the monovalent pooled harvest for the chemicals used for disruption and purification.14).0 per cent of the intended amount. determine the amount of antimicrobial preservative by a suitable chemical method. FINAL BULK VACCINE Appropriate quantities of the monovalent pooled harvests are blended to make the final bulk vaccine.2. Description. 11). The confidence interval (P = 0. (2) the strain or strains of influenza virus used to prepare the vaccine. (4) the haemagglutinin content in micrograms per virus strain per dose. such as chickembryo fibroblasts or chick kidney cells obtained from chicken flocks free from specified pathogens .1 per cent v/v at any time during inactivation. Inactivated Influenza Vaccine (Whole Virion) Influenza Vaccine (Whole Virion.95) of the assay is not greater than 80. Mycoplasmas (2. The final vaccine represents one passage from the working seed lot. the concentration does not exceed 0. Carry out the test for sterility using 10 ml for each medium. Working seed lots represent not more than fifteen passages from the approved reassorted virus or the approved virus isolate. Inactivated) is a sterile. Haemagglutinin antigen. The stated amount of haemagglutinin antigen for each strain present in the vaccine is 15 µg per dose.2.4). The haemagglutinin and neuraminidase antigens of each seed lot are identified as originating from the correct strain of influenza virus by suitable methods.INACTIVATED INFLUENZA VACCINE (WHOLE VIRION) IP 2007 immunodiffusion test (2. (3) the method of inactivation.14). it is held at a temperature of 5±3°. Determine the content of haemagglutinin antigen by an immunodiffusion test (2. aqueous suspension of a strain or strains of influenza virus. the virus of each strain is grown in the allantoic cavity of eggs derived from specific pathogen free flocks. The lower confidence limit (P = 0.0 per cent of the amount stated on the label for each strain. The label states (1) that the vaccine has been prepared on eggs. The virus is inactivated by a method that has been demonstrated on three consecutive batches to be consistently effective for the manufacturer. The inactivation process shall also have been shown to be capable of inactivating avian leucosis viruses and mycoplasmas. An antimicrobial agent may be added at the time of harvest. the process should cause minimum alteration of the haemagglutinin and neuraminidase antigens. Carry out the test at 20° to 25°. The inactivation process shall have been shown to be capable of inactivating the influenza virus without destroying its antigenicity. would comply with the test for safety and efficacy.0 per cent of the estimated content. Only a working virus seed lot that complies with the following requirements may be used in the preparation of the monovalent pooled harvest. If the monovalent pooled harvest is stored after inactivation. type A or B.95) of the estimate of haemagglutinin antigen content is not less than 80. recommends new strains corresponding to prevailing epidemiological evidence.0 per cent to 125. If formaldehyde solution is used. Carry out the test for mycoplasmas using 10 ml. the allantoic fluids are harvested and combined to form a monovalent pooled harvest. by comparison with an appropriate haemagglutinin antigen reference preparation. unless clinical evidence supports the use of a different amount. the concentration does not exceed 0. After incubation at a controlled temperature.2. by comparison with a haemagglutinin antigen reference 58 Production The production method is validated to demonstrate that the product.2. Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine.7. SEED LOT The production of vaccine is based on a seed-lot system.For production. At no stage in the production is penicillin or streptomycin used. if necessary. PROPAGATION AND HARVEST An antimicrobial agent may be added to the inoculum. Substrate for virus propagation Influenza virus seed to be used in the production of vaccine is propagated in fertilised eggs from chicken flocks free from specified pathogens or in suitable cell cultures. MONOVALENT POOLED HARVEST To limit the possibility of contamination. the monovalent pooled harvest is concentrated and purified by high-speed centrifugation or other suitable method. inactivation is initiated as soon as possible after preparation. (5) the season during which the vaccine is intended to protect. Sterility (2. if tested. Before or after the inactivation process.2 g/l of formaldehyde at any time during inactivation.14). Labelling. if betapropiolactone is used. or a mixture of strains of the two types grown individually in eggs derived from specific pathogen free flocks or cell culture and inactivated in such a manner that their antigenic properties are retained. Choice of vaccine strain The World Health Organisation reviews the world epidemiological situation annually and. The origin and passage history of virus strains shall be approved by the competent authority. . Bacterial endotoxins (2. determine the amount of antimicrobial preservative by a suitable chemical method.20). Carry out the test described below under Tests.11).14). Antimicrobial preservative.5 ml of the allantoic fluid from each surviving embryo and pool the fluids.2.0 per cent to 125. the allantoic fluid from each surviving embryo and examine each individual harvest for live virus by a haemagglutination test.1). Total protein (2. Sterility (2.IP 2007 JAPANESE ENCEPHALITIS VACCINE (HUMAN) preparation or with an antigen preparation calibrated against it. (4) the haemagglutinin content in micrograms per virus strain per dose.2. Inoculate 0. they may be omitted on the final lot. carry out for that fluid a further passage in eggs and test for haemagglutination. The content is not less than 85. Carry out the test for sterility using 10 ml for each medium.0 per cent of the quantity stated on the label. FINAL BULK VACCINE Appropriate quantities of the monovalent pooled harvests are blended to make the final bulk vaccine. Tests Viral inactivation.0 per cent and not greater than 115. The test is not valid unless at least eight of the ten embryos survive. but in any case.0 per cent of the estimated content. Provided that the test for viral inactivation has been performed with satisfactory results on each monovalent pooled harvest and that the tests for free formaldehyde.14) on the first three monovalent pooled harvests from each working seed lot.49). Ovalbumin. Description.2. Not more than 1 µg of ovalbumin per human dose. Identification The assay serves to confirm the antigenic specificity of the vaccine.95) of the estimate of haemagglutinin antigen content is not less than 80. The lower confidence limit (P = 0. Where applicable. Complies with the test for sterility. no haemagglutination occurs. tamper-proof containers. Maximum 0. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. If haemagglutination is found for any of the fluids. not more than 100 µg of protein per virus strain per human dose and not more than a total of 300 µg of protein per human dose. determined by a suitable technique using a suitable reference preparation of ovalbumin. Antimicrobial preservative. The confidence interval (P = 0. Neuraminidase antigen. determine the amount of antimicrobial preservative by a suitable chemical method. Not more than 100 IU of endotoxin per human dose.2. Carry out test for sterility using 10 ml of bulk for each sterility medium. Harvest 0. Carry out the test at 20° to 25°. Free formaldehyde (2. Carry out the test at 20° to 25°.95) of the assay is not greater than 80. by comparison with an appropriate haemagglutinin antigen reference preparation. The content is not less than the minimum amount shown to be effective and is not greater than 115.3.2 ml of the pooled fluid into a further ten fertilised eggs and incubate at 33° to 37° for 3 days. Sterility (2. The vaccine is a slightly opalescent liquid. Complies with the test for abnormal toxicity. Viral inactivation.2 ml of the vaccine into the allantoic cavity of each of ten fertilised eggs and incubate at 33° to 37° for 3 days. (2) the strain or strains of influenza virus used to prepare the vaccine. The presence and type of neuraminidase antigen are confirmed by suitable enzymatic or immunological methods (2. Not more than six times the total haemagglutinin content of the vaccine as determined in the assay. Harvest about 0.11). Labelling.2.2. ovalbumin and total protein have been performed with satisfactory results on the final bulk vaccine.11).1 ml of 59 Japanese Encephalitis Vaccine (Human) Japanese Encephalitis Vaccine for human use is a liquid or freeze dried preparation of Japanese encephalitis virus grown in approved substrate and inactivated by a validated method.0 per cent of the intended amount. The test is not valid unless at least eight of the ten embryos survive.2. Assay Determine the content of haemagglutinin antigen by an immunodiffusion test (2. Sterility (2.3). . FINAL LOT The final bulk vaccine is distributed aseptically into sterile. Only a final lot that is satisfactory with respect to each of the requirements given below under Tests and Assay may be released for use. (3) the method of inactivation. The label states (1) that the vaccine has been prepared on eggs. The containers are closed so as to prevent contamination.0 per cent of the amount stated on the label for each strain. Inoculate 0. Abnormal toxicity (2. (5) the season during which the vaccine is intended to protect.2 g/l. Where applicable.3. 60 Identification Each working seed lot is identified as Japanese encephalitis virus using specific antibodies by an approved method. The National Regulatory Authority shall determine the acceptable number of passages from the master virus seed lot to produce working virus seed lots. Inactivation is confirmed by carrying out an amplification test for residual infectious Japanese encephalitis virus. b) Cell culture vaccine All processing of the cell bank and subsequent cell cultures are done under aseptic conditions in an area where no other cells are handled. The cell culture media may contain a pH indicator such as phenol red.03 ml is inoculated intracerebrally into each of the 10 mice weighing between 12 and 15 g. The mice are observed for 14 days for symptoms caused by Japanese encephalitis virus. SEED LOT The strain of Japanese encephalitis virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Substrate for virus propagation The virus is propagated in an approved cell substrate like a Vero cell line. the concentration shall at no time exceed 1:2000. No Japanese encephalitis virus is detected. The content of residual hostcell DNA. the virus harvest is inactivated by a validated method at a fixed.15). Alternatively. Residual host-cell DNA (2. If formalin is used.JAPANESE ENCEPHALITIS VACCINE (HUMAN) IP 2007 Production General provisions The vaccine is produced on the basis of virus seed lot system The production method shall have been shown to yield consistently the vaccines that comply with the tests for immunogenicity. should not be greater than 10 ng per single human dose if cells are used in the production. and mice showing symptoms of Japanese encephalitis virus are sacrificed and virus presence is confirmed by immunofluorescence test. Maintain the cultures for a further period of 14 days and then examine the cell cultures for Japanese encephalitis virus using an immunofluorescence test. during or after any concentration or purification. Extraneous agents (2.7. concentrated and inactivated by addition of formalin or any other suitable inactivating agent. the media may contain human albumin. The vaccine may be issued in single or multidose containers.3).7. would comply with the test for safety and efficacy. The working seed lot complies with the tests for the virus seed lots. Virus harvests are pooled. Inactivation. The method shall have been shown to be capable of inactivating Japanese encephalitis virus without destruction of the immunogenic activity. Inoculate a quantity of inactivated viral suspension equivalent to not less than 25 doses into cell cultures of the same type as those used for production of the vaccine. Multiple harvests from the same production cell culture may be pooled and considered as a single harvest.2. PROPAGATION AND HARVEST a) Mouse brain vaccine The vaccine is prepared by using a seed-lot system. . 5 ml of each culture fluid is pooled on day 14 and 21 and 0. but the final medium for maintaining cell growth during virus multiplication does not contain animal serum. It may contain a suitable preservative. Virus concentration. Only an inactivated viral suspension that complies with the following tests may be used in the preparation of the final bulk vaccine. Not less than 500 ml of the cell cultures employed for vaccine production are set aside as uninfected cell cultures (control cells). No Japanese encephalitis virus shall be detected. if tested. The virus suspension is harvested on one or more occasions during incubation. Serum proteins. if present are reduced to an acceptable level by suitable method of purification. Virus harvests that comply with the tests given under Identification and Virus concentration are pooled in the preparation of the inactivated viral harvest. well defined stage of the process which may be before. Serum and trypsin used in the preparation of cell suspension and media are shown to be free from infectious extraneous agents. determined using a suitable method. Control cells. Approved animal (but not human) serum may be used in the media. Only a working seed lot that complies with the following tests may be used for virus propagation. Purification and inactivation The virus harvest may be concentrated and/or purified by suitable methods. The virus concentration of each working seed lot is determined by a cell culture method using immunofluoresence or any other approved method. safety and stability.3). An approved strain of virus is grown by inoculating intracerebrally into healthy mice. The production method is validated to demonstrate that the product. The control cells of the production cell culture from which the single harvest is derived should comply with the test for identification and with the tests for extraneous agents (2. Make a passage after 7 days. An approved stabilizer may be added to maintain the activity of the product.11). pool the separated serum from each group and inactivate the sera by heating at 56° for 30 minutes.0 per cent of the intended amount. After incubation for an appropriate time (about 61 Identification The vaccine is shown to contain Japanese encephalitis virus antigen by a suitable immunochemical method using specific antibodies. Inject intraperitoneally in two doses of 0. wash them in chilled sterile saline solution to remove blood clots.14).2. determine the amount of antimicrobial preservative by a suitable chemical method. The inactivated sera may be stored at -20°. Complies with the test for sterility. The content is not less than 85. 1:160. Sterility (2.11). Determine the amount of antimicrobial preservative by a suitable chemical method. the Assay also serves to identify the vaccine.1).2. determined by a suitable immunochemical method (2. Tests Complies with the test for Inactivation under final Purification and Inactivation. Dilute the supernatant with Hanks’ balanced salt solution containing 5 per cent calf serum so as to contain about 200 Plaque-Forming Units (PFU) of the virus per 0. Sterility (2. The content is not less than the minimum amount shown to be effective and is not greater than 115.5 ml each at 7-day interval into at least 20 mice of 4 weeks of age. Determination of potency Prepare appropriate dilutions of the vaccine under examination and of the Standard Preparation in a suitable medium. The containers are then sealed so as to prevent contamination. Not more than 50 ng per single human dose. Provided that the test for inactivation has been carried out with satisfactory results on the inactivated virus suspension and the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine. Complies with the test for abnormal toxicity.15). Free formaldehyde (2. Standard preparation The Standard preparation is a freeze-dried Japanese encephalitis virus vaccine the potency of which has been determined in relation to the Japanese encephalitis reference vaccine obtained from the National Institute of Health.3. Inoculate the mixture into cell cultures and overlay the infected cells with 1 per cent agar. if necessary. Thiomersal can be used as preservative.. Where applicable.01 per cent w/v.4 ml. 1:40. Not more than 10 ng per single human dose.3. Bleed each mouse 7 days after the second injection. Antimicrobial preservative.2. 1:640 etc. Not more than 0. Bovine serum albumin (for cell culture vaccine).3). Harvest their brains aseptically. Centrifuge the emulsion at 2000 g for 30 minutes. e.2. Dilute the sera appropriately. Tests and Assay may be released for use. Less than 25 IU per single human dose. Biological assay Potency of Japanese encephalitis virus vaccine is determined by titrating the neutralizing antibodies produced in the immunized mice by plaque reduction method or serum neutralization test (SNT) using appropriate cell culture. Residual host-cell DNA (for continuous cell line vaccines) (2. Only a final lot that complies with each of the tests given under Identification. Homogenize the brains with Hanks’ balanced salt solution containing 5 per cent calf serum to make a 10 per cent emulsion. Antimicrobial preservative. Abnormal toxicity (2. Sacrifice the animals after they show characteristic symptoms of encephalitis and become moribund. Prepare a working pool of the challenge virus strain by inoculating intracerebrally 0.2. Formaldehyde (2.20). alternatively. . Japan.0 per cent of that stated on the label.01 per cent w/v. Carry out test for sterility using 10 ml of bulk for each sterility medium.g.2.20). Not more than 0.0 per cent and not greater than 115. Tokyo. these tests may be omitted on the final lot. mix with an equal volume of the challenge virus suspension and incubate at 37° for 90 minutes for neutralization. Bacterial endotoxins (2.IP 2007 JAPANESE ENCEPHALITIS VACCINE (HUMAN) FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated viral suspensions.03 ml of 100 fold dilution of the standard strain in Hanks’ balanced salt solution containing 5 per cent calf serum into a suitable number of 2-day old suckling mice. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Suggested method Preparation of challenge virus suspension The approved challenge virus strain is stored in freeze-dried form or in liquid form stored below -70° . 95) of the logarithm of the virus concentration are greater than ± 0. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. The reconstituted vaccine complies with the test for sterility.3. Sterility (2.0 log10 lower than that of the unheated vaccine. the second viral component infects susceptible cell cultures. following test for thermal stability and with each of the tests given below under Identification and Tests and Assay may be released for use.Titrate the vaccine for infective measles virus at least in triplicate. determined by a suitable immunochemical method (2.1). Thermal stability. FINAL BULK VACCINE Virus harvests for each component are pooled and clarified to remove cells. the minimum measles virus concentration stated on the label is not less than 1x103 CCID50 per single human dose. The test is not valid unless (a) the mean number of plaques obtained with the Standard preparation is between 100 and 150 per dish and (b) the potency of the vaccine under examination is not less than that of the Standard preparation. the virus concentration of the heated vaccine is not more than 1. using at least eight cell cultures for each dilution 0. Bovine serum albumin. semi-micro determination of water or by any suitable validated method.43).2.2. The estimated measles virus concentration is not less than that stated on the label. stain the cells and count the number of plaques formed on the cultures to obtain the plaque reduction rates for the vaccine under examination and the Standard preparation.2. (2) the strain of virus used. would comply with the test for safety and efficacy. Calculate the neutralizing antibody titres for each group using standard statistical methods (5.MEASLES AND RUBELLA VACCINE (LIVE) IP 2007 48 hours). A suitable stabilizer may be added and the pooled harvests diluted as appropriate. it is no longer able to infect cell cultures susceptible to these viruses. Measles vaccine (Live) RS is suitable for use as a reference preparation. Complies with the test for abnormal toxicity. Carry out test for sterility using 10 ml of bulk for each sterility medium.5 log10 62 Production General provisions The two components are prepared as described in the monographs on Measles vaccine (live) and Rubella vaccine (live) and comply with the tests prescribed therein.3.14). Suitable quantities of the pooled harvest for each component are mixed. Abnormal toxicity (2. Tests Water (2.11).11). Mix the vaccine with a sufficient quantity of antibodies specific for rubella virus. a minimum virus concentration for release of the product is established such as to ensure. The label states (1) the biological origin of the cells used for the preparation of the vaccine. determined by Karl Fischer. Labelling. The assay is not valid if the confidence limits (P = 0. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37° for 7 days and for vaccine stored at 2° to 8°. it may be omitted on the final lot.5 log10 step or by a method of equal precision. Identification Measles and Rubella Vaccine (Live) Measles and Rubella Vaccine (Live) is a freeze-dried preparation of suitable attenuated strains of measles virus and rubella virus grown in suitable cell cultures.2. When the vaccine reconstituted as stated on the label is mixed with quantities of specific antibodies sufficient to neutralize any one viral components. with the . Maintain samples of the final lot of freezedried vaccine in the dry state at 37° for 7 days. Use an appropriate virus reference preparation to validate each assay.7). The vaccine is reconstituted immediately before use to give a clear liquid that may be coloured owing to the presence of a pH indicator. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine. if tested. FINAL LOT For each component. Titrate the vaccine for infective rubella virus at least in triplicate. The production method is validated to demonstrate that the product. Sterility (2. Not more than 3. in the light of stability data. Only a final lot that complies with the tests for minimum virus concentration of each component for release. When the vaccine reconstituted as stated on the label is mixed with antibodies specific for measles virus and rubella virus. For each component. B. Not more than 50 ng per single human dose.0 per cent. Assay A. using at least eight cell cultures for each 0. that the minimum concentration stated on the label will be present at the end of the period of validity. the minimum rubella virus concentration stated on the label are not less than 1x103 CCID50 per single human dose. Unless otherwise justified and authorized. a cellbank system. The working seed lot complies with the tests for seed lots. as stated on the label. Only a single harvest that complies with the following tests may be used in the preparation of the final bulk vaccine.7. Not less than 500 ml of the production cell culture is set aside as uninfected cell cultures (control cells). if the virus is propagated in human diploid cells. even with authorized exceptions. The production method is validated to demonstrate that the product. Substrate for virus propagation The virus is propagated in human diploid cells or in cultures of chick embryo cells derived from a chicken flock free from specified pathogens. The master/working seed lot complies with the test for neurovirulence of live virus vaccines. using specific antibodies. (2) the type and origin of the cells used for the preparation of the vaccine. SEED LOT The strain of measles virus used in the production of measles vaccine shall be identified by historical records that include 63 Identification The single harvest contains virus that is identified as measles virus by serum neutralisation in cell culture. the number of passages beyond the level used for clinical studies shall not exceed five. but the final medium for maintaining cell growth during virus multiplication does not contain animal serum.3).95) of the logarithm of the virus concentration are greater than ± 0. Virus concentration. Production General provisions The production of vaccine is based on a virus seed-lot system and. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration. using specific antibody. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled.7. (3) the minimum virus concentration for each component of the vaccine. . The virus concentration in the single harvest is determined as prescribed under Assay to monitor consistency of production and to determine the dilution to be used for the final bulk vaccine. or below -60° if not freeze-dried. (5) that the vaccine must not be given to a pregnant woman and that a woman should not become pregnant within two months after having the vaccine. The estimated rubella virus concentration is not less than that stated on the label. Labelling.3. Measles Vaccine (Live) Measles Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of measles virus.7. Suitable animal (but not human) serum may be used in the growth medium. Macaca and Cercopithecus monkeys susceptible to measles virus are suitable for the test. Extraneous agents (2. Rubella vaccine (Live) RS is suitable for use as a reference preparation. the virus in the final vaccine shall have undergone no more passages from the master seed lot than were used to prepare the vaccine shown in clinical studies to be satisfactory with respect to abnormal toxicity and efficacy. Only a seed lot that complies with the following tests may be used for virus propagation. The viral suspensions are harvested at a time appropriate to the strain of virus being used. would comply with the tests for abnormal toxicity and efficacy. Identification The master and working seed lots are identified as measles virus by serum neutralization in cell culture. Serum and trypsin used in the preparation of cell suspensions and culture media are shown to be free from extraneous agents. information on the origin of the strain and its subsequent manipulation. It is preferable to have a substrate free from antibiotics during production. to give a clear liquid that may be coloured owing to the presence of a pH indicator. Extraneous agents (2. Use an appropriate virus reference preparation to validate each assay. if tested. The virus concentration of the master and working seed lots is determined to monitor consistency of production. (4) the time within which the vaccine must be used after reconstitution.IP 2007 MEASLES VACCINE (LIVE) dilution step or by a method of equal precision. Virus seed lots are prepared in large quantities and stored at temperatures below -20° if freeze-dried. Virus concentration. Complies with the test for extraneous agents. Neurovirulence (2.3). The assay is not valid if the confidence limits (P = 0. The label states (1) the strains of virus used in the preparation of the vaccine. The vaccine is reconstituted immediately before use.5). MEASLES, MUMPS AND RUBELLA VACCINE (LIVE) IP 2007 Control cells. If human diploid cells are used for production, the control cells comply with the test for identification and extraneous agents. FINAL BULK VACCINE Virus harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). The final bulk vaccine complies with the test for sterility carried out using 10 ml for each medium. FINAL LOT A minimum virus concentration for release of the product is established so as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration for release, with the following requirement for thermal stability and with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37° for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37° for 7 days and for vaccine stored at 2° to 8°. The virus concentration of the heated vaccine is not more than 1.0 log10 lower than that of the unheated vaccine. Assay Titrate the vaccine for infective virus at least in triplicate, using at least five cell cultures for each 0.5 log10 dilution step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated virus concentration is not less than that stated on the label; the minimum virus concentration stated on the label is not less than 1 × 103 CCID50 per human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration is greater than ± 0.3. Measles vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strain of virus used for the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration; (4) the time within which the vaccine must be used after reconstitution. Measles, Mumps and Rubella Vaccine (Live) Measles, Mumps and Rubella Vaccine (Live) is a freeze-dried preparation of suitable attenuated strains of measles virus, mumps virus and rubella virus grown in suitable cell cultures. The vaccine is reconstituted immediately before use to give a clear liquid that may be coloured owing to the presence of a pH indicator. Production General provisions The three components are prepared as described in the monographs on Measles Vaccine (Live), Mumps Vaccine (Live) and Rubella Vaccine (Live) and comply with the tests prescribed therein. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. FINAL BULK VACCINE Virus harvests for each component are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Suitable quantities of the pooled harvest for each component are mixed. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. 64 Identification When the vaccine reconstituted as stated on the label is mixed with specific measles antibodies, it is no longer able to infect susceptible cell cultures. Tests Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Sterility ( 2.2.11). The reconstituted vaccine complies with the test for sterility. Abnormal toxicity ( 2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). IP 2007 MENINGOCOCCAL POLYSACCHARIDE VACCINE FINAL LOT For each component, a minimum virus concentration for release of the product is established such as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration of each component for release, with the following requirement for thermal stability and with each of the requirements given below under Identification and Tests may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37° for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37° for 7 days and for vaccine stored at 2 to 8°. The virus concentration of the heated vaccine is not more than 1.0 log10 lower than that of the unheated vaccine. is not less than 1x103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than ± 0.3. Measles vaccine (Live) RS is suitable for use as a reference preparation. B. Mix the vaccine with a sufficient quantity of antibodies specific for measles virus and rubella virus. Titrate the vaccine for infective mumps virus at least in triplicate, using at least eight cell cultures for each dilution 0.5 log10 step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated mumps virus concentration is not less than that stated on the label; the minimum mumps virus concentration stated on the label is not less than 5 x 103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than ± 0.3. Mumps vaccine (Live) RS is suitable for use as a reference preparation. C. Mix the vaccine with a sufficient quantity of antibodies specific for mumps virus. Titrate the vaccine for infective rubella virus at least in triplicate, using at least eight cell cultures for each dilution 0.5 log10 step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated rubella virus concentration is not less than that stated on the label; the minimum rubella virus concentration stated on the label is not less than 1x103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than ± 0.3. Rubella vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strains of virus used in the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration for each component of the vaccine; (4) the time within which the vaccine must be used after reconstitution; (5) that the vaccine must not be given to a pregnant woman and that a woman should not become pregnant within two months after having the vaccine. Identification When the vaccine reconstituted as stated on the label is mixed with antibodies specific for measles virus, mumps virus and rubella virus, it is no longer able to infect cell cultures susceptible to these viruses. When the vaccine reconstituted as stated on the label is mixed with quantities of specific antibodies sufficient to neutralize any two viral components, the third viral component infects susceptible cell cultures. Tests Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Sterility (2.2.11). The reconstituted vaccine complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Assay A. Mix the vaccine with a sufficient quantity of antibodies specific for mumps virus and rubella virus.Titrate the vaccine for infective measles virus at least in triplicate, using at least eight cell cultures for each dilution 0.5 log10 step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated measles virus concentration is not less than that stated on the label; the minimum measles virus concentration stated on the label 65 Meningococcal Polysaccharide Vaccine Meningococcal Polysaccharide Vaccine is a freeze-dried preparation of one or more purified capsular polysaccharides obtained from one or more suitable strains of Neisseria meningitidis group A, group C, group Y and group W135 that are capable of consistently producing polysaccharides known to be safe and effective in man. MENINGOCOCCAL POLYSACCHARIDE VACCINE IP 2007 N. meningitidis group A polysaccharide consists of partly O-acetylated repeating units of N-acetylmannosamine, linked with 1α→6 phosphodiester bonds. N. meningitidis group C polysaccharide consists of partly O-acetylated repeating units of sialic acid, linked with 2α→9 glycosidic bonds. N. meningitidis group Y polysaccharide consists of partly O-acetylated alternating units of sialic acid and D-glucose, linked with 2α→6 and 1α→4 glycosidic bonds. N. meningitidis group W135 polysaccharide consists of partly O-acetylated alternating units of sialic acid and D-galactose, linked with 2α→6 and 1α→4 glycosidic bonds. medium. The liquid media used and the final medium are semisynthetic and free from substances precipitated by cetrimonium bromide (hexadecyltrimethylammonium bromide) and do not contain blood-group substances or highmolecular-mass polysaccharides. The bacterial purity of the culture is verified by microscopic examination of Gram-stained smears and by inoculation into appropriate media. The cultures are centrifuged and the polysaccharides precipitated from the supernatant by addition of cetrimonium bromide. The precipitate obtained is harvested and may be stored at or below -20° awaiting further purification. PURIFIED POLYSACCHARIDES The polysaccharides are purified, after dissociation of the complex of polysaccharide and cetrimonium bromide, using suitable procedures to remove successively nucleic acids, proteins and lipopolysaccharides. The purification step consists of ethanol precipitation of the polysaccharides or purification with chloroform and n-butanol or by cold phenol treatment, which are then dried and stored at or below -20°. The loss on drying is determined by thermogravimetry, Karl Fischer or any other suitable method and the value is used to calculate the results of the other chemical tests with reference to the dried substance. Only purified polysaccharides that tested comply with the following requirements may be used in the preparation of the final bulk vaccine. Protein (2.7.1). Not more than 10 mg of protein per gram of purified polysaccharidefor group A and C organisms and less than 50 mg of protein per gram of polysaccharide for group Y and W135 calculated using bovine plasma albumin as a reference or other methods approved by National Regulatory Authority. Nucleic acids (2.7.1). Not more than 10 mg of nucleic acids per gram of purified polysaccharide, calculated with reference to the dried substance. O-Acetyl groups (2.7.1). Not less than 2 mmol of O-acetyl groups per gram of purified polysaccharide for group A, not less than 1.5 mmol per gram of polysaccharide for group C, not less than 0.3 mmol per gram of polysaccharide for groups Y and W135, all calculated with reference to the dried substance. Phosphorus (2.7.1). Not less than 80 mg of phosphorus per gram of group A purified polysaccharide, calculated with reference to the dried substance. Sialic acid (2.7.1). Not less than 800 mg of sialic acid per gram of group C polysaccharide and not less than 560 mg of sialic acid per gram of purified polysaccharide for groups Y and W135, all calculated with reference to the dried substance. Use the following reference solutions: 66 Production General provisions Production of the meningococcal polysaccharides is based on a well defined seed-lot system. The method of production shall have been shown to yield consistently meningococcal polysaccharide vaccines of satisfactory immunogenicity and safety for man. The production method is validated to demonstrate that the product, if tested, would comply with the test of abnormal toxicity for antisera and vaccines. SEED LOT The strains of N. meningitidis used for the master seed lots shall be identified by historical records that include information on their origin and by their biochemical, serological, physicochemical or molecular characteristics. Cultures from the working seed lot shall have the same characteristics as the strain that was used to prepare the master seed lot. The strains have the following characteristics: a) Colonies obtained from a culture are round, uniform in shape and smooth with a mucous, opalescent, greyish appearance. b) Gram staining reveals characteristic Gram-negative diplococci in ‘coffee-bean’ arrangement. c) e) The oxidase test is positive. Suspensions of the culture agglutinate with specific antisera of known titre. d) The culture utilizes glucose and maltose. PROPAGATION AND HARVEST The working seed lots are cultured on solid media that do not contain blood-group substances or ingredients of mammalian origin. The inoculum may undergo one or more subcultures in liquid medium before being used for inoculating the final 0 per cent m/m of groupheterologous N. Use a column 0.2 mol/kg and a pH of 7.050 µg of polysaccharide for a bivalent vaccine.5 mg of each polysaccharide in a volume of about 1. determination of calcium is carried out on the purified polysaccharide by a suitable method.5 mg of polysaccharide in a volume of about 1. 67 b) 75.1).4.4. Group Y polysaccharide. b) 0. Calcium.0 per cent of Group C polysaccharide is eluted before K0 of 0. Identification Carry out an identification test for each polysaccharide present in the vaccine by a suitable immunochemical method (2.14) to establish the elution pattern of the different polysaccharide(s). Apply to the column about 2. Sterility (2. At least 65.5.11).2. Apply to the column about 2.100 µg of polysaccharide for a tetravalent vaccine.g. Abnormal toxicity (2. In addition. Complies with the test for sterility.075 µg of polysaccharide for a trivalent vaccine. Group W135 polysaccharide. meningitidis groups are dissolved in a suitable solvent that may contain a stabilizer.2. the percentages eluted before this distribution coefficient are within the limits approved for the particular product.0 per cent of group C polysaccharide. Water (2. Carry out test for sterility using 10 ml of bulk for each sterility medium.2.2 mol/kg and a pH of 7. d) 0.025 µg of polysaccharide for a monovalent vaccine. 75.16).0 to 7. 0. The vaccine complies with the test if: a) 65.IP 2007 MENINGOCOCCAL POLYSACCHARIDE VACCINE Group C polysaccharide. the content is within the limits approved for the product. MALLS) or any other suitable method.14). A 150 mg/l solution of Nacetylneuraminic acid. Collect fractions of about 2. meningitidis polysaccharide.0 to 7.025 µg of polysaccharide for a monovalent vaccine. Molecular size.5. Complies with the test for pyrogens. multiple angle LASER light scattering.11). The containers are then closed so as to avoid contamination. A solution containing 95 mg/l of Nacetylneuraminic acid and 55 mg/l of glucose. b) 0.5 ml and elute at about 20 ml/h.8).2.2.5 ml and determine the content of polysaccharide by a suitable method. Use a column about 0.0 per cent of group A polysaccharide. A solution containing 95 mg/l of N-acetylneuraminic acid and 55 mg/l of galactose.50 is reached. If a calcium salt is used during purification.0 per cent of group W135 polysaccharide is eluted before a distribution coefficient (K0) of 0. using agarose for chromatography or cross-linked agarose for chromatography either alone or in combination with light scattering and refractive index detector (e. Only a final lot that is satisfactory with respect to each of the requirements prescribed below under Identification. Pyrogens (2.0 per cent of Group A polysaccharide is eluted before K0 of 0.9 m long and 16 mm in internal diameter equilibrated with a solvent having an ionic strength of 0.8).5 ml and determine the content of polysaccharide by a suitable method. Not more than 3.50.100 µg of polysaccharide for a tetravalent vaccine.0 per cent of group Y polysaccharide and 80. 80.2.2. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers.50. it shall be shown that there is not more than 1. d) 0. Identification and serological specificity The identity and serological specificity are determined by a suitable immunochemical method (2. Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final lot.075 µg of polysaccharide for a trivalent vaccine.0 per cent. Complies with the test for abnormal toxicity.50.2.0 per cent of Group Y & W135 polysaccharide is eluted before K0 of 0. Examine by gel filtration or high performance size-exclusion chromatography (HPSEC) (2. . Identity and purity of each polysaccharide shall be confirmed. Collect fractions of about 2. Inject each of the rabbit with 1ml per kg body weight of solution containing a) c) 0. of moisture content by thermogravimetery. Examine by gel filtration or size-exclusion chromatography (2.43). Molecular size. Pyrogens (2. Inject each of the rabbit with 1ml per kg body weight of solution containing a) c) 0. Tests Sterility (2.050 µg of polysaccharide for a bivalent vaccine. Karl Fischer or any other suitable method.3.14). c) 80. FINAL BULK VACCINE One or more purified polysaccharides of one or more N.16).5 ml and elute at about 20 ml/h. Use cross-linked agarose for chromatography and apply a suitable immunochemical method (2. Tests and Assay may be released for use.9 m x 15 mm equilibrated with a solvent having an ionic strength of 0. Complies with the test for pyrogens. 0. To determine sialic acid.1) to determine the content of polysaccharide C. Virus seed lots are prepared in large quantities and stored at temperatures below -20° if freeze-dried. The master/working seed lot complies with the test for neurovirulence of live virus vaccines.0 per cent of the quantity of each polysaccharide stated on the label. the number of passages beyond the level used for clinical studies shall not exceed five. For a divalent vaccine (group A + group C). The vaccine complies with the test if K0 for the principal peak is a) not greater than 0.0 per cent and not more than 130. if tested. Not less than 500 ml of the production cell culture is set aside as uninfected cell culture (control cells).1) to determine the content of polysaccharide A and measurement of sialic acid (2.7. shown in clinical studies to be satisfactory with respect to safety and efficacy even with authorized exceptions. For a tetravalent vaccine (group A + group C + group Y + group W135) a suitable immunochemical method (2.5). The vaccine is reconstituted immediately before use to give a clear liquid that may be coloured owing to the presence of a pH indicator. use cross linked agarose for chromatography R1 and apply a suitable immunochemical method (2.7. The production method is validated to demonstrate that the product. Substrate for virus propagation The virus is propagated in human diploid cells or in primary cultures of chick embryo cells derived from a chicken flock free from specified pathogens. or below -60° if not freeze-dried. use as reference solution a 150 mg/l solution of Nacetylneuraminic acid. the virus in the final vaccine shall have undergone no more passages from the master seed lot than were used to prepare the vaccine .7.14) to establish the elution pattern of the different polysaccharides. Extraneous agents (2. The virus concentration of the master and working seed lots is determined to ensure consistency of production. SEED LOT The strain of mumps virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Production General provisions The production of vaccine is based on a virus seed-lot system and. b) not greater than 0. The production method shall have been shown to yield consistently live mumps vaccines of adequate immunogenicity and safety in man. using specific antibodies. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration. Identification The master and working seed lots are identified as mumps virus by serum neutralisation in cell culture.MUMPS VACCINE (LIVE) IP 2007 For a tetravalent vaccine (group A + group C + group Y and group W135). (2) the number of µg of polysaccharide per human dose.68 for group W135 polysaccharide. The vaccine contains not less than 70. c) not greater than 0.3).57 for group Y polysaccharide. 68 Mumps Vaccine (Live) Mumps Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of mumps virus. The label states (1) the group or groups of polysaccharides (A. Suitable animal (but not human) serum may be used in the growth media.7. Virus concentration.2. if the virus is propagated in human diploid cells. Serum and trypsin used in the preparation of cell suspensions and culture media are shown to be free from extraneous agents.2. To avoid unnecessary use of monkeys in the test for neurovirulence. Y or W135) present in the vaccine. Only a seed lot that complies with the following tests may be used for virus propagation. Neurovirulence (2. The working seed lot complies with the tests for seed lots. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. The viral suspensions are harvested at a time appropriate to the strain of virus being used. would comply with the tests for safety and efficacy. a cellbank system. It is preferable to have a substrate free from antibiotics during production.70 for group A and group C polysaccharide. Assay Carry out an assay as stated under each polysaccharide present in the vaccine. C. Macaca and Cercopithecus monkeys are suitable for the test. Labelling. use measurement of phosphorus (2. Unless otherwise justified and authorised.14) is used with a reference preparation of purified polysaccharide for each group. Only a final lot that complies with the tests for minimum virus concentration for release. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Each strain is grown for 24 to 72 hours in a liquid medium or on a solid medium. Sterility ( 2.IP 2007 PERTUSSIS VACCINE Only a single harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. The final bulk vaccine complies with the test for sterility. The virus concentration of the vaccine exposed to 37° for 7 days is not more than 1. Not more than 50 ng per single human dose. Abnormal toxicity (2. in the light of stability data. Strains.9 per cent w/v solution of sodium chloride or other suitable 69 Identification When the vaccine is reconstituted as stated on the label is mixed with specific mumps antibodies. The virus concentration in the single harvest is determined as prescribed under Assay to monitor consistency of production and to determine the dilution to be used for the final bulk vaccine.2.1). Complies with the test for sterility.11). pertussis with known origin and history are used. the minimum virus concentration stated on the label is not less than 5 x 103 CCID50 per human dose. Control cells.2.3. Assay Titrate the vaccine for infective virus at least in triplicate.11). culture medium and cultivation method are chosen in such a way that agglutinogens 1. The estimated virus concentration is not less than that stated on the label. 2 and 3 are present in the final vaccine. using specific antibodies. Pertussis Vaccine Pertussis Vaccine is a sterile saline suspension of inactivated whole cells of one or more strains of Bordetella pertussis. washed to remove substances derived from the medium and suspended in a 0.43). Sterility (2. Mumps vaccine (Live) RS is suitable for use as a reference preparation. (3) the minimum virus concentration and. The control cells comply with a test for extraneous agents (2. . Labelling.7. FINAL LOT A minimum virus concentration for release of the product is established such as to ensure. Thermal stability.3). using at least five cell cultures for each 0. it is no longer able to infect susceptible cell cultures. The assay is not valid if the confidence limits (P = 0.2. Identification The single harvest contains virus that is identified as mumps virus by serum neutralization in cell culture. The bacteria are harvested.14). the liquid medium used in the final cultivation stage does not contain blood or blood products.2. One or more strains of B. Complies with the test for abnormal toxicity. pertussis suspension Production is based on a seed-lot system. Sterility (2. carried out using 10 ml for each medium. determined by Karl Fischer. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine. Use an appropriate virus reference preparation to validate each assay. Not more than 3.3. Tests Water (2. Single harvest complies with sterility test should be processed further.11). Production General provisions Inactivated B. that the minimum concentration stated on the label will be present at the end of the period of validity. Virus concentration. FINAL BULK VACCINE Single harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabiliser may be added and the pooled harvests diluted as appropriate. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37° for 7 days and for vaccine stored at 2° to 8°. it may be omitted on the final lot. The label states (1) the strain of virus used for the preparation of the vaccine. Human blood or blood products are not used in any culture media.0 log10 lower than that of the unheated vaccine.5 log10 dilution step or by a method of equal precision. determined by a suitable immunochemical method (2. Maintain samples of the final lot of freezedried vaccine in the dry state at 37° for 7 days.2. semi-micro determination of water or by any suitable validated method. with the following requirement for thermal stability and with each of the requirements given below under Identification and Tests and Assay may be released for use.95) of the logarithm of the virus concentration is greater than 0. (4) the time within which the vaccine must be used after reconstitution. (2) the type and origin of the cells used for the preparation of the vaccine. Bovine serum albumin.0 per cent. Carry out test for sterility using 10 ml of bulk for each sterility medium. calibrated in comparison to International standard. administered intracerebrally. 72 hours and 7 days after the injection. tamper-proof containers. If 2 or more strains of B. which consists of a quantity of dried pertussis vaccine. (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60. Sterility (2. consisting of a freeze dried vaccine or another suitable preparation. and (c) not more than 5. Tests Specific toxicity Use not less than 10 healthy mice each weighing between 14 . Only a final lot that is satisfactory with respect to each of the requirements given below under Identification. Use healthy mice of a suitable strain. they may be omitted on the final lot. Inject each mouse of the vaccine group intraperitoneally with 0. Allow the animals access to food and water for at least 2 hours before injection and during the test.2. in 70 Identification Identify pertussis vaccine by agglutination of the bacteria in the vaccine by antisera specific to B. Free formaldehyde (2. free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine.3. For this comparison. to be used as challenge strain). pertussis are used.5 ml. to 16 g for the vaccine group and for the saline control. Single harvests are not used for the final bulk vaccine unless they have been shown to contain B.0 per cent of the vaccinated mice die during the test. FINAL LOT The final bulk vaccine is distributed aseptically into sterile.20).18323. pertussis (e. from time to time. Distribute the mice randomly. calibrated in International Units. The opacity of the suspension is determined not later than 2 weeks after harvest by comparison with the reference preparation of Opacity and used as the basis of calculation for subsequent stages in vaccine preparation. The amount is not less than 85. Maximum 0. The suspension is maintained at 5 + 3° for a suitable period to diminish its toxicity. Antimicrobial preservative.5 ml of a 0. containing a quantity of the vaccine equivalent to not less than half the single human dose.0 per cent and not greater than 115. the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. The containers are closed so as to prevent contamination. determine the amount of antimicrobial preservative by a suitable chemical method. Provided the tests for specific toxicity.11).PERTUSSIS VACCINE IP 2007 isotonic solution. preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. pertussis is tested using a suitable culture medium. with the dose of a reference preparation. Weigh the groups of mice immediately before the injection. Where applicable. Antimicrobial preservative.11). Sterility (2. pertussis. Where applicable. Freedom from live B. are required. Suitable antimicrobial preservatives may be added.0 per cent of that per control mouse.0 per cent and not more than 115. Complies with the test for sterility. determine the amount of antimicrobial preservative by a suitable chemical method. Reference preparation The reference preparation is an International standard of Pertussis vaccine. FINAL BULK VACCINE Suitable quantities of the inactivated single harvests are pooled to prepare the final bulk vaccine.0 per cent of the intended amount. Use mice of the same sex or distribute males and females equally between the groups. Assay Carry out the assay of Pertussis Vaccine as described below : Biological assay of pertussis vaccine The potency of PertussisVaccine is determined by comparison of the dose necessary to protect mice against the effects of a lethal dose of Bordetella pertussis challenge culture. as the parent strain and to be free from contaminating bacteria and fungi. The International Unit is the activity contained in a stated amount of the International standard. pertussis cells with the same characteristics with regard to growth and agglutinogens.9 per cent w/v sterile solution of sodium chloride. The content is not less than 85. The equivalence in International Units of the International Standard is stated by the World Health Organization. The bacteria are killed and detoxified in controlled conditions by means of a suitable chemical agent or by heating or by a combination of these methods.2 g/l. The bacterial concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. The test may be repeated and the results of the tests combined. the Standard preparation of Pertussis vaccine & a suitable strain of B.g. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. weighing between 13 and 16 g from the same stock. The vaccine complies with the test if (a) at the end of 72 h the total mass of the group of vaccinated mice is not less than that preceding the injection. Tests and Assay may be released for use.0 per cent of the intended amount. Inject each mouse of the control group with 0.2. required to give the same level of protection. IP 2007 PNEUMOCOCCAL POLYSACCHARIDE VACCINE six to eight groups of not less than 16 and not more than 24 and four groups of 10. B. into each mouse one dose of the dilution. from the dilution selected for challenge. if the test results of a statistically valid assay show that the estimated potency of the vaccine is not less than 4.03 ml. in a volume of not more than 0. The bacterial purity of the culture is verified . lies between the largest and the smallest doses given to the mice.5 IU.1 IU and 0. Labelling. Incubate the plates at 37° for 48 to 72 hours and calculate the number of colony forming units (CFUs). aliquots of challenge suspension frozen in liquid nitrogen with a suitable preservative like 10 per cent DMSO may be used. 0. Select a suitable strain of B. 1/40 and 1/200 of the human dose of the vaccine under examination) and (0. each dose being contained in a volume. suggested dilutions are (1/8.g. The challenge should be completed within 2 to 2. a dose of 0. in terms of significance of the scope of dose response curve. pertussis (e. Monovalent bulk polysaccharides The bacteria are grown in a suitable liquid medium that does not contain blood-group substances or high-molecular-mass polysaccharides. In the same way. for LD50 titration of challenge preparation.5 hours of preparation. three dilutions of the reference vaccine and similar dilutions of the vaccine under examination.85 per cent w/v of sodium chloride and having a pH of 7. Exclude any mouse from consideration. calculate the potency of vaccine under examination. in the estimate of potency. not exceeding 0. inject 10 human dose into each guineapig and observe for 12 days. of the reference preparation). The mice should all be of the same sex or the males and females should be distributed equally between the groups.5 ml. The label states (1) the minimum number of International Units per single human dose. inject intracerebrally.2 or in another suitable solution. For each dilution use 16 to 24 mice and inject intraperitoneally.000 organisms and 100 to 1000 LD50 per dose. The production method is validated to demonstrate that the product.00. Pneumococcal Polysaccharide Vaccine Pneumococcal Polysaccharide Vaccine consists of a mixture of equal parts of purified capsular polysaccharide of various serotype antigens prepared from suitable pathogenic strains of Streptococcus pneumoniae in different desired combinations whose capsules have been shown to be made up of polysaccharides that are capable of inducing satisfactory levels of specific antibodies in man. to avoid heterogenicity. Use at least. b) the number of animals. Seed a suitable highest dilution of the challenge suspension. Half of the groups of 16 to 24 should receive the reference preparation and the other half should receive the vaccine under examination. approximately 1. 71 The test is not valid unless: a) for both the vaccine under examination and the reference preparation. (3) that the vaccine is not to be frozen. the ED50 (protective dose).0 to 7. but when more than one test is performed the results of all valid tests must be combined.0 IU per single human dose and the lower fiducial limit (P = 0. into each of two B. casamino acid) and 0. The production method shall have been shown to yield consistently pneumococcal polysaccharide vaccines of acceptable immunogenicity and safety in man. In each case the dilutions are so selected that the dilution protecting 50 per cent of the mice (ED50) is as near as possible to middle of the dilution range.03 ml of the challenge dilution randomly. (2) that the vaccine must be shaken before use.02 IU or any other suitable standardized dilutions. Production General provisions Production of the vaccine is based on a well defined seed-lot system for each type. Alternatively. The challenge should contain. Count the number of mice surviving in each of the groups. After 14 to 17 days of immunization. Make two subcultures after reviving the strain on a suitable medium (e. before and after challenge. 18323). On the basis of the numbers of animals surviving in each of the groups of 16 to 24 mice. that dies within 3 days of challenge.0 IU.G medium plates. The test may be repeated once. The four groups of 10 each should be used for the LD50 titration of challenge suspension. would comply with the tests for abnormal toxicity of vaccines for human use. against the potency of reference preparation. after 14 days.g. inject 4 groups of 10 control mice each. Determine the opacity of the suspension by using 5th International reference preparation for opacity (10 OU) and/or spectophotometerically.G. if tested. It contains upto 23 immunochemically different capsular polysaccharides listed in the Table 1. For example. prepared by a series of dilutions. Calculate the potency of the vaccine by Probit analysis and LD 50 of the challenge suspension by Reed and Munch Method. which die in the four groups of 10 injected with the challenge suspension and its dilutions indicate that the challenge dose contains 100 to 1000 LD50 and 1 LD50 contains not more than 300 colony forming units. medium) and suspend the harvested growth in a solution containing 1 per cent w/v of casein hydrolysate (e.g.95) of estimated potency is not less than 2.02 to 0. c) and the statistical analysis shows no deviation from linearity or parallelism. d) the vaccine passes the requirements for potency. modified as follows for the tesonn guinea-pig. into each immunized mouse. capable of causing the death of mice within 14 days of intracerebral injection. 0-7.0 0-1.5-4 1-3 0-1.5 0-2.15 < 0.0 0-1.7.3.7.7.15 < 0.5 0-2 0-1 0-2 Phosphorus Molecular size K0 Nitrogen CL-4B** CL-2B*** 0-1. Impurities are removed by such techniques as fractional precipitation.7.5 0.0-4.25 < 0.0-5.5 0-1.5 0-1.20 < 0.0 0-1. The monovalent bulk polysaccharide is stored at a suitable temperature in conditions that avoid the uptake of moisture.65 < 0. The purified polysaccharides comply with the following tests as applicable: Protein (2. Hexosamine (2.30). 72 determined by the methods prescribed below.7.5-3.6-3.20 < 0. Comply with the test for uronic acids.9 3.5 <2 2.5-3. washed.55 < 0.50 ≥ 15 Uronic acids ≥ 45 ≥ 15 ≥ 40 ≥ 12 ≥ 40 ≥ 20 ≥ 15 ≥ 13 ≥ 25 ³ 20 ³ 15 ≥ 28 ≥ 13 ≥ 12 ≥9 ≥ 25 ≥ 20 ≥ 15 ≥ 20 ≥ 14 ≥ 20 ≥ 20 ≥ 25 ≥ 37 HexoMethylsamines pentoses ≥ 38 O-acetyl Groups ≥ 1. Comply with the test for hexosamine.4-4.5 1.5-6.0 < 0.1). Comply with the test for methylpentoses. and dried in a vacuum to a residual moisture content shown to be favourable to the stability of the polysaccharide.0 0-1.30 < 0.0 0-1. enzymatic digestion and ultrafiltration.5-6 0-1 0-1 4-6 2.40 < 0.5-2.45 < 0.0 0-1.1).5 3-5 1. Uronic acids (2.5 1. .5 0-1 0.0 1.60 < 0.8 ≥ 12 ≥ 12.5 0-3.0-5. Nucleic acids (2.0 3.15 < 0.1). Comply with the test for phosphorus.5 <2 <2 <2 <2 <2 <2 <2 <2 <1 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 Total 3. The residual moisture content is determined by drying under reduced pressure over diphosphorus pentoxide or by thermogravimetric analysis and the value obtained is used to calculate the results of the tests shown below with reference to the dried substance.0 0-1 2.5-5.0 2.4-3.1). Comply with the test for protein. Only a monovalent bulk polysaccharide that complies with the following requirements may be used in the preparation of the final bulk vaccine.0 0-1.0 0-1.1).0 0-2 1.1).5 <2 <5 <2 <2 <2 <7 <3 <3 <5 <3 <2 <3 <2 <3 <2 <2 <2 < 2.Specifications on monovalent bulk polysaccharides (per cent contents): Molecular Type* 1 2 3 4 5 6B 7F 8 9N 9V 10A 11A 12F 14 15B 17A or 17F 18C 19A 19F 20 22F 23F 33F Proteins Nucleic acids <2 <2 <5 <3 < 7.50 < 0.45 < 0. Total nitrogen (2.60 < 0. Comply with the test for nucleic acids.15 < 0.0 3.5-3 0.2-4 0. The polysaccharide is obtained by fractional precipitation.5 2.0-4.7.15 < 0.5-4. Methylpentoses (2.20 < 0. are shown in the Table 1.PNEUMOCOCCAL POLYSACCHARIDE VACCINE IP 2007 Table 1.5 2.45 < 0.15 < 0.15 < 0.0 0-1. Percentage contents of components. Comply with the test for total nitrogen.55 < 0.5 ≥ 12 * The different types are indicated using the Danish nomenclature ** Cross linked agarose for chromatography R *** Cross linked agarose for chromatography R1 and the culture is inactivated with phenol.5-4. Phosphorus (2. 4. Antimicrobial preservative. Carry out test for sterility using 10 ml of bulk for each sterility medium. The confidence interval (P = 0. Molecular size is determined by gel filtration or high performance size-exclusion chromatography (HPSEC) (2. (2) the total amount of polysaccharide in the container.5 µg/ml of each polysaccharide.4.5 g/l. Specificity For establishing the specificity. Tests Sterility (2. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.0 per cent of the intended amount. Pyrogens (2. Complies with the test for pyrogens.11). Where applicable. The content is not less than 85.5 to 7. Molecular size.14). pH (2.5 ml contains 25 µg of each polysaccharide. including factor sera for types within groups. . Complies with the test for sterility. physicochemical or immunochemical method (2. Abnormal Toxicity (2. the assay may be replaced by a qualitative test that identifies each polysaccharide. on each monovalent bulk polysaccharide used in the preparation of the final lot. provided that an assay has been performed 73 Poliomyelitis Vaccine (Inactivated) Poliomyelitis Vaccine (Inactivated) is a liquid preparation of suitable strains of human polioviruses 1. Phenol (2. The solution is sterilized by filtration through a bacteriaretentive filter. and purified polysaccharides of each type as standards.0 per cent and not greater than 115. determine the amount of antimicrobial preservative by a suitable chemical method. Complies with the test for abnormal toxicity with the following modifications. Identification The assay also serves to identify the vaccine.2. Inject each of the rabbit with 1 ml of a dilution of the vaccine containing 2.4.3. Not more than 2.95) of the assay is not less than 80. The vaccine contains not less than 70.0 per cent of the quantity stated on the label for each polysaccharide. Identification Confirm the identity of the monovalent bulk polysaccharide by immunochemical method (2.16) using cross linked Agarose for chromatography R or chromatography Agarose for chromatograph R1.2.1).1). including factor sera for distinguishing types within groups.2. Comply with the test for O-acetyl groups.0 per cent of the estimated content.11).7.0 and not more than 120.2. either alone or Multiple angle light laser scattering (MALLS) or any other suitable method. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification. Inject 10 human doses each in two guinea pigs weighing between 250 and 350 g by intraperitoneal route and observe for 12 days.0 per cent of the intended amount. Assay Determine the content of each polysaccharide by a suitable biochemical.11). Tests and Assay may be released for use. Comply with the test for sterility.36). using antisera specific for each polysaccharide contained in the vaccine. no reaction should occur.IP 2007 POLYSACCHARIDE VACCINE (INACTIVATED) O-Acetyl groups (2. they may be omitted on the final lot.5 µg/ml. Sterility (2. 4.2. The label states (1) the number of µg of each polysaccharide per human dose. The content is not less than 85.2. Sterility (2. The polysaccharides are tested at a concentration of 50 µg/ml using a method capable of detecting 0. determine the amount of antimicrobial preservative by a suitable chemical method. FINAL BULK VACCINE The final bulk vaccine is obtained by aseptically mixing the different polysaccharide powders.24). When consistency of production has been established on a suitable number of consecutive batches.14)(except for polysaccharides 7F. 2 and 3 grown in suitable cell cultures and inactivated by a validated method. Provided that the tests for phenol and for antimicrobial preservative have been carried out with satisfactory results on the final bulk vaccine.2. Production General provisions The production method should consistently yield vaccines of acceptable safety and immunogenicity in man. when the antigens are tested against all the antisera specific for the other polysaccharides of the vaccine. An antimicrobial preservative may be added. The uniform mixture is aseptically dissolved in a suitable isotonic solution so that one human dose of 0. Labelling.0 per cent and not more than 130. Where applicable. 14 and 33F). FINAL LOT The final bulk vaccine is distributed and filled aseptically into sterile containers (vials or ampoules).8).0 per cent and not greater than 115. Antimicrobial preservative. unless otherwise justified and authorised.POLYSACCHARIDE VACCINE (INACTIVATED) IP 2007 Production of the vaccine is based on a virus seed-lot system. if primary. subject to approval by the competent authority. it is not to be used nor are any of the remaining monkeys of the group concerned unless it is evident that their use will not impair the safety of the product. closed colonies of monkeys bred in captivity. secondary or tertiary monkey kidney cells. a previously approved seed lot prepared using virus passaged in cells from wild monkeys may. simian virus 40. Kidneys that show no pathological signs are used for preparing cell cultures. If primary. the virus in the final vaccine shall not have undergone more passages from the master seed lot than was used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. it shall be demonstrated by suitable validation tests that cell cultures beyond the passage level used for production are free from tumorigenicity. filoviruses and herpesvirus B (Cercopithecine herpesvirus 1).7. The supplier of animals is certified by the competent authority. secondary or tertiary monkey kidney cells complies with the requirements given below under Virus Propagation and Harvest for single harvests produced in such cells. Virus concentration. disrupt the cells before carrying out the test. secondary or tertiary monkey kidney cells are used. Monitored. If secondary or tertiary cells are used. The following special requirements for the substrate for virus propagation apply to primary.7. not from animals caught in the wild. Primary. secondary or tertiary monkey kidney cells. Extraneous agents (2. The animals used are of a species approved by the competent authority. Kidney cells used for vaccine production and control are derived from monitored. Monkeys used in the preparation of kidney cell cultures for production and control of the vaccine. the monkeys are also shown to be free from antibodies to herpesvirus B (Cercopithecine herpesvirus 1) infection. SEED LOT Each of the three strains of poliovirus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Monkey cell cultures for vaccine production. Unless otherwise justified and authorised. be used for vaccine production if historical data on safety justify this.2). Monkeys from which kidneys are to be removed are thoroughly examined. The primary monkey kidney cell suspension complies with the test for mycobacteria . The monkeys used are shown to be tuberculin-negative and free from antibodies to simian virus 40 (SV40) and simian immunodeficiency virus. A working seed lot produced in primary. closed colonies of monkeys. if tested. In addition.3). would comply with the test for safety and efficacy. Freedom from extraneous agents is achieved by the use of animals maintained in closed colonies that are subject to continuous and systematic veterinary and laboratory monitoring for the presence of infectious agents. Human herpesvirus 1 has been used as an indicator for freedom from herpesvirus B antibodies on account of the danger of handling herpesvirus B 74 (Cercopithecine herpesvirus 1). If a monkey shows any pathological lesion relevant to the use of its kidneys in the preparation of a seed lot or vaccine. Cell lines are used according to a cell-bank system. secondary or tertiary monkey kidney cells have been used for isolation of the strain.7.2) or in primary. measures are taken to ensure that the strain is not contaminated with simian viruses such as simian immunodeficiency virus. The working seed lot complies with the requirements for seed lots for virus vaccines. The virus concentration of each working seed lot is determined to define the quantity of virus to be used for inoculation of production cell cultures. Each group of cell cultures derived from a single monkey forms a separate production cell culture giving rise to a separate single harvest. production complies with the requirements indicated below. If Macaca spp. The production method is validated to demonstrate that the product. have not been previously employed for experimental purposes. in good health and. monkeys are used for production. secondary or tertiary monkey kidney cells. . All the operations described in this section are conducted outside the area where the vaccine is produced. in a continuous cell line (2. Each monkey is tested serologically at regular intervals during a quarantine period of not less than 6 weeks imposed before entering the colony and then during its stay in the colony. 2 or 3 by virus neutralisation in cell culture using specific antibodies. Identification Each working seed lot is identified as human poliovirus 1. The monkeys are kept in groups in cages. Only a working seed lot that complies with the following requirements may be used for virus propagation. Substrate for virus propagation The virus is propagated in a human diploid cell line (2. particularly for evidence of tuberculosis and herpesvirus B (Cercopithecine herpesvirus 1) infection. Approved animal serum (but not human serum) may be used in the cell culture media.7.IP 2007 POLYSACCHARIDE VACCINE (INACTIVATED) PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells or viruses are being handled. Control cells. secondary or tertiary monkey kidney cells are used.4). The dilution of supernatant in the nutrient medium is not greater than 1:4 and the area of the cell layer is at least 3 cm2 per ml of inoculum. . The cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. Neutralise the sample by a high-titre antiserum against the specific type of poliovirus. The control cells of the production cell culture comply with a test for Identification (if a cell-bank system is used for production) and with the requirements for extraneous agents. carried out using 10 ml. test a sample of at least 10 ml of the single harvest for the absence of SV40 virus and other extraneous agents. Only a purified monovalent harvest that complies with the following requirements may be used for the preparation of the inactivated monovalent harvest. where continuous cell lines in a fermenter are used for production. or other cells shown to be at least as sensitive for SV40.11). accidental reasons. The tests for Identification and Sterility may be carried out instead on the purified. where primary. pooled monovalent harvest. Mycoplasmas (2. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from extraneous agents. Virus concentration. At the end of this period. The single harvest complies with the test for sterility. Test in rabbit kidney cell cultures. accidental reasons. Incubate the cultures at 37° and observe for 14 days. by the method described under Test in Rabbit Kidney Cell Cultures. the tests in cell cultures are carried out as shown below under Test in Rabbit Kidney Cell Cultures and Test in Cercopithecus Kidney Cell Cultures). 200 × 106 cells are set aside to prepare control cells. Set aside one or more containers of each batch of cells with the same medium as non-inoculated control cells. the purification process shall have been shown to reduce consistently the content of substrate-cell DNA to not more than 500 pg per single human dose. Sterility (2. PURIFICATION AND PURIFIED MONOVALENT HARVEST Several single harvests of the same type may be pooled and may be concentrated. secondary or tertiary monkey kidney cells are used for production. The test is not valid if more than 20 per cent of the control cells are discarded for non-specific. Identification The single harvest is identified as containing human poliovirus 1. Virus concentration. The virus concentration is determined by titration of infectious virus. where primary. The virus concentration of each single harvest is determined by titration of infectious virus in cell cultures. If continuous cell lines are used for production. After demonstration of consistency of production at the stage of the single harvest. Test in rabbit kidney cell cultures. Test in Cercopithecus kidney cell cultures. secondary or tertiary monkey kidney cells are used for production. The single harvest complies with the test for mycoplasmas. the test for virus concentration may be carried out instead on the purified. a cell sample equivalent to at least 500 ml of the cell suspension. make at least one subculture of fluid in the same cell culture system and observe both primary cultures and subcultures for an additional 14 days. is taken to prepare control cell cultures. Test in Cercopithecus kidney cell cultures. pooled monovalent harvest. Test a sample of at least 10 ml of the pooled supernatant fluid from the control cultures for the absence of SV40 virus and other extraneous agents by inoculation onto cell cultures prepared from the kidneys of cercopithecus monkeys. Only a single harvest that complies with the following requirements may be used in the preparation of the vaccine. The monovalent harvest or pooled monovalent harvest is purified by validated methods. The test is not valid if more than 20 per cent of the control cell cultures are discarded 75 for non-specific. secondary or tertiary monkey kidney cells are used for production. Incubate the cultures at 37° and observe for at least 2 weeks.2. at the concentration employed for vaccine production. Where primary. Test the sample in primary cercopithecus kidney cell cultures or cells that have been demonstrated to be at least as susceptible for SV40. Where primary. Test a sample of at least 10 ml of the pooled supernatant fluid from the control cultures for the absence of herpesvirus B (Cercopithecine herpesvirus 1) and other viruses in rabbit kidney cell cultures. Identification The virus is identified by virus neutralisation in cell cultures using specific antibodies or by determination of D-antigen. test a sample of at least 10 ml of the single harvest for the absence of herpesvirus B (Cercopithecine herpesvirus 1) and other viruses in rabbit kidney cell cultures as described for the control cells. Not less than 500 ml of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells). 2 or 3 by virus neutralisation in cell cultures using specific antibodies. carried out using 10 ml for each medium. corresponding to at least 1500 human doses. To avoid failures in inactivation caused by the presence of virus aggregates. during validation studies. Test for effective inactivation. FINAL LOT Only a final lot that complies with each of the requirements given below under Identification. Antimicrobial preservative. inactivation is started within a suitable period. an inactivation curve with at least four points (for example. 24. the test for inactivation is carried out on that pool rather than on the final bulk vaccine. If the final bulk vaccine is prepared from a trivalent pool of inactivated monovalent harvests. it may be omitted on the final lot. Observe the subcultures for at least 2 weeks. Make not fewer than two passages from each container. Inoculate the samples in cell cultures such that the dilution of vaccine in the nutrient medium is not greater then ¼ and the area of the cell layer is at least 3 cm2 per ml of inoculum. a sample of at least 1500 ml or.11). Sterility (2.2 g/l. 2 and 3 by a suitable immunochemical method such as the determination of D-antigen by enzyme-linked immunosorbent assay (ELISA). determine the amount of antimicrobial preservative by a suitable chemical . Provided that the tests for free formaldehyde and antimicrobial preservative and the in vivo assay have been performed with satisfactory results on the final bulk vaccine. for the passages. Before addition of any antimicrobial preservative. a test for effective inactivation is carried out on this pool instead of on the final bulk vaccine.2. they may be omitted on the final lot. Antimicrobial preservative. test the susceptibility of the cell culture used by inoculation of live poliovirus of the same type as that present in the inactivated monovalent harvest. The content is not less than 85. time 0. of the prior filtration.11). determine the amount of antimicrobial preservative by a suitable chemical method. At the end of the observation period. No sign of poliovirus multiplication is present in the cell cultures. Set aside one or more containers with the same medium as noninoculated control cells. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. INACTIVATION AND INACTIVATED MONOVALENT HARVEST Several purified monovalent harvests of the same type may be mixed before inactivation.2. the equivalent of 1500 doses is tested for residual live poliovirus in cell cultures. filtration is carried out before and during inactivation. Inactivation. The inactivated monovalent harvest complies with the test for sterility. Only an inactivated monovalent harvest that complies with the following requirements may be used in the preparation of a trivalent pool of inactivated monovalent harvests or a final bulk vaccine. preferably not more than 24 h and in any case not more than 72 h. The virus suspension is inactivated by a validated method that has been shown to inactivate poliovirus without destruction of immunogenicity. as described for the inactivated monovalent harvest.0 per cent and not greater than 115. to the total protein content (specific activity) of the purified monovalent harvest is within the limits approved for the particular product.14) is within the limits approved for the particular preparation. FINAL BULK VACCINE The final bulk vaccine is prepared directly from the inactivated monovalent harvests of human polioviruses 1. Tests and Assay may be released for use. and 96 h) is established showing the decrease in concentration of live virus with time. use cell culture supernatant and inoculate as for the initial sample. Tests Free formaldehyde (2. Identification The vaccine is shown to contain human polioviruses 1. one at the end of the observation period and the other 1 week before. carried out using 10 ml for each medium. Carry out test for sterility using 10 ml of bulk for each sterility medium. Where applicable.2. Where applicable. The content of D-antigen determined by a 76 suitable immunochemical method (2. 2 and 3 or from a trivalent pool of inactivated monovalent harvests. Take one sample not later than three-quarters of the way through the inactivation period and the other at the end.0 per cent of the intended amount. the presence of an excess of formaldehyde at the end of the inactivation period is verified. 48. If a trivalent pool of inactivated monovalent harvests is used. Observe the cell cultures for at least 3 weeks. for a purified and concentrated vaccine. Maximum 0. If formaldehyde is used for inactivation.14). D-antigen content. verify the absence of residual live poliovirus by inoculation on suitable cell cultures of two samples of each inactivated monovalent harvest.POLYSACCHARIDE VACCINE (INACTIVATED) IP 2007 Specific activity.20). determined by a suitable immunochemical method (2. Provided that the test for bovine serum albumin has been performed with satisfactory results on the trivalent pool of inactivated monovalent harvests or on the final bulk vaccine. Sterility (2. A stabiliser and an antimicrobial preservative may be added. The ratio of the virus concentration or the Dantigen content. After neutralisation of the formaldehyde with sodium bisulphite (where applicable).3.2. .0 per cent of the intended amount. expressed in units of D-antigen per single human dose. The midpoint on a log 2 scale of the minimum and maximum geometric mean titres of the series of 3 or more tests is used as the cutoff value. 2 and 3 by a suitable immunochemical method (2. The dose range is chosen such that a dose response to all 3 poliovirus types is obtained. The 77 number of animals per group must be sufficient to obtain results that meet the validity criteria. The capacity of the vaccine to induce the formation of neutralizing antibodies is determined in-vivo by one of the following methods: Test in chicks or guinea-pigs. Rather. Test on rats. Complies with the test for sterility. Inoculate the mixtures into cell cultures for the detection of unneutralised virus and read the results up to 7 days after inoculation. determined by a suitable immunochemical method (2. Results are read following fixation and staining after 7 days of incubation at 35°.49).0 per cent of the animals. the potency of the vaccine is not significantly less than that of the reference preparation. the content. after validation. Poliomyelitis Vaccine.IP 2007 POLIOMYELITIS VACCINE. (2) the statistical analysis shows no significant deviation from linearity or parallelism. For each group of animals. Due to interlaboratory variation. Neutralising titres against all 3 polivirus types are measured separately using 100 CCID50 of the Sabin strains as challenge viruses. Labelling. LIVE (ORAL) method. For each of the 3 poliovirus types. groups of 10 rats are usually sufficient although valid results may be obtained with fewer animals per group. 2 and 3. In vivo test. Prepare a suitable series of at least three dilutions of the vaccine under examination using a suitable buffered saline solution. using a parallel-line model. (2) the nominal amount of virus of each type (1. Bleed the animals after 20 to 22 days. Carry out in parallel a control test using a suitable reference preparation.2.11).3).14). Mix 100 CCID50 of virus with the dilution of serum and incubate at 37° for 4 h 30 min to 6 h. For a valid antibody assay. Not more than 50 ng per single human dose. Not more than 5 IU per human dose.2. The weight of individual animal must not vary by more than 10. The vaccine complies with the test if a dilution of 1 in 100 or more produces an antibody response for each of the three types of virus in 50.5 ml per rat is used. Keep at 5 ± 3° for 12 to 18 h. Sterility (2. Bovine serum albumin. Examine the sera for the presence of neutralising antibody. each weighing between 250 and 350 g. For each type.3.0 per cent from the group mean.0 per cent of the estimated potency.0 per cent and 400. 2 and 3). prepared in a form suitable for oral administration. Live (Oral) Oral Poliomyelitis Vaccine is a preparation of approved strains of live attenuated poliovirus type 1. note the number of sera which have neutralising antibody and calculate the dilution of the vaccine giving an antibody response in 50.0 per cent and not greater than 115. expressed with reference to the amount of D-antigen stated on the label. Vero or Hep2 as indicator cells.14) using an appropriate reference preparation calibrated in D-antigen units. A suitable in vivo assay method consists of intramuscular injection into the hind limb(s) of not fewer than 3 dilutions of the vaccine under examination and a reference vaccine. 2 or 3 grown in in vitro cultures of approved cells. Inject 0. (3) the fiducial limits of the estimated relative potency fall between 25. the cut-off values are determined for each laboratory based on a minimum series of 3 tests with the reference vaccine. using a separate group for each dilution of vaccine.2. Not more than 10 µg of protein nitrogen per human dose. The content is not less than 85.5 ml of the dilutions intramuscularly into groups of ten 3-week-old chickens or groups of ten guinea-pigs. at a dilution of 1 in 4. As a measure of consistency of production. The test is not valid unless (1) for both the test and reference vaccines the ED50 lies between the smallest and the largest doses given to the animals. Use of 4 dilutions is often necessary to obtain valid results for all 3 serotypes. containing any one type or any combination of the three types of Sabin strains.0 per cent of the animals. Assay D-antigen content. Bleed the animals on the fifth or sixth day after the injection and separate the sera. Protein content (2. For the probit method it is necessary to establish a cut-off neutralising antibody titre for each poliovirus type to define a responder. The label states (1) the types of poliovirus contained in the vaccine. is within the limits approved for the particular product. using for each dilution a group of 10 specific pathogen-free rats of the suitable strain. (3) the cell substrate used to prepare the vaccine. it is not possible to define cut-off values that could be applied by all laboratories. Bacterial endotoxins ( 2. and neutralization conditions of 3 h at 35° to 37° followed by 18 h at 2° to 8°. The potency is calculated by comparison of the preparation of responders for the vaccine under examination and the reference vaccine by the probit method or. An inoculum of 0. to each of the human polioviruses 1.2. determine the D-antigen content for human polioviruses 1. the titre of each challenge virus must be shown to be within the range of 10 to 1000 CCID50 and the neutralizing antibody titre of a control serum must be within 2 twofold dilutions of the geometric mean titre of the serum. until the monkeys are used. SEED LOT The strains of poliovirus used shall be identified by historical records that include information on the origin and subsequent manipulation of the strains. If kidneys from near-term monkeys are used. Primary monkey cells. If primary monkey kidney cells are used. using specific antibodies. Identification Each working seed lot is identified as poliovirus of the given type. even after completion of the quarantine period.7.7.2) or in primary monkey kidney cells (including serially passaged cells from primary monkey kidney cells). Each group of cell cultures derived from a single monkey or from fetuses from no more than ten near-term monkeys is prepared and tested as an individual group. All the operations described in this section shall be conducted outside the areas where the vaccine is produced. Cell lines are used according to a cell-bank system. and not previously employed for experimental purposes shall be used. The monkeys used shall be shown to be free from antibodies to simian virus 40 (SV40) and simian immunodeficiency virus. Kidneys that show no pathological signs are used for preparing cell cultures. the maintenance medium after virus inoculation shall contain no added serum. If a monkey shows any pathological lesion relevant to the use of its kidneys in the preparation of a seed lot or vaccine. Working seed lots are prepared by a single passage from a master seed lot and at an approved passage level from the original Sabin virus. Unless otherwise justified and authorised. LIVE (ORAL) IP 2007 Production General provisions The vaccine strains and the production method should consistently yield vaccines that are both immunogenic and safe in man. the room that housed the group shall be thoroughly cleaned and decontaminated before being used for a fresh group. If at any time during the quarantine period the overall death rate of a shipment consisting of one or more groups reaches 5 per cent (excluding deaths from accidents or where the cause was specifically determined not to be an infectious disease). the virus concentration is the basis for the quantity of virus used in the neurovirulence test. After the last monkey of a group has been taken. and having no contact with other monkeys during the quarantine period. Continuous cell lines are approved by the competent authority.2) or in continuous cell lines (2. If the vaccine is prepared in monkey kidney cell cultures. Only a virus seed lot that complies with the following requirements may be used for virus propagation. Virus for the preparation of vaccine is grown by aseptic methods in such cultures. Substrate for virus propagation The virus is propagated in human diploid cells (2. Not more than two monkeys shall be housed per cage and cage-mates shall not be interchanged. with separate feeding and cleaning facilities. The following special requirements for the substrate for virus propagation apply to primary monkey cells. monkeys from that entire shipment shall continue in quarantine from that time for a minimum of 6 weeks. the monkeys shall also be shown to be free from antibodies to cercopithecid herpesvirus 1 (B virus). Human herpesvirus has been used as an indicator for freedom from B virus antibodies on account of the danger of handling cercopithecid herpesvirus 1 (B virus). Monkey kidney cell cultures for vaccine production. production complies with the requirements indicated below. Determined by the method described below. healthy monkeys kept in one room. Virus seed lots are prepared in large quantities and stored at a temperature below -60°. serially passaged monkey kidney cell cultures from primary monkey kidney cells may be used for virus propagation. the mother is quarantined for the term of pregnancy. The production of vaccine is based on a virus seed-lot system. If Macaca spp. If the monkeys are from a colony maintained for vaccine production. If animal serum is used in the propagation of the cells. it shall not be used. Virus concentration. as in quarantine. nor shall any of the remaining monkeys of the quarantine group concerned be used unless it is evident that their use will not impair the safety of the product. Adequate precautions shall be taken to prevent cross-infection between cages. The groups shall be kept continuously in isolation. are used for production. Monkeys used for preparation of kidney cell cultures and for testing of virus.POLIOMYELITIS VACCINE. A quarantine group is a colony of selected. particularly for evidence of tuberculosis and cercopithecid herpesvirus 1 (B virus) infection. The monkeys shall be kept in well-constructed and adequately ventilated animal rooms in cages spaced as far apart as possible. . in good health. the virus in the final vaccine shall not have undergone more than two passages from the master seed lot. The monkeys shall be kept in the country of manufacture of the vaccine in quarantine groups for a period of not less than 6 weeks before use. animals of a species approved by the competent authority. otherwise the monkey kidney cells are not propagated in series. 78 Monkeys from which kidneys are to be removed shall be anaesthetised and thoroughly examined. a sample of at least 30 ml of the pooled fluid removed from the cell cultures of the kidneys of each single monkey or from fetuses from not more than ten near-term monkeys is divided into two equal portions. Cell cultures. control cell cultures are maintained at 33° to 35° for the relevant incubation periods. If the working seed lot is produced in primary monkey cells. Working seed lot shall be free from detectable DNA sequences from simian virus 40 (SV40) Neurovirulence (2.IP 2007 POLIOMYELITIS VACCINE. evidence is found of the presence in a cell culture of any extraneous agent.2) it complies with the requirements for seed lots for virus vaccines. The cultures are incubated at a temperature of 35° to 37° and are observed for a total period of at least 4 weeks. it is equal to the probability of false rejection on the occasion of a first test (i. apply to control cells when the vaccine is produced in primary monkey cells The virus suspension is harvested not later than 4 days after virus inoculation. the seed lot shall cease to be used in vaccine production if the frequency of failure of the monovalent pooled harvests produced from it is greater than predicted statistically. This statistical prediction is calculated after each test on the basis of all the monovalent pooled harvests tested. On the day of inoculation with virus seed. The subcultures are also observed for at least 2 weeks. the entire group of cultures concerned shall be rejected. Only a single virus harvest that complies with the following requirements may be used in the preparation of the monovalent 79 pooled harvest. 1 per cent). as that used for vaccine production. provided that it does not contain SV40 antibody. Serum may be added to the original culture at the time of subculturing.6). If the working seed lot is produced in human diploid cells (2. PROPAGATION AND HARVEST All processing of the cell-banks and subsequent cell-cultures is done under aseptic conditions in an area where no other cells are handled. where primary monkey cells are used. The cell-culture medium may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration.e. At least one bottle of each kind of cell culture remains uninoculated to serve as a control. each cell culture is examined for degeneration caused by an infective agent. If the test is carried out only by the manufacturer. where necessary. tested in monkey kidney cell cultures from another species so that tests on the pooled fluids are done in cell cultures from at least one species known to be sensitive to SV40. suckling mice and guinea-pigs given under Tests for extraneous agents in viral vaccines for human use. it complies with the requirements given below under Propagation and Harvest and Monovalent Pooled Harvest and with the tests in adult mice. Control cells. The pooled fluid is inoculated into bottles of these cell cultures in such a way that the dilution of the pooled fluid in the nutrient medium does not exceed 1 in 4. in this examination.3). the temperature is maintained constant to ±0.7. Each master and working seed lot complies with the test for neurovirulence of poliomyelitis vaccine (oral) in monkeys. After inoculation of the production cell culture with the virus working seed lot. a test in a second species is not required. Complies with tests for extraneous agents. but the final medium for maintaining cell growth during virus multiplication does not contain animal serum. given below.5°. LIVE (ORAL) Extraneous agents (2. Not less than 5 per cent and not more than 1000 ml of the cell cultures employed for vaccine production are set aside as uninfected cell cultures (control cells). If. The virus concentration of virus harvests is determined as prescribed under Assay to monitor consistency of production and to determine the dilution to be used for the final bulk vaccine. The other portion of the pooled fluid is. but not the same animal. The following special requirements apply to virus propagation and harvest in primary monkey cells.7.3 ). Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from live extraneous agents.7. The area of the cell sheet is at least 3 cm2 per ml of pooled fluid. Genetic markers. One portion of the pooled fluid is tested in monkey kidney cell cultures prepared from the same species. Virus concentration. Animal serum may be used in the propagation of the cells.7. Extraneous agents ( 2. at least one subculture of fluid is made from each of these cultures in the same cell culture system. within the range 33° to 35°. the test slides are provided to the control authority for assessment. If the monkey species used for vaccine production is known to be sensitive to SV40. but the maintenance medium after inoculation of test material contains no added serum except as described below. provided that the serum does not contain SV40 antibody. as shown below. special requirements.7. Primary monkey cells. shown to be suitable. . inoculated cells are maintained at a fixed temperature.2) or in continuous cell lines (2. During this observation period and after not less than 2 weeks’ incubation. The control cells of the production cell culture from which the virus harvest is derived comply with a test for identity and with the requirements for extraneous agents or. Furthermore. the probability of false rejection on retest being negligible. On the day of inoculation with the virus working seed lot. Each working seed lot is tested for its replicating properties at temperatures ranging from 36° to 40° as described under Monovalent Pooled Harvest. It is preferable to have a substrate free from antibiotics during production. Approved animal (but not human) serum may be used in the media. Tests for haemadsorbing viruses. At the end of the observation period. A sample of at least 10 ml of each single harvest is neutralised by a type-specific poliomyelitis antiserum prepared in animals other than monkeys. similar samples of the pooled fluid are taken and the tests referred to in this section in the two kinds of monkey kidney cell culture and in the rabbit cell cultures are repeated. which may be held at 4°. Tests for extraneous agents in viral vaccines for human use. In preparing antisera for this purpose. the control cell cultures are 80 examined for degeneration caused by an infectious agent. LIVE (ORAL) IP 2007 Fluorescent-antibody techniques may be useful for detecting SV40 virus and other viruses in the cells. Single harvests Tests for neutralised single harvests in monkey kidney cell cultures. evidence is found of the presence of an extraneous agent. The cultures are incubated at a temperature of 35° to 37° and observed for at least 2 weeks. the single harvest from the whole group of cell cultures concerned is rejected. If this examination or any of the tests required in this section shows evidence of the presence in a control culture of any extraneous agent. the poliovirus grown in the corresponding inoculated cultures from the same group shall be rejected. Tests for other extraneous agents. in these tests. Control cell cultures. Half of the neutralised suspension (corresponding to at least 5 ml of single harvest) is tested in monkey kidney cell cultures prepared from the same species. with the exception of the sample for the test for B virus. a sample of at least 20 ml of the pooled fluid from each group of control cultures is taken and tested in two kinds of monkey kidney cell culture. The other half of the neutralised suspension is tested. These control cell cultures are incubated in the same conditions as the inoculated cultures for at least 2 weeks and are examined during this period for evidence of cytopathic changes. If the presence of cercopithecid herpesvirus 1 (B virus) is demonstrated. and then only with the approval of the competent authority. the samples of pooled cell-culture fluid shall be kept at a temperature of -60° or below. A further sample of 10 ml of the pooled fluid removed from the cell cultures on the day of inoculation with the seed lot virus is tested for the presence of extraneous agents by inoculation into human cell cultures sensitive to measles virus. At the time of harvest. Human herpesvirus has been used as an indicator for freedom from B virus inhibitors on account of the danger of handling cercopithecid herpesvirus 1 (B virus). the manufacture of oral poliomyelitis vaccine shall be discontinued and the competent authority shall be informed. Serum used in the nutrient medium of these cultures shall have been shown to be free from inhibitors of B virus. . the immunising antigens used shall be prepared in non-simian cells. accidental reasons. but not the same animal. If. At the time of harvest or within 4 days of inoculation of the production cultures with the virus working seed lot. At the end of the observation period for the original control cell cultures. Manufacturing shall not be resumed until a thorough investigation has been completed and precautions have been taken against any reappearance of the infection. A sample of 2 per cent of the pooled fluid is tested in each of the cell culture systems specified. the remaining control cell cultures are similarly tested. The area of the cell sheet is at least 3 cm2 per ml of pooled fluid.POLIOMYELITIS VACCINE. as that used for vaccine production. as described above under Cell cultures. The tests are not valid if more than 20 per cent of the culture vessels have been discarded for non-specific accidental reasons by the end of the respective test periods. On the day of inoculation with the virus working seed lot 25 per cent (but not more than 2500 ml) of the cell suspension obtained from the kidneys of each single monkey or from not more than ten near-term monkeys is taken to prepare uninoculated control cell cultures. If these tests are not done immediately. in monkey kidney cell cultures from another species so that the tests on the neutralised suspension are done in cell cultures from at least one species known to be sensitive to SV40. the production cell cultures shall not be used and the measures concerning vaccine production described above must be undertaken. At least one bottle of the cell cultures remains uninoculated to serve as a control. provided that the test is done not more than 7 days after it has been taken.3). At the end of the observation period. The sample is inoculated into bottles of these cell cultures in such a way that the dilution of the pooled fluid in the nutrient medium does not exceed 1 in 4. if necessary. The fluids collected from the control cell cultures at the time of virus harvest and at the end of the observation period may be pooled before testing for extraneous agents. A further sample of at least 10 ml of the pooled fluid is tested for cercopithecid herpesvirus 1 (B virus) and other viruses in rabbit kidney cell cultures. The tests are not valid if more than 20 per cent of the control cell cultures have been discarded for non-specific. or within 7 days of the day of inoculation of the production cultures with the working seed lot. a sample of 4 per cent of the control cell cultures is taken and tested for haemadsorbing viruses. as described above.7. If the presence of Cercopithecid herpesvirus 1 (B virus) is demonstrated. The tests are made as described in (2. Virus concentration The virus concentration is determined by the method described below and serves as the basis for calculating the dilutions for preparation of the final bulk. of 81 Identification Each monovalent pooled harvest is identified as poliovirus of the given type.0 log of its value at 36°. for the quantity of virus used in the neurovirulence test and to establish and monitor production consistency. If growth at 40° is so low that a valid comparison cannot be established.0 log greater than that determined at 40°. MONOVALENT POOLED HARVEST Monovalent pooled harvests are prepared by pooling a number of satisfactory single harvests of the same virus type. The TgPVR21 transgenic mouse model provides a suitable alternative to the monkey neurovirulence test for neurovirulence testing of types 1. Neurovirulence (2.5 kg. A sample of the monovalent pooled harvest is tested for cercopithecid herpesvirus 1 (B virus) and other viruses by injection of not less than 100 ml into not fewer than 10 healthy rabbits each weighing between 1. provided that it does not contain SV40 antibody. Quality and Safety of Biologicals. a temperature in the region of 39. Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. Each rabbit receives not less than 10 ml and not more than 20 ml.0° to 39. If the titres obtained for one or more of the reference viruses are not concordant with the expected values.6). that single harvest is rejected.5 and 2. The tests are not valid if more than 20 per cent of the culture vessels have been discarded for non-specific accidental reasons by the end of the respective test periods.5° is used. Each monovalent pooled harvest complies with the test for neurovirulence of poliomyelitis vaccine (oral). During this observation period and after not less than 2 weeks’ incubation. Serum may be added to the original cultures at the time of subculturing. Additional tests are made for extraneous agents on a further sample of the neutralised single harvests by inoculation of 10 ml into human cell cultures sensitive to measles virus. . Animal serum may be used in the propagation of the cells.IP 2007 POLIOMYELITIS VACCINE. the test must be repeated. Geneva. If the test is carried out only by the manufacturer. The test is carried out using a standard operating procedure approved by the competent authority.0 to 5. but the maintenance medium.and rct/40+ strains of poliovirus of the same type. No indication of the presence of retrovirus is found. LIVE (ORAL) The neutralised suspensions are inoculated into bottles of these cell cultures in such a way that the dilution of the suspension in the nutrient medium does not exceed 1 in 4. the causes of these change are investigated. The following special requirements apply to monovalent pooled harvests derived from primary monkey cells. If the cytopathic changes are shown to be due to unneutralised poliovirus. the test is repeated. Fluorescent-antibody techniques may be useful for detecting SV40 virus and other viruses in the cells. A ratio of the replication capacities of the virus in the monovalent pooled harvest is obtained over a temperature range between 36° and 40° in comparison with the seed lot or a reference preparation for the marker tests and with appropriate rct/40. Primary monkey cells. Retroviruses. for both the virus in the harvest and the appropriate reference material. The monovalent pooled harvest passes the test if. Test on rabbits.7. At least one bottle of each type of cell culture remains uninoculated to serve as a control and is maintained by nutrient medium containing the same concentration of the specific antiserum used for neutralisation. at least one subculture of fluid is made from each of these cultures in the same cell-culture system. Genetic markers. the acceptable minimum reduction is determined for each virus strain at a given temperature. the test slides are provided to the competent authority for assessment. The subcultures are also observed for at least 2 weeks. The area of the cell sheet is at least 3 cm2 per ml of neutralised suspension. The cultures are incubated at a temperature of 35° to 37° and observed for a total period of at least 4 weeks. using specific antiserum. at which temperature the reduction in titre of the reference material must be in the range 3. contains no added serum other than the poliovirus neutralising antiserum. provided that the serum does not contain SV40 antibody. 2 or 3 live poliomyelitis vaccines (oral) in transgenic mice susceptible to poliovirus) is available from WHO. The incubation temperatures used in this test are controlled to within ±0. except as described below. Each monovalent pooled harvest is filtered through a bacteria-retentive filter. after the inoculation of the test material. 2 or 3 vaccines once a laboratory qualifies as being competent to perform the test and the experience gained is to the satisfaction of the competent authority. the titre determined at 36° is at least 5. Monovalent pooled harvests from continuous cell lines may be purified. If there is evidence of the presence of SV40 or other extraneous agents attributable to the single harvest. The monovalent pooled harvest is examined using a reverse transcriptase assay. If any cytopathic changes occur in any of the cultures.1°. A suitable procedure (Neurovirulence test of type 1. The test is not valid if more than 20. Measure the rectal temperature of each animal on each working day for 6 weeks.0 infectious virus units (CCID50) per single human dose for type 1. confirmatory serological tests are carried out on the blood of the affected animals. Test on guinea-pigs. using specific antibodies. Use an appropriate virus reference preparation to validate each assay.11). after the first day of the test. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more satisfactory monovalent pooled harvests and may contain more than one virus type.5 log10 infectious virus units (CCID50) per single human dose. Examine the cultures on days 7 to 9. In addition. At the end of the observation period. The label states (1) the types of poliovirus contained in the vaccine. Tests and Assay may be released for use. Identification The vaccine is shown to contain poliovirus of each type stated on the label. The monovalent pooled harvest passes the test if none of the rabbits shows evidence of infection with B virus or with other extraneous agents or lesions of any kind attributable to the bulk suspension. At the end of the observation period carry out autopsy on each animal. (b) the virus concentration of the reference preparation differs by more than 0. (3) the cell substrate used for the preparation of the vaccine. For a trivalent vaccine. (4) that the vaccine is not to be injected. not less than 1 × 105.0 infectious virus units (CCID50) for type 2. Complies with the test for sterility. The rabbits are observed for at least 3 weeks for death or signs of illness. and the remainder subcutaneously. (2) the minimum amount of virus of each type contained in one single human dose.11).2. LIVE (ORAL) IP 2007 which 1 ml is given intradermally at multiple sites. Expose samples of the final lot at 37° for 48 hours. using appropriate type-specific antiserum (or preferably a monoclonal antibody) to neutralise each of the other types present. The estimated difference between the total virus concentration of the unheated and heated vaccines is not greater than 0.05 ml of each of the selected dilutions of virus followed by a suitable cell suspension of the Hep-2 (Cincinnati) line.2. . and not less than 1 × 105. Groups of eight to twelve flat-bottomed wells in a microtitre plate are inoculated with 0. If any signs of infection with Marburg virus are noted. the measures concerning vaccine production described above under Cell cultures are taken. the minimum virus titres are decided by the competent authority.1 ml of the monovalent pooled harvest by intracerebral injection and 0. each weighing between 350 and 450 g. Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final lot. Examine by autopsy all animals that. If the vaccine contains more than one poliovirus type. Tests Sterility (2. Determine the total virus concentration as described under Assay in parallel for the heated vaccine and for unheated vaccine. the estimated mean virus titres must be: not less than 1 × 106. Suitable flavouring substances and stabilisers may be added. Assay Titrate for infectious virus at least in triplicate using the method described below. Labelling. die or are killed because they show disease or show for three consecutive days a body temperature higher than 39°. carry out histological examination to detect infection with Marburg virus. Sterility (2. inject a suspension of liver or spleen tissue or of blood intraperitoneally into not fewer than three guinea-pigs. Complies with the test for sterility. 82 FINAL LOT Only a final lot that complies with the following requirement for thermal stability and is satisfactory with respect to each of the requirements given below under Identification. titrate each type separately.95) of the logarithm of the virus concentration is greater than ±0.0 per cent of the inoculated rabbits show signs of intercurrent infection during the observation period. in addition. carry out a passage from these animals to not fewer than five guineapigs using blood and a suspension of liver or spleen tissue. If the presence of B virus is demonstrated. Administer to not less than five guineapigs.5 ml by intraperitoneal injection and observe as described above for 2 to 3 weeks. 0. The assay is not valid if (a) the confidence interval (P = 0. and the brain and organs removed for detailed examination to establish the cause of death. For monovalent or divalent vaccine.0 per cent of the guinea-pigs survive to the end of the observation period and remain in good health and no animal shows signs of infection with filoviruses virus. administer to not fewer than five guinea-pigs 0. Measure the rectal temperature of the latter guinea-pigs for 2 to 3 weeks. The plates are incubated at a suitable temperature. Thermal stability.5 ml by intraperitoneal injection.8 infectious virus units (CCID50) for type 3. All rabbits that die after the first 24 h of the test and those showing signs of illness are examined by autopsy.3.POLIOMYELITIS VACCINE. The monovalent pooled harvest complies with the test if not fewer than 80.5 log CCID50 from the assigned value. Method. immunofluoresence or any other approved method. The virus suspension is harvested on one or more occasions during incubation.IP 2007 RABIES VACCINE. Serum proteins. The eggs are inoculated with virus seed by the yolk sac route. Virus concentration. Extraneous agents (2. strain of fixed rabies virus grown in an approved cell culture/ embryos of duck/chicken and inactivated by a validated method. The production method shall have been shown to yield consistently vaccines that comply with the requirements for immunogenicity. The production method is validated to demonstrate that the product. The infected sterile living embryos are harvested. The control cells of the production cell culture from which the single harvest is derived should comply with a test for identification and with the requirements for extraneous agents (2. The vaccine complies with the General Requirements of Vaccines for Human Use. The cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. Viral harvests that comply with the following requirements are pooled in the preparation of the inactivated viral harvest. Virus concentration. and stabilizer with aseptic precautions. or in duck embroys or in cultures of chicken embryos derived from a flock certified as free from specified pathogens(2. Working seed lots are prepared by not more than five passages from the master seed lot.3).7.3).7.2). supernatants are collected and stored as raw virus harvest at a suitable temperature. a cell-bank system shall be followed. the media may contain human albumin. the virus harvest is inactivated by a validated method at a fixed. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures are done under aseptic conditions in an area where no other cells are handled. Identification The single harvest contains virus that is identified as rabies virus using specific antibodies by an approved method. SEED LOT The strain of rabies virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. It may be coloured owing to the presence of a pH indicator. safety and stability. and other extraneous agents. if tested. Titrate for infective virus in cell cultures or by any other approved method. HUMAN Rabies Vaccine. The working seed lot complies with the requirements for the virus seed lots. if present are reduced to an acceptable level by a suitable method of purification. Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. Substrate for virus propagation The virus is propagated in any suitable approved cell substrate like a human diploid cell line (2.7. but the final medium for maintaining cell growth during virus multiplication does not contain animal serum.7. minced and emulsified in suitable diluent. Control cells. Unless otherwise justified and authorized. Only a working seed lot that complies with the following requirements may be used for virus propagation. Human Rabies Vaccine for Human use is a freeze-dried or liquid (adsorbed) preparation of a suitable approved. When vaccine is prepared in embryonated eggs. The freeze-dried vaccine is reconstituted immediately before use as stated on the label to give a clear or slightly opalescent solution/suspension. Control eggs. the egg proteins are minimized by an appropriate method of purification. the virus in the final vaccine shall not have undergone more passages from the master seed lot than was used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. Control eggs shall be tested for freedom from haemagglutinating agents. Production General provisions The vaccine is produced on the basis of virus seed lot system and if a cell line is used for virus propagation. would comply with the test for safety and efficacy. PURIFICATION AND INACTIVATION The virus harvests may be concentrated and/or purified by suitable methods. The titre is used to monitor consistency of production.7). Serum and trypsin used in the preparation of cell suspension and media are shown to be free from infectious extraneous agents. Emulsions are centrifuged. Approved animal (but not human) serum may be used in the media. well defined stage of the process which may 83 Identification Each working seed lot is identified as rabies virus using specific antibodies by an approved method. a continuous cell line. The virus concentration of each working seed lot is determined by a cell culture method using . Not less than 500 ml of the cell cultures employed for vaccine production are set aside as uninfected cell cultures (control cells). 8). Inoculate intracerebrally 0. Determine the antigen content by a suitable approved in vitro or in vivo method. If betapropiolactone is used. during or after any concentration or purification.03 ml is inoculated intracerebrally into each of the 10 mice weighing 12 to 15 g.2. The content should be within the limits approved for the particular product. Inactivation.11). The mice are observed for 14 days for symptoms caused by rabies virus and mice showing symptoms of rabies are sacrificed and virus presence is confirmed by an immunoflurescence test or tested for live virus in cell culture by immunofluorescence test or method of equal sensitivity. The containers are then sealed so as to prevent contamination and the introduction of moisture. alternatively. Thiomersal can be used as preservative. Sterility (2. Residual host-cell DNA (2. No rabies virus is detected. Alternatively. the Assay also serves to identify the vaccine. inject into each rabbit a single human dose of the vaccine diluted to ten times its volume. it may be omitted from the test on final lot. Not more than 3. Make a passage after 7 days. No rabies virus should be detected. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers and can be freeze-dried in case of lyophilized products. Bacterial endotoxins (2. . and mice showing symptoms of rabies are sacrificed and virus presence is confirmed by immunoflurescence test or tested for live virus in cell culture by immunofluorescence test or method of equal sensitivity. Inactivation is confirmed by carrying out an amplification test for residual infectious rabies virus.3). No rabies virus should be detected. 5 ml of each culture fluid is pooled on days 14 and 21 and 0.0 per cent determined by an approved method. Only a final lot that complies with each of the requirements given below under Identification. Cell culture vaccines Only an inactivated viral suspension that complies with the following requirements may be used in the preparation of the final bulk vaccine. The method shall have been shown to be capable of inactivating rabies virus without destruction of the immunogenic activity.RABIES VACCINE.11).03 ml into each of 20 mice weighing between 12 and 15 g. Antigen content. Sterility (2. No rabies virus is detected.43). Neither symptoms of disease in the central nervous system nor death occurs in any of the animal within 14 days.2. The mice are observed for 14 days for symptoms caused by rabies virus. Complies with the test for sterility. Alternatively. Tests Inactivation.2. Inactivation is confirmed by carrying out Mice Inoculation Test for residual infectious rabies virus. Carry out test for sterility using 10 ml of bulk for each sterility medium. Water (2. Provided that the test for inactivation has been carried out with satisfactory results on the inactivated virus suspension and the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine. these tests may be omitted on the final lot. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated viral suspensions. Embryonated egg vaccine Only concentrated viral suspensions that comply with the test for sterility. Unless otherwise justified and authorized. inject 0. Inoculate a quantity equivalent to not less than 25 human doses of vaccine into cell cultures of the same type as those used for production of the vaccine. Tests and Assay may be released for use. Less than 25 IU per single human dose. Maintain the culture for a further 14 days and then examine the cell culture for rabies virus using an immunofluorescence test. not more than 4 days after inactivation or on the sample frozen after inactivation and stored at -70º.2. HUMAN IP 2007 be before. should not be greater than 10 ng per single human dose. the content of residual host-cell DNA.2. Identification The vaccine is shown to contain rabies virus antigen by a suitable immunochemical method using specific antibodies. Pyrogens (2.15). Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.3. If a continuous cell line is used for virus propagation. If the inactivation test is already performed on inactivated virus used for final lot. Inoculate a quantity of inactivated viral suspension equivalent to not less than 25 vaccine doses into cell cultures of the same type as those used for production of the vaccine. determined using a suitable method.03ml of the vaccine intracerebrally into each of the 10 mice weighing between 12 and 15g. Make a passage after 7 days. the concentration shall at no time exceed 1:3500. Complies with the test for pyrogens. antigen content and endotoxin requirements may be used for preparation of bulk for inactivation. An approved stabilizer may be added to 84 maintain the activity of the product during and after freezedrying. not more than four days after inactivation or on the sample frozen 4 days after inactivation are stored at –70°. Maintain the cultures for a further period of 14 days and then examine the cell cultures for rabies virus using an immune fluorescence test. This may not be mandatory for lot release. The sealed ampoules or plastic vials can be stored at –60º or below. When stored under prescribed conditions. After 7 days.03 ml by standard statistical methods. The potency determined by method described under “Assay” of a sample of the preparation under examination after storage at 37° for 4 weeks is not less than 2. inject 0. drawn from a uniform stock three to four weeks old. The equivalence in international units of the international standard is stated by the World Health Organization. Distribute the mice into six groups of at least 16 mice each and four groups of 10 mice each and must be of the same sex or the sexes must be equally distributed among the groups. Prepare ten fold serial dilutions of the standard challenge virus suspension. The content is not less than 85. A 10 per cent suspension of the brains is prepared in a suitable diluent approved by the competent authority and thoroughly homogenised. determined by a suitable technique using a suitable reference preparation of ovalbumin. Residual host-cell DNA (for continuous cell line vaccines) (2. Using the four groups of 10 mice each. the supernatant liquid is distributed into sterile vials and freeze dried.15). Standard challenge virus suspension. distributed into sterile ampoules or sterile plastic vials and sealed.1). Should not be greater than 10 ng per single human dose.0 per cent and not greater than 115.03 ml of the virus suspension intracerebrally into each mouse.2. Reconstitute the standard preparation with a suitable diluent. Inject intraperitoneally each mouse in each group with dilutions of the vaccine and reference preparation and repeat the injections. Use mice of a suitable strain. Prepare at least three 5-fold serial dilutions of the solution of the standard preparation and three 5-fold serial dilutions of the vaccine under examination. Throughout the test all mice that die before the fifth day after challenge are excluded from the test and all mice that die with 85 signs of rabies between the fifth and fourteenth day after challenge are counted as failing to resist the challenge. weighing between 11 and 15 g. once the consistency of the product is approved by National Regulatory Authority.9). Antimicrobial preservative. the potency of which has been determined in relation to the International standard. Aluminium content (for gel absorbed vaccine) (2. determined by a suitable immunochemical method (2. The strain of mice suitable for the test is such that when 0. Ovalbumin (for egg based vaccines). HUMAN Abnormal toxicity (2. determine the amount of antimicrobial preservative by a suitable chemical method.03 ml containing 5 to 50 LD50 of the challenge virus suspension is injected intracerebrally per mouse there is 100 per cent mortality. A working pool of the challenge virus strain is prepared by injecting intracerebrally 0.2. calibrated in international units. They are then washed in chilled saline solution to remove blood clots. Storage time needs to be validated by the manufacturer.5 units per single human dose. prepare identical dilutions of the vaccine and . For both. Alternatively. The animals when moribund after showing characteristic signs of rabies are sacrificed and their brains harvested aseptically. For this comparison. Bovine serum albumin (for cell culture vaccine). After centrifuging lightly. Allocate one dilution to each of the six groups of 16 mice each. the serial dilutions should be prepared in such a way that the lowest dilution protects more than 50 per cent of the injected mice. a reference preparation of rabies vaccine. The sealed and freeze-dried supernatant liquid containing vials are stored at -20°. Not more than 1 µg of ovalbumin per human dose.25 mg per single human dose. Assay The Standard preparation is the international standard or another suitable preparation. When stored under prescribed conditions the virus titre of the freeze-dried preparation may be expected to be maintained for not less than 3 years. Not more than 50 ng per single human dose. Test animals. Calculate the virus titre of the standard challenge virus suspension in LD50 per dose of 0.IP 2007 RABIES VACCINE. Virus titre of the challenge virus. the washed brains are homogenised in a suitable diluent approved by the competent authority to give 10 per cent suspension. Not more than 1. and a suitable preparation of rabies virus for use as the challenge preparation are necessary. the standard preparation and the preparation under examination. Determination of potency of the vaccine. Where applicable.2. Complies with the test for abnormal toxicity. Accelerated degradation. the virus titre may be expected to be maintained for not less than one year. The test described below uses a parallel-line model with at least three points for the vaccine under examination and the reference preparation. Observe the mice for 14 days.3.03ml of a 10 fold dilution of the CVS strain of rabies virus in 2 per cent v/v sterile inactivated normal horse serum in water for injection or another suitable diluent approved by the competent authority into a suitable number of test animals. The potency of rabies vaccine is determined by comparing the dose necessary to protect mice against a lethal intracerebral dose of rabies vaccine necessary to provide the same protection. The international unit is the activity contained in a stated quantity of the international standard. It is then centrifuged lightly. using a different group for each suspension.0 per cent of the intended amount.14). 03 ml of the standard challenge virus suspension such that on the basis of preliminary titration. (b) there is not deviation from linearity or parallelism of the dose response lines. within 28 days of inoculation. The virus concentration of the master and working seed lots is determined to ensure consistency of production. Unless otherwise justified and authorised. (c) the titre of the challenge virus suspension lies between 5 to 50 LD50. or below -60° if not freeze-dried.5 IU per single human dose. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. Only a single harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. 0. Substrate for virus propagation The virus is propagated in human diploid cells (2. but the final medium for maintaining cell growth during virus multiplication does not contain animal serum. The production method is validated to demonstrate that the product. Calculate the potency of the preparation under examination by standard statistical methods.RUBELLA VACCINE (LIVE) IP 2007 reference preparation and repeat the injections.7. Neurovirulence (2.3). Serum and trypsin used in the preparation of cell suspensions and culture media are shown to be free from extraneous agents. The master/working seed lot complies with the test for neurovirulence of live virus vaccines. Extraneous agents (2. information on the origin of the strain and its subsequent manipulation.5). using specific antibodies. SEED LOT The strain of rubella virus used in the production of rubella vaccine shall be identified by historical records that include 86 Identification The single harvest contains virus that is identified as rubella virus by serum neutralization in cell culture. Not less than 500 ml of the production cell culture is set aside as uninfected cell culture (control cells). the 50 per cent protective dose lies between the largest and smallest doses given to the mice. The vaccine is reconstituted immediately before use to give a clear liquid or may be coloured owing to the presence of a pH indicator.7. After a further 7 days. Macaca and Cercopithecus monkeys are suitable for the test. the virus in the final vaccine shall have undergone no more passages from the master seed lot than were used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. The temperature of incubation is controlled during the growth of the virus. Production General provisions The production of vaccine is based on a virus seed-lot system and a cell-bank system. The vaccine complies with the test if the estimated potency is not less than 2. To avoid unnecessary use of monkeys in the test for neurovirulence virus seed lots are prepared in large quantities and stored at temperatures below -20° if freeze-dried. the confidence limit (P = 0. would comply with the test for safety and efficacy. Only a seed lot that complies with the following tests may be used for virus propagation. inject each vaccinated mouse intracerebrally with 0. Observe the mice for 14 days and record the number of mice surviving the challenge in each group.0 per cent and not more than 400 per cent of the estimated potency.7. It is preferable to have a substrate free from antibiotics during production. The label states the biological origin of the cells used for the preparation of the vaccine. The virus suspension is harvested.95) are not less than 25. Suitable animal (but not human) serum may be used in the growth medium. Identification The master and working seed lots are identified as rubella virus by serum neutralisation in cell culture.03 ml contains between 5 to 50 LD50. The production method shall have been shown to yield consistently live rubella vaccines of adequate immunogenicity and safety in man.2). on one or more occasions. using specific antibodies. Labelling. The working seed lot complies with the tests for seed lots. Virus concentration. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration. The virus concentration in the single harvest is determined as prescribed under Assay to monitor . Virus concentration. The test is not valid unless (a) for both the preparation under examination and the standard preparation. Rubella Vaccine (Live) Rubella Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of rubella virus. Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. if tested. . The label states (1) the strain of virus used for the preparation of the vaccine. Extraneous agents (2.3. the purity of each culture is tested and contaminated cultures are discarded. (5) that the vaccine must not be given to a pregnant woman and that a woman must not become pregnant within two months. The reconstituted vaccine complies with the test for sterility. it may be omitted on the final lot. Tetanus Vaccine (Adsorbed) Tetanus Vaccine (Adsorbed) is a preparation of tetanus formol toxoid adsorbed on mineral carrier.2. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37° for 7 days and for vaccine stored at 2° to 8°. The formol toxoid is prepared from the toxin produced by the growth of Clostridium tetani. Not more than 50 ng per single human dose.3). Abnormal toxicity (2.1). with the following requirement for thermal stability and with each of the requirements given below under Identification and Tests may be released for use.11). The assay is not valid if the confidence limits (P=0. Only a final lot that complies with the tests for minimum virus concentration for release. Single harvests may be pooled to prepare the bulk purified toxoid. Assay Titrate the vaccine for infective virus at least in triplicate. FINAL LOT A minimum virus concentration for release of the product is established such as to ensure. (3) the minimum virus concentration. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Carry out test for sterility using 10 ml of bulk for each sterility medium. would comply with the following test. The virus concentration of the heated vaccine is not more than 1.5 log10 dilution step or by a method of equal precision.43).7. semi-micro determination of water or by any suitable validated method. Use an appropriate virus reference preparation to validate each assay. (2) the type and origin of the cells used for the preparation of the vaccine. Sterility (2. Water (2. The control cells comply with a test for identification and with the tests for extraneous agents (2. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine. if tested. determined by Karl Fischer. Control cells.2. The single harvest complies with the tests for extraneous agents. Toxin-containing culture medium is collected aseptically. Thermal stability. in the light of stability data. Rubella vaccine (Live) RS is suitable for use as a reference preparation.3).0 per cent. At the end of cultivation. Tests Sterility (2. the minimum virus concentration stated on the label is not less than 1 × 103 CCID50 per human dose. from which toxoid is prepared. FINAL BULK VACCINE Single harvests that comply with the above tests are pooled and clarified to remove cells. restored by deliberate reselection. The production method is validated to demonstrate that the product.IP 2007 TETANUS VACCINE (ADSORBED) consistency of production and to determine the dilution to be used for the final bulk vaccine. The estimated virus concentration is not less than that stated on the label.11). seed cultures are managed in a defined seed-lot system in which toxinogenicity is conserved and. that the minimum concentration stated on the label will be present at the end of the period of validity. (4) the time within which the vaccine must be used after reconstitution. where necessary.2. determined by a suitable immunochemical method (2.7.2. it is no longer able to infect susceptible cell cultures. A highly toxinogenic strain of Clostridium tetani with known origin and history is grown in a suitable liquid medium. BULK PURIFIED TOXOID For the production of tetanus toxin. Maintain samples of the final lot of freezedried vaccine in the dry state at 37° for 7 days. Bovine serum albumin. The toxin content (Lf per ml) is checked to monitor consistency of production.14). Complies with the test for abnormal toxicity. Labelling. using at least five cell cultures for each 0. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Not more than 3.95) of the logarithm of the virus concentration is greater than ± 0. 87 Identification When the vaccine reconstituted as stated on the label is mixed with specific rubella antibodies. Production General provisions The maximum number of Lf per single human dose of tetanus vaccine is 25.0 log10 lower than that of the unheated vaccine.3. The following method. determine the amount of antimicrobial preservative by a suitable chemical method. FINAL LOT The final bulk vaccine is distributed aseptically into sterile. Not less than 1. Free formaldehyde (2. that have not previously been treated with any material that will interfere with the test. Antigenic purity. tamper-proof containers.2.4.2 g/l.000 Lf per mg of protein nitrogen. or dies from. 6.11).9). Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Where applicable. if more than one animal dies in the second test. Absence of tetanus toxin.25 mg per single human dose. Identification Tetanus toxoid is identified by a suitable immunochemical method (2. Alternatively. The toxoid complies with the test if neither sample produces any sign of a toxic reaction attributable to tetanus toxin. Complies with the test for abnormal toxicity for antisera and vaccine. The purified toxin is detoxified with formaldehyde by a method that avoids destruction of the immunogenic potency of the toxoid and reversion of toxoid to toxin. Irreversibility of toxoid.24). Inject five times the dose stated on the label subcutaneously or intraperitoneally into each of five guineapigs. each weighing 250 to 350 g.11).TETANUS VACCINE (ADSORBED) IP 2007 The toxin is purified to remove components likely to cause adverse reactions in humans. Carry out test for sterility using 10 ml of bulk for each sterility medium. Where applicable. is given as an example. Certain antimicrobial preservatives. they may be omitted on the final lot. 88 . pH (2. repeat the test once. Maximum 0. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide.0 Sterility (2. If more than one animal dies from non-specific causes. Complies with the test for sterility. Inject subcutaneously at least 500 Lf of purified toxoid in a volume of 1 ml into each of five healthy guinea-pigs. Divide the dilution into two equal parts.2. Sterility (2.2. Sterility (2. Abnormal toxicity (2. Maintain at 37° for about 16 hours and centrifuge until a clear supernatant liquid is obtained.2 g/l. particularly on exposure to heat. such as inoculation into mice or guinea-pigs. Tests and Assay may be released for use. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification. Antimicrobial preservative. determine the amount of antimicrobial preservative by a suitable chemical method. the resulting mixture is approximately isotonic with blood.1). If more than one animal dies from non-specific causes within this period repeat the test. prepare a dilution of the bulk purified toxoid containing the same toxoid concentration as the final vaccine. Test both dilutions by a suitable sensitive assay for active tetanus toxin. Free formaldehyde (2.0 per cent of the intended amount. purification may be carried out after detoxification. Specific toxicity. The containers are closed so as to prevent contamination.0 per cent and not greater than 115. Maximum 1.11).3.3.2. adversely affect the antigenic activity and must not be used.3. Antimicrobial preservative. If within 21 days of the injection any of the animals shows signs of or dies from tetanus. Carry out test for sterility using 10 ml of bulk for each sterility medium. None of the guinea-pigs shows any symptoms of.20).1). particularly those of the phenolic type. applicable to certain vaccines. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent solution. the toxoid does not comply with the test. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent.2. Keep one of them at 2° to 8° and the other at 37° for 6 weeks. The content is not less than 85. the toxoid does not comply with the test. Only bulk purified toxoid that complies with the following requirements may be used in the preparation of the final bulk vaccine.0 per cent and not greater than 115. Tests Aluminium (2. Provided the tests for antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine. tetanus within 21 days. Abnormal toxicity (2.0 per cent of the intended amount. Maximum 0. giving a precipitate.20). None of the second group of animals shows symptoms of tetanus or dies from tetanus or any other cause within 21 days. Using the buffer for the final vaccine. The content is not less than 85.2. Complies with the test for abnormal toxicity for antisera and vaccine. Suitable antimicrobial preservatives may be added. The clear supernatant liquid reacts with a suitable tetanus antitoxin.14).0 and 7. allocate the challenge toxin solution and the three dilutions made from it one to each of the four groups of five guinea-pigs and inject subcutaneously 1. 50.and 160-fold with the same buffer solution. Determination of potency. prepare from the challenge toxin by dilution with phosphate buffered saline pH 7. (c) the fiducial limits of assay lie between 50. it is not necessary to verify the paralytic dose for every assay. Select a preparation of tetanus toxin containing not less than 50 times the 50 per cent paralytic 89 dose per ml.5-fold steps and in which the dilutions of intermediate concentration. Challenge toxin. include four further groups of five guinea-pigs to serve as unvaccinated controls.0 per cent of the estimated potency.IP 2007 TETANUS VACCINE (ADSORBED) Potency of tetanus component Assay. the dilutions form a series differing by not more than 2. Use healthy mice from the same stock. Preparation of the challenge toxin solution. Determine by either of the following methods. are necessary. The test is not valid unless (a) for both the vaccine under examination and the Standard preparation. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardised. healthy guinea-pigs weighing between 250 and 350 g.0 per cent confidence interval of estimated potency is less than 40 IU per single human dose then the limits of the 95. the number of paralysed animals among the four groups of five injected with the challenge toxin solution and its dilutions indicate that the challenge was approximately 50 times the 50 per cent paralytic dose. If the challenge toxin preparation has been shown to be stable.5 Unit of tetanus antitoxin per ml. The test may be repeated any number of times but when more than one test is performed the results of all valid tests must be combined in the estimate of potency. collect the serum from each animal and carry out the biological test for tetanus antitoxin.4 a challenge toxin solution containing fifty times the 50 per cent paralytic dose per ml. weighing . for use as a challenge toxin. Distribute them into six groups of sixteen each. Immediately prior to use. For this comparison. (b) Test on mice Test animals. Use healthy guinea-pigs from the same stock and weighing between 250 and 350 g.0 per cent confidence interval of the estimater of potency shall be within 50 to 200 per cent of the estimated potency unless the lower limit of the 95. one-tenth of the stated human dose diluted to 1 ml with saline solution into each of 9 normal. Allocate the six dilutions one to each of the six groups of sixteen guinea-pigs and inject subcutaneously 1. the potency of which has been determined in relation to the International standard.0 per cent confidence interval of the estimated potency is greater than 40 IU per single human dose. Suggested method (a) Test on guinea-pigs Test animals. for each. Not more than 2 weeks after the second injection. After 28 days inject each animal subcutaneously with 1. protect approximately 50 per cent of the animals from the paralytic effects of the subcutaneous injection of the quantity of tetanus toxin prescribed for this test. (2) Carry out the biological assay of adsorbed Tetanus Vaccine (Adsorbed) described below. adsorbed or another suitable preparation. Sera of at least 6 guinea-pigs out of 9 should contain not less than 0. dilute portions of this challenge toxin solution 16-. Count the number of guinea-pigs without paralysis 5 days after injection of the challenge toxin and calculate the potency of the vaccine under examination relative to the potency of the Standard preparation on the basis of the number of animals without paralysis in each of the six groups of sixteen.0 ml of each dilution into each guinea-pig in the group to which that dilution is allocated. when injected subcutaneously in 1-ml volumes into guineapigs.0 ml of each toxin solution into each guineapig in the group to which that toxin solution is allocated. Examine the guinea-pigs twice daily. (d) the statistical analysis shows no deviations from linearity or parallelism. The guinea-pigs should all be of the same sex or the males and females should be distributed equally between the groups.0 per cent and 200. using standard statistical methods. If necessary. If necessary. If the lower limit of the 95. the Standard preparation of adsorbed tetanus toxoid and a suitable preparation of tetanus toxin. remove and kill all animals showing definite signs of tetanus paralysis. the 50 per cent protective doses lie between the largest and smallest doses of the preparations given to the guinea-pigs. Biological assay of adsorbed tetanus vaccine The potency of adsorbed tetanus vaccine is determined by comparing the dose of the vaccine required to protect guineapigs or mice from the paralytic effects of a subcutaneous injection of tetanus toxin with the dose of the Standard preparation needed to give the same protection. described under Tetanus antitoxin or any other method approved by National Regulatory Authority. Prepare in saline solution three dilutions of the vaccine under examination and three dilutions of a solution of the Standard preparation such that. Standard preparation The Standard preparation is International standard for Tetanus toxoid. (1) Inject subcutaneously on each of two occasions separated by an interval of not more than 4 weeks. (b) if applicable.0 ml of the challenge toxin solution containing fifty times the 50 per cent paralytic dose. the 50 per cent protective doses lie between the largest and smallest doses of the preparations given to the mice.4 a challenge toxin solution containing fifty times the 50 per cent paralytic dose in each 0. Carbonate coating buffer pH 9. Dissolve with stirring 80. Count the number of mice without paralysis 4 days after injection of the challenge toxin and calculate the potency of the vaccine under examination relative to the potency of the Standard preparation on the basis of the numbers of animals without paralysis in each of the six groups of sixteen. dissolve 10 mg of .5 ml. Peroxidase-conjugated rabbit or goat antibody directed against guinea-pig IgG. 50. (b) if applicable.3 g of disodium hydrogen phosphate dihydrate and 2. columns 1-12. Dilute to 10 times its volume with water before use. Determination of antibody titre. A positive guinea-pig serum control and a negative guinea-pig serum control are included on each plate to monitor the assay performance. the number of paralysed animals among the four groups of six injected with the challenge toxin solution and its dilutions indicate that the challenge was approximately 50 times the 50 per cent paralytic dose.5-fold steps and in which the dilutions of intermediate concentration. Determination of potency. Dilutions of test and reference sera are made on ELISA plates coated with tetanus toxoid.5 ml of the challenge toxin solution containing fifty times the 50 per cent paralytic dose. At least 40. Select a preparation of tetanus toxin containing not less than 100 times the 50 per cent paralytic dose per ml. Distribute them into six groups of sixteen each. allocate the challenge toxin solution and the three dilutions made from it one to each of the four groups of six mice and inject subcutaneously 0.51 g of citric acid in 1000 ml of water and adjust the solution to pH 4. (c) the fiducial limits of the assay lie between 50. for each.TETANUS VACCINE (ADSORBED) IP 2007 between 14 and 20 g. The test is not valid unless (a) for both the vaccine under examination and the Standard preparation. The mice should all be of the same sex or the males and females should be distributed equally among the groups.93 g of sodium hydrogen carbonate in 1000 ml of water. include four further groups of six mice to serve as unvaccinated controls.0 g of sodium chloride. the dilutions form a series differing by not more than 2. 2. Shortly before use. After 28 days inject each animal subcutaneously with 0. The ELISA and ToBI tests shown below are given as examples of immunochemical methods that have been found suitable for the determination of antibody tire. (d) the statistical analysis shows no deviation from linearity or parallelism.0 per cent yield of serum is obtained by this procedure.0 with a 400 g/l solution of sodium hydroxide.5 ml of each dilution into each mouse in the group to which the dilution is allocated.5 g/l of polysorbate 20 and 25 g/l of dried skimmed milk. If necessary.5 ml of each toxin solution into each mouse in the group to which that toxin solution is allocated.6. Citric acid solution. Invert the tubes containing blood samples 6 times and allow to stand at 37° for 2 h. rows A-H. Tetanus toxoid. Peroxidase substrate. Peroxidase-conjugated rabbit or goat antibody directed against guinea-pig-IgG is added followed by a peroxidase substrate. 14.5 ml volumes into mice. Diluent block buffer.0 per cent of the estimated potency. Clostridium tetani guinea-pig antiserum (for vaccines-human use) reference preparation (positive control serum).4 (PBS). Determination of antibody titre in guinea-pig serum by enzyme-linked immunosorbent assay (ELISA). Dissolve 10. (c) Determination of antibodies in guinea-pigs Preparation of serum samples. Allocate the six dilutions one to each of the six groups of sixteen mice and inject subcutaneously 0.59 g of anhydrous sodium carbonate and 2. centrifuge at room temperature at 800 g for 20 min. transfer the serum to sterile tubes and store at a temperature below -20°. Preparation of the challenge toxin solutions. dilute portions of this challenge toxin solution 16-. For preparation of serum samples. protect approximately 50 per cent of the animals from the paralytic effects of the subcutaneous injection of the quantity of tetanus toxin prescribed for this test. The test may be repeated any number of times but when more than one test is performed the results of all valid 90 tests must be combined in the estimate of potency. Reagents and equipment ELISA plates: 96 wells. Peroxidase conjugate. Distribute into 150 ml bottles and sterilise by autoclaving at 121° for 15 min.0 g of potassium dihydrogen phosphate. PBS containing 0. Store at room temperature to prevent crystallisation. Phosphate buffered saline pH 7.5 g/l of polysorbate 20. then at 4° for 2 hours. when injected subcutaneously in 0. Dissolve 1. Washing buffer. the following technique has been found suitable.0 per cent and 200. PBS containing 0. If necessary.and 160-fold with the same buffer solution.0 g of potassium chloride in 1000 ml of water. Optical density is measured and the relative antibody titre is calculated using the usual statistical methods. Prepare in saline solution three dilutions of the vaccine under examination and three dilutions of a solution of the Standard preparation such that. Immediately prior to use prepare from the challenge toxin by dilution with phosphate buffered saline pH 7. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardised. Challenge toxin. using standard statistical methods. 10 ml of sodium acetate buffer pH 5.0 g of sodium chloride. Place 100 µI of PBS in each well.55 g of disodium hydrogen phosphate dihydrate and 4. Discard 100 µl from the last column so that all wells contain 100 µl. Incubate at 37° in a humid atmosphere for 1 h. 6 g/l solution of tetramethylbenzidine in alcohol. do not stack more than 4 plates high.5 using a saturated solution of citric acid monohydrate and dilute to 1000 ml with water. Dissolve 90. Wash the plates thoroughly with washing buffer. Tetanus toxin or toxoid is added to serial dilutions of test and reference sera.6 and autoclave at 121° for 20 min. Allow to stand at room temperature. 20.5 g/l of polysorbate 80. Prepare 0. make twofold serial dilutions from row B down the plate to row H by transferring 100 µl to the following well. The wells of row 11 are a positive control. Incubate at 37° for 2 h. A positive control serum and a negative control serum are included on each plate to monitor assay performance. 91 Flat-bottomed ELISA plates. Diluent buffer. for 30 min. Immediately before use add 5 µl of strong hydrogen peroxide solution. Wash thoroughly with washing buffer.2'-azinobis(3-ethylbenzothiazoline-6sulphonate) (ABTS) in 20 ml of citric acid solution. Optical density is measured and the antibody titre is calculated using the usual statistical methods. by transfer of 100 µI to the following well. Place 100 µl of reference guineapig tetanus antitoxin in the first well of a row.39 g of sodium hydrogen carbonate and 0. Sodium acetate buffer pH 5. Phosphate buffered saline pH 7. Tetramethylbenzidine solution. Reagents and equipment Round-bottomed. Peroxidaseconjugated equine anti-tetanus IgG is added followed by a peroxidase substrate. rigid polystyrene microtitre plates. Allow to stand overnight at 4° in a humid atmosphere. The substance dissolves within 30-40 min at room temperature.5. Read the plates at 405 nm in the same order as addition of substrate was made.2 g of anhydrous sodium acetate in 900 ml of water. Incubate in a humid atmosphere at 37° for 1 h. Place 100 µl of undiluted test sera in the first well of the required number of rows. Autoclave at 100° for 60 min. Add 100 µI of peroxidase substrate to each well. Block buffer. 2. wash the plates thoroughly with washing buffer. Coat the ELISA plates: immediately before use make . On the following day.67 ml of tetramethylbenzidine solution and 20 µI of strong hydrogen peroxide solution. To avoid interference from temperature gradient. adjust to pH 5. Block the plates by addition of 100 µl of diluent block buffer to each well.5 g of anhydrous sodium carbonate. the serum/ antigen mixtures are incubated overnight. Add 40 µl of PBS to the wells of column 12 (negative control). positive control serum to columns 2 and 3 and test sera to columns 4-12 and add 100 µI of each serum to the first 2 wells of the column to which it is allocated. positive control serum (from about 0. Dissolve 135. Carbonate buffer pH 9. Mix 90 ml of water. Peroxidase substrate. adjust to pH 9. Washing solution.2 (PBS). Peroxidase-conjugated equine anti-tetanus IgG.5.6. Method The description below is given as an example of a suitable plate lay-out but others may be used. Incubate in a humid atmosphere at 37° for 1 h.5 Lf/ml in carbonate coating buffer).5 g/l of polysorbate 80. Add 40 µl of this solution to all wells except those column 12. Prepare suitable dilutions of negative control serum. Clostridium tetani guinea-pig antiserum (for vaccines-human use) reference preparation. Using a multichannel micropipette. PBS containing 5 g/l of bovine albumin. Wash the plates thoroughly with washing solution. protected from light. Tetanus toxin or tetanus toxoid. Determination of antibody titre in guinea-pig serum by toxinor toxoid-binding inhibition (ToBI). Wells 1A-H are for negative control serum and wells 2A-H and 3A-H are for positive control serum for assay monitoring. Tap water containing 0.80 g of sodium dihydrogen phosphate monohydrate in water and dilute to 15 litres with the same solvent. Prepare a suitable dilution (a 1 in 2000 dilution has been found suitable) of peroxidase conjugate in diluent block buffer and add 100 µI to each well. the mixtures are transferred to an ELISA plate coated with tetanus antitoxin. 1. except those of row A.01 IU/ml) and test sera.2 g of sodium azide in 1000 ml of water. Allocate the negative control serum to column 1. Method Block the round-bottomed polystyrene microtitre plates by placing in each well 150 µI of block buffer.1 Lf/ml solution of tetanus toxin or toxoid using PBS a diluent. PBS containing 5 g/l of bovine albumin and 0. Equine anti-tetanus IgG. Place 100 µI of diluent block buffer in each well of the plates. Dissolve 1. Wells 4-12A-H are for test samples. Using a multi channel micropipette.IP 2007 TETANUS VACCINE (ADSORBED) diammonium 2. Cover the plates with a lid or sealer. To determine unbound toxin or toxoid. Discard 100 µl from the last row so that all wells contain 100 µI. make it two fold serial dilutions across the plate (up to column 10). Shake the plates gently and cover them with lids. Wash the plates thoroughly with washing buffer. Coat each well of the ELISA plates with 100 µl of tetanus toxoid solution (0. The production method shall have been shown to yield 92 Identification The single harvest is shown to contain tick-borne encephalitis virus by a suitable immunochemical method. (4) that the vaccine must be shaken before use. The colour changes from blue to yellow. Cover the plates with a lid. Block the plates by placing in each well 125 µl of block buffer. SEED LOT The strain of virus used is identified by historical records that include information on the origin of the strain and its subsequent manipulation. if tested. Add 100 µl of the dilution to each well and cover the plates with a lid. wash the ELISA plates thoroughly with washing solution. Stop the reaction at a given time (within 10 min) by the addition of 100 µl of 2 M sulphuric acid to each well in the same order as the addition of substrate. The virus in the final vaccine shall not have undergone more passages from the master seed lot than the virus in the vaccine used in clinical trials. Serum and trypsin used in the preparation of cell suspensions and media used must be shown to be free from extraneous agents. Wash the ELISA plates thoroughly with washing solution.4. Substrate for virus propagation The virus is propagated in chick embryo cells prepared from eggs derived from a chicken flock free from specified pathogens (2.7.5 h. Labelling. Cover the plates with lids.TICK-BORNE ENCEPHALITIS VACCINE (INACTIVATED) IP 2007 a suitable dilution of equine anti-tetanus IgG in carbonate buffer pH 9. Incubate at 37° in a humid atmosphere for 1. Virus seed lots are stored at or below -60°. would comply with the test for abnormal toxicity and immunogenicity. preferably using monoclonal antibodies. Make a suitable dilution (a 1 in 4000 dilution has been found suitable) of the peroxidaseconjugated equine anti-tetanus IgG in diluent buffer. At least 500 ml of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells). To avoid temperature gradient effects. Production General provisions The vaccine complies with the General Requirements of Vaccines for Human Use. (2) the minimum units per single human dose or the minimum International Units per single human dose if potency test done by challenge method or both. Production of the vaccine is based on a virus seed lot system. Transfer 100 µl of the preincubation mixture from the polystyrene plates to the corresponding wells of the ELISA plates. the tests in cell cultures are carried out in human and simian cells only. Tick-borne Encephalitis Vaccine (Inactivated) Tick-borne Encephalitis Vaccine (Inactivated) is a liquid preparation of a suitable strain of tick-borne encephalitis virus grown in cultures of chick-embryo cells or other suitable cell cultures and inactivated by a suitable. PROPAGATION AND HARVEST All processing of the cell cultures if performed under aseptic conditions in an area where no other cells are being handled. Add 100 µl of peroxidase substrate to each well.6 and add 100 µl to all wells. Measure the absorbance (2. starting with column 12 and then from 1 to 11. Each seed lot complies with the requirements for extraneous agents in viral vaccines for human use. (d) Any other validated serological assay in guinea-pigs/mice approved by National Regulatory Authority.7.7) at 450 nm immediately after addition of the sulphuric acid or maintain the plates in the dark until reading. validated method. Only a single harvest that complies with the following requirements may be used in the preparation of the inactivated harvest. Wash the plates thoroughly with washing solution. do not stack more than 4 plate high. Only a seed lot that complies with the following requirements may be used for virus propagation. (5) that the vaccine is not to be frozen. The cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. preferably using . The label states (1) the human dose (ml). Incubate at 37° in a humid atmosphere for 2 h. Incubate the plates at room temperature. On the following day. Identification Each seed lot is identified as containing the vaccine strain of tick-borne encephalitis virus by a suitable immunochemical method. (3) the name and the amount of the adsorbent and preservative. The virus concentration of each seed lot is determined by titration in suitable cell cultures to monitor consistency of production. A blue colour develops. consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. Virus concentration. Wash the ELISA plates thoroughly with washing solution.7) or in other suitable cell cultures. Incubate the 2 series of plates overnight in a humid atmosphere at 37°. The production method is validated to demonstrate that the product.3). Extraneous agents (2. Incubate at 37° in a humid atmosphere for 1 h. Maximum 1.2.03 ml intracerebrally into each of not fewer than ten mice about 4 weeks old. Free formaldehyde (2. Inoculate a quantity of the inactivated harvest equivalent to not less than ten human doses of vaccine in the final lot into primary chicken fibroblast cell cultures. Sterility ( 2. Residual infective virus.14).3). they may be omitted on the final lot. Sterility (2. Pyrogens (2.11). Determine the antigen content of the purified. Assay The potency is determined by comparing the dose necessary to protect a given proportion of mice against the effects of a lethal dose of tick-borne encephalitis virus. Determine the virus concentration by titration in suitable cell culture to monitor consistency of production. Control cells. Complies with the test for sterility. Tests Aluminium (2.8). Virus concentration. Complies with the test for pyrogens.9).2. Sterility (2. inactivated harvest that complies with the following requirements way be used in the preparation of final bulk vaccine.1 g/l.3.14). If the vaccine is produced using a cell-bank system.11). No cytopathic effect is detected at the end of the incubation period. 93 Specific activity. Bovine serum albumin. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more purified. Complies with the test for mycoplasmas carried out using 1 ml for each medium. per kg of body mass. as part of the validation studies. The control cells comply with the tests for extraneous agents (2.11). administered intraperitoneally. bovine serum albumin (where applicable) and pyrogens and the assay have been carried out satisfactory results on the final bulk vaccine.20). Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final lot. Incubate at 37 ± 1° for 14 days. if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Collect the culture fluid and inoculate 0. If formaldehyde is used for inactivation.4). Only a purified. inactivated harvests. Inject into each rabbit. determined by a suitable immunochemical method (2. preferably by continuous-flow.2. viral aggregates are removed by filtration immediately before the inactivation process.IP 2007 TICK-BORNE ENCEPHALITIS VACCINE (INACTIVATED) monoclonal antibodies. an inactivation curve is plotted representing residual live virus concentration measured on not fewer than three occasions. Tests and Assay may be released for use. the control cells comply with a test for identification. inactivated harvest by a suitable immunochemical method (2.2.11).2. Complies with the test for sterility carried out using 10 ml for each medium. Sterility (2. FINAL LOT Only a final lot that is satisfactory with respect to each of the requirements given below under Identification. Observe the mice for 14 days. carried out using 10 ml for each medium. Determine the total protein content by a suitable method.2. Identification The vaccine is shown to contain tick-borne encephalitis virus antigen by a suitable immunochemical method using specific antibodies or by the mouse immunogenicity test described under Assay. the method shall have been shown to be consistently capable of inactivating tick-borne encephalitis virus without destroying the antigenic and immunogenic activity.7.7. Purification Several inactivated single harvests may be pooled before concentration and purification by suitable methods. Complies with the test for sterility. one dose of vaccine.3. the vaccine contains not more than 50 ng per single human dose. They show no evidence of tick-borne encephalitis virus infection. sucrose density-gradient centrifugation. with the quantity of a reference preparation . Carry out test for sterility using 10 ml of bulk for each sterility medium.25 mg per single human dose. Inactivation To avoid interference. Only an inactivated harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. is within the limits approved for the specific product. Provided that the tests for free formaldehyde.2. If bovine serum albumin has been used during production. Maximum 0. the presence of an excess of free formaldehyde is verified at the end of the inactivation process. calculated as the antigen content per unit mass of protein. The specific activity. or by virus neutralization in cell cultures. or other cells shown to be at least as sensitive to tick-borne encephalitis virus with not less than 3 cm2 of cell sheet per ml of inoculum. Mycoplasma (2. The virus suspension is inactivated by a validated method. Determination of potency of the vaccine.2 ml of this virus suspension intraperitoneally into each vaccinated mouse. Tuberculin Purified Protein Derivative Tuberculin PPD.7). and inject intraperitoneally into each mouse 0. straw-coloured liquid. Phenol is not added to preparations that are to be freeze-dried. or dry cream-coloured powder. (2) for both the vaccine under examination and the reference preparation the 50.2 ml of the dilution allocated to its group. in order to comply with validity criteria four to five dilutions will usually be necessary.2 ml of the challenge suspension or the dilution allocated to its group. is separated by precipitation. Calculate the results by the usual statistical methods for an assay with quantal responses (5. one to each of the five groups of ten mice. Inject 0. (3) the statistical analysis shows a significant slope and no significant deviation from linearity and parallelism of the doseresponse lines. (4) the fiducial limits (P = 0.TUBERCULIN PURIFIED PROTEIN DERIVATIVE IP 2007 of tick-borne encephalitis vaccine necessary to provide the same protection. The active fraction in the filtrate. Allocate the challenge suspension and the four dilutions. Include all valid tests to estimate the 94 mean potency and the fiducial limits (P = 0.1 ml constitutes one intradermal dose containing appropriate number of Units.* * To ensure availability of a preparation of uniform potency. compute weighed means with the inverse of the squared standard error as weights. Use mice of the same sex or distribute males and females equally between groups. and a suitable stabiliser may be added. The test is not valid unless (1) the concentration of the challenge virus is not less than 100 LD50. Distribute the mice into not less than six groups of a suitable size to meet the requirements for validity of the test. Prepare dilutions such that the most concentrated suspension is expected to protect more than 50 per cent of the animals and the least concentrated suspension less than 50. Validity criteria. The preparation is free from mycobacteria. complies with the following requirements.95) for the mean potency. Use healthy mice weighing between 11 and 17 g derived from the same stock.2 ml. it should yield a ready-to-use preparation when reconstituted as per manufacturer’s instructions.Denmark as a powder to be reconstituted as stated on the label.0 per cent. Production It is prepared from the water-soluble fraction obtained by heating in free-flowing steam or in an autoclave and subsequently filtering cultures of the mycobacteria grown in a suitable liquid medium. . use not fewer than four groups of ten mice. prepare a series of not fewer than three dilutions of the challenge virus suspension at not greater than one-hundredfold intervals. or pellet. To verify the challenge dose. A colourless or pale. The final sterile product is distributed into sterile glass containers which are then sealed so as to prevent microbial contamination or alternatively it is freeze-dried and the containers subsequently sealed. reconstituted if necessary as stated on the label. for titration of the challenge suspension. Tuberculin Purified Protein Derivative for Human Use Tuberculin Purified Protein Derivative is a preparation made from the heat-treated products of growth and lysis of one or more strains of Mycobacterium tuberculosis that reveal delayed hypersensitivity in animals sensitised by a microorganism of the same species.0 per cent and not more than 300. it is in a ready-touse form and 0. Prepare not less than three suitable dilutions of the vaccine under examination and of the reference preparation. which is predominantly protein. The preparation.0 per cent protective dose (PD50) lies between the largest and smallest doses given to the mice. Seven days later make a second injection using the same dilution scale. such as 0. Potency requirement. The vaccine complies with the test if the estimated potency is not less than that approved by the competent authority. For this comparison an approved reference preparation and a suitable preparation of tick-borne encephalitis virus from an approved strain for use as the challenge preparation are necessary. If issued as a liquid. based on data from clinical efficacy trials. Allocate each dilution to a different group of mice and inject subcutaneously into each mouse 0. The preparation may be issued either as a sterile liquid or as a freeze-dried product. Tuberculin Purified Protein Derivative is produced and issued by the Statens Serum Institute . 14 days after the second injection prepare a suspension of the challenge virus containing not less than 100 LD50 in 0. Description. Calculations. An antimicrobial preservative that does not give rise to false positive reactions. (2) the type of cells used for production of the vaccine. The following is cited as an example of a method that has been found suitable for a given vaccine. The label states (1) the strain of virus used in preparation. washed and redissolved. If issued as a freezedried product.5 per cent w/v of phenol. Observe the animals for 21 days after the challenge and record the number of mice that die in the period between 7 and 21 days after the challenge.0 per cent of the estimated potency. Selection and distribution of test animals.95) are not less than 33. Labelling. Storage.5 to 7. No growth of mycobacteria should occur. B. When similar injections are administered to nonsensitised guinea-pigs no such reactions occur. It should not be allowed to freeze.36). Tuberculin Purified Protein Derivative in freeze-dried form complies with the following additional requirements. Inject 5.1 mg per ml of heat-inactivated. derivative with the dose of the appropriate Standard Preparation necessary to give the same effect. Not less than 1 month and not more than 6 months later carry out the following test : Shave the flanks to provide space for at least three reactions on each side. (4) the name and proportion of any added substances. Sensitising effect.24). at intervals of 5 days.0 ml intraperitoneally or subcutaneously into each of two guinea-pigs weighing between 300 and 400 g. Carry out tests for live mycobacteria in the preparation under examination using suitable culture media. Carry out the biological assay of tuberculin purified protein derivative described below. tuberculosis (supplied in ampoules containing 5. Biological assay The potency of tuberculin purified protein derivative is determined by comparing the dose necessary to reveal delayed hypersensitivity in guinea-pigs or other animals hypersensitised with mycobacteria of the same type as that used in the preparation of the tuberculin purified protein . When progressively increasing doses are injected intradermally into specifically sensitised guinea-pigs.IP 2007 TUBERCULIN PURIFIED PROTEIN DERIVATIVE Identification A. consisting of PPD derived from cultures of M. The label states (1) the number of Units per dose of 0. Kill the animals and carry out an autopsy.2. Live mycobacteria A. Dilute the preparations so that the lesions produced are 8 to 25 mm in diameter. Phenol (if present) (2.11).5 ml of a solution containing 1. Store in light-resistant containers at a temperature between 2° and 8°.000 Units) or another suitable preparation the potency of which has been determined in relation to the International Standard. Use phosphate-buffered saline pH 7. dried mycobacteria of the same type as that used in the preparation of tuberculin. (2) the total volume in the container (for liquid preparation). (5) the species or strain used. Two to three weeks after the third injection inoculate the same dose intradermally into these animals and into a control group of three guinea-pigs of the same weight but that have not had any previous injections of tuberculin. purified protein derivative (PPD). varying from erythema to necrosis.00. a dose of the preparation under examination containing about 500 Units in a volume of 0. weighing not less than 250 g and which have not previously been treated with any material which will interfere with the test.5 ml of a suspension in a suitable mineral oil with or without emulsifier and containing 0. Standard Preparation The Standard Preparation is the 1st International Standard for Tuberculin. Inject intradermally into each of three guinea-pigs three times.1 ml. B. (6) the storage conditions. No guinea-pig shows signs of infection with mycobacteria. Observe the animals for not less than 42 days. (3) the nature and quantity of the reconstituting liquid (for the freeze-dried preparation). The potency test described below serves as a test for identity if it is performed on material from the final containers. Sterility (2. The estimated potency is not less 80 per cent and not more than 125 per cent of the stated potency. the highest dose being about 10 times as strong as the lowest.3. (8) that care 95 Tests pH (2. (7) the date after which the contents are not intended to be used. No harmful effects are produced within 7 days.4 containing 0. in a Latin square design.005 per cent w/v of polysorbate 80 to prepare the dilutions of the Standard preparation and of the preparation under examination and to reconstitute freeze-dried preparations containing no stabiliser. Method Sensitise 12 guinea-pigs each weighing not less than 400 g by the intramuscular injection of a total of about 0.5 per cent w/v.00.1 or 0. Inject subcutaneously into 2 healthy guinea-pigs. Inject each dose intradermally in the same volume (0. Use at least three doses of the Standard preparation and at least three doses of the preparation under examination. The reactions of the two groups are not significantly different after 48 to 72 hours. Complies with the tests for sterility. Measure the diameters of the lesions after 24 to 48 hours and calculate the result of the test using standard statistical methods on the basis that the lesion diameters are directly proportional to the logarithm of the concentration of the tuberculin. reactions occur at the points of injection. but not more than a total of 12 injection sites per animal. Potency.4.000 Units per ml. Labelling. 6. established in 1951.5. The fiducial limits of error are not less than 64 per cent and not more than 156 per cent of the stated potency. Allocate the doses to the available sites in a random manner. Not more than 0.1 ml or per ml or per mg. Toxicity. mammalian. 0.2 ml) at the sites to which it has been allocated. Light blue. Live (Oral) Typhoid Vaccine (Live. transparent due to lysis of cells.0 per cent of galactose and bromoethymol blue. LIVE (ORAL) IP 2007 should be taken to avoid inhaling the powder (for the freezedried preparation).0. the strain does not contain Vi antigen. Production Choice of vaccine strain The main characteristic of the strains is the defect of the enzyme uridine diphosphate-galactose-4 epimerase. the growth conditions.16). Before carrying out the measurement. is 6. pH (2. Identification Culture bacteria are examined on an agar medium containing 1.8 to 7. It contains the flagellar H:d antigen and does not produce hydrogen sulphide on Kligler iron agar. . subcultured once and are then grown in a suitable medium containing 0. transparent due to lysis of cells. No yellow colonies (galactose-fermenting) should be found. Light blue. Serological characteristics Strain Ty 21a grown in a synthetic medium without galactose does not agglutinate to specific anti-O:9 antiserum. Strain Ty 21a agglutinates to H:d flagellar antiserum. The working seed lots represent not more than one subculture from the master seed lot. This property serves to distinguish Ty 21a from other galactose-epimerase-negative S. Only a master seed lot that complies with the following requirements may be used in the preparation of working seed lots. typhi strains. The final vaccine represents not more than four subcultures from the original vaccine on which were made the laboratory and clinical tests showing the strain to be suitable. the vaccine complies with the tests stated under Capsules. Whatever the growth conditions.0 per cent of the bacteria and to a water content shown to be favourable to the stability of the vaccine.0 per cent of galactose. Biochemical markers Strian Ty 21a does not produce hydrogen sulphide on Kligler iron agar. The harvest must be free from contaminating micro-organisms. galactokinase and galactose-1phosphate uridyl-transferase are reduced by 50 to 90 per cent. FREEZE-DRIED HARVEST The harvest is mixed with a suitable stabilizer and freeze-dried by a process that ensures the survival of at least 10.1 to 0. Only a single harvest that complies with the following requirements may be used for the preparation of the freezedried harvest. Strain Ty 21a grown in medium free of galactose shows only the rough (R) type of lipopolysaccharide. Cells of strain Ty 21a lyse if grown in the presence of 1. The activities of galactopermease. Only a freeze-dried harvest that complies with the following tests may be used for the preparation of the final bulk.4. no activity of the enzyme uridine diphosphate-galactose-4-epimerase is found in the cytoplasm of strain Ty 21a compared to strain Ty 2.5 is obtained and correct the reading to take account of the dilution. strain Ty 21a) is a freeze-dried preparation of the live Salmonella typhi strain Ty 21a grown in a suitable medium. Typhoid (Strain Ty 21a) Vaccine.5. should be found.24).0 per cent of galactose and bromothymol blue.0 per cent of galactose. measured at 546 nm. The strain agglutinates to anti-O:9 antiserum only if grown in medium containing galactose. strain Ty 21a does not agglutinate to Vi antiserum. PROPAGATION AND HARVEST The bacteria from the working seed lot are multiplied in a preculture. When presented in capsules.001 per cent of galactose at 30° for 13 to 15 hours. Optical density The optical density of the culture. SEED LOT The vaccine is prepared using a seed-lot system. dilute the culture so that a reading in the range 0.TYPHOID (STRAIN TY 21A) VACCINE. No yellow colonies (galactose-fermenting) should be found. No antimicrobial preservative is added to the vaccine. The strain is nonvirulent for mice. should be found. Galactose metabolism In a spectrophotometric assay. 6. Cell growth Strain Ty 21a cells lyse when grown in the presence of 1. Biosynthesis of lipopolysaccharide Lipopolysaccharides are extracted by the hot-phenol method and examined by size-exclusion chromatography (2. Whatever 96 Identification Culture bacteria on an agar medium containing 1. concave colonies.5 to 11. Oral.4. The bacteria are harvested. concave colonies. which may contain blood-group substances. Only a final lot that is satisfactory with respect to Identification.0 per cent of galactose and bromothymol blue. Number of live bacteria Carry out the test using not less than five dosage units. (b) the culture utilises glucose without production of gas.43). which is used to inoculate the final medium. Water (2. typhi. No pathogenic bacterium. (c) colonies on agar are oxidasenegative. Capsular polysaccharide is a partly 3-O-acetylated repeated units of 2-acetylamino-2-deoxy-D-galactopyranuronic acid with á-(1?4)-linkages.5 to 4. typhi used for the master seed lot shall be identified by historical records that include information on its origin and by its biochemical and serological characteristics. The liquid medium used and the final medium are semi-synthetic.9). Carry out the test using suitable selective media. and no Salmonella other than strain Ty 21a are found. typhi strain Ty 21a per gram. Only a final bulk that complies with the following requirement may be used in the preparation of the final lot. SEED LOT The strain of S. Not less than 40 × 109 live S. Tests Microbial contamination (2.0 per cent. incubate at 36 ± 1° for 20 to 36 hours. serological or molecular methods. Determine the total viable count using the plate-count method. typhi strain Ty 21a per gram. 11 Labelling. 1. The number of contaminating microorganisms per dosage unit is not greater than 102 bacteria and 20 fungi.2. Typhoid Polysaccharide Vaccine Typhoid Polysaccharide Vaccine is a preparation of purified Vi capsular polysaccharide obtained from Salmonella typhi Ty2 strain or some other suitable strain of known origin and history that has the capacity to produce Vi polysaccharide. Staphylococcus aureus. typhi Ty 21a bacteria per dosage unit. FINAL BULK VACCINE The final bulk vaccine is prepared by aseptically mixing one or more freeze-dried harvests with a suitable sterile excipient. Pseudomonas aeruginosa.0 per cent. Only a strain that has the following characteristics may be used in the preparation of the vaccine: (a) stained smears from a culture are typical of S. shall be demonstrated along with adequate documentation. Homogenise the contents of the dosage units in a 0. Tests and Number of live bacteria will be released for use. Immediately after homogenization prepare a suitable dilution of the suspension using cooled diluent and inoculate brain heart infusion agar. physicochemical.5 to 4. particularly Escherichia coli. the inoculum obtained is transferred to a liquid medium. if tested. FINAL LOT The final bulk vaccine is distributed under aseptic conditions into capsules with a gastro-resistant shell or into suitable containers.43). or a liquid medium. Light blue. Water (2. except that in the determination of number of live bacteria each dosage unit must contain not less than 4 x 109 live bacteria.4. (2) that the vaccine is for oral use only. The method of production shall have been shown to yield consistently Vi polysaccharide typhoid vaccines of adequate immunogenicity and safety in man. No yellow colonies (galactose-fermenting) should be found. and which have been characterized by suitable biochemical. determined by the semimicro determination of water or any other validated method. The production method is validated to demonstrate that the product. Production General provisions The production of Vi polysaccharide is based on a defined seed-lot system. Cultures from the working seed lot shall have the same characteristics as the strain that was used to prepare the master seed lot. would comply with the tests for abnormal toxicity. 1. . free from substances that are precipitated by cetrimonium bromide and do not contain blood-group substances or high-molecular-mass 97 Identification Culture bacteria from the vaccine under examination on an agar medium containing 1. Number of live bacteria. (d) a suspension of the culture agglutinates specifically with an appropriate Vi antiserum or colonies form haloes on an agar plate containing a suitable Vi antiserum.9 per cent w/v solution of sodium chloride at 4° using a mixer in a cold room with sufficient glass beads to emerge from the liquid.4. should be found.IP 2007 TYPHOID POLYSACCHARIDE VACCINE Number of live bacteria Not less than 1 x 10 live S. PROPAGATION AND HARVEST The working seed lot is cultured on a solid medium. The vaccine contains not less than 2 x 109 live S. The label states (1) the minimum number of live bacteria per dosage unit. concave colonies transparent due to lysis of cells. The polysaccharide is precipitated in its salt form and dried at 5+3°. Identification Carry out an identification test using a suitable immunochemical method (2.2.2.7. 0.24). Not more than 20 mg per gram of polysaccharide. Bacterial endotoxins (2.1). Examine by gel filtration or size-exclusion chromatography (2. Where applicable. calculated with reference to the dried substance.5 g/l. calculated with reference to the dried substance. Not less than 50 per cent of the polysaccharide is found in the pool containing fractions eluted before K0 = 0. determine the amount of antimicrobial preservative by a suitable chemical method.1).2. O-Acetyl groups (2. Use a column 0.1).5 ml. Determine O-acetyl groups on the two pools.11).1). 98 Identification Carry out an identification test using a suitable immunochemical method (2. using suitable procedures to eliminate successively nucleic acids.2. The content is not less than 85.0 per cent of the intended amount. after dissociation of the polysaccharide/cetrimonium bromide complex. Complies with the tests for sterility. Provided the tests for free formaldehyde and antimicrobial preservative have been carried out on the final bulk vaccine.2. FINAL LOT The final bulk vaccine is distributed and filled aseptically into sterile containers. the content is not more than 2.14).1).3). FINAL BULK VACCINE One or more batches of purified Vi polysaccharide are dissolved in a suitable solvent. The containers are closed so as to prevent contamination.5 ml.4. which may contain an antimicrobial preservative. Carry out test for sterility using 10 ml of bulk for each sterility medium. Molecular size. The culture is then inactivated at the beginning of the stationary phase by the addition of formaldehyde.5 to 7. Determine by a suitable method.2.7. O-Acetyl groups (2.085 (± 25 per cent) µmol per dose (25 µg of polysaccharide). The vaccine contains minimum of 25 µg purified ViPolysaccharide per dose of 0. . Determine the point corresponding to K0 = 0.11). pH (2.16) using cross-linked agarose for chromatography.7. they may be omitted on the final lot.0 per cent and not greater than 115.7. Nucleic acids (2. The loss on drying is determined by thermogravimetry. Tests and Assay and with the requirements for Bacterial endotoxins may be released for use. the content should not be more than 150 IU per mg of polysaccharide. unless it has been demonstrated that they are removed by the purification process.0 to 7. If phenol has been used in the preparation. the powder obtained constitutes the purified Vi polysaccharide.5.4.25 and make two pools consisting of fractions eluted before and after this point. Bacterial cells are eliminated by centrifugation.5. the polysaccharide is precipitated from the culture medium by addition of hexadecyltrimethylammonium bromide (cetrimonium bromide). so that the volume corresponding to one dose contains 25 µg of polysaccharide and the solution is isotonic with blood (250 mosm/kg to 350 mosm/kg). Not less than 2 mmol per gram of polysaccharide. proteins and lipopolysaccharides. The precipitate is harvested and may be stored at or below -20° before purification. Test solution.TYPHOID POLYSACCHARIDE VACCINE IP 2007 polysaccharides.2 mol per kg and a pH of 7. Not more than 10 mg per gram of polysaccharide.9 m long and 16 mm in internal diameter equilibrated with a solvent having an ionic strength of 0. Only a final lot that is satisfactory with respect to each of the requirements prescribed below under Identification. Abnormal toxicity (2. Apply about 5 mg of polysaccharide in a volume of 1 ml to the column and elute at about 20 ml/h. Sterility (2. The bacterial purity of the culture is verified by microscopic examination of Gramstained smears and by inoculation into appropriate media. 6. Complies with the test for abnormal toxicity. Karl Fischer or any other suitable method and is used to calculate the results of the chemical tests shown below with reference to the dried substance. Only those pools of purified Vi polysaccharide that comply with the following requirements may be used in the preparation of the final bulk: Protein (2. Collect fractions of about 2.14). Place 3 ml of the vaccine in each of three tubes (two reaction solutions and one correction solution).25. Tests Sterility (2. Purified Vi Polysaccharide The polysaccharide is purified. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. calculated with reference to the dried substance. Make up the volume in each tube to 3 ml with water. The vaccine is reconstituted as stated on the label to give a uniform suspension containing not less than 5 x 108 and not more than 1 x 109 bacteria per human dose. If phenol has been used in the preparation.150 g of acetylcholine chloride in 10 ml of water (stock solution containing 15 g/l of acetylcholine chloride).2M hydrochloric acid to each tube. typhi such as.0 per cent of the intended amount. The human dose does not exceed 1. The bacteria are inactivated by acetone.3. if tested. Antimicrobial preservative. Labelling. Add 0.2.2.5 ml. anti-Vi agglutinins.5 ml of the working dilution.0 per cent to 120. Prepare a blank using 3 ml of purified water. 1.5 ml of a 20 per cent w/vl solution of ferric chloride in 0.0 per cent.14) using a reference purified polysaccharide. The label states (1) the number of micrograms of polysaccharide per human dose (25 µg). subtract the absorbance of the corresponding correction solution. The bacteria are inactivated either by acetone or by .0 per cent and not greater than 115. For each reaction solution. by formaldehyde. If phenol has been used in the preparation. stopper the tubes and shake vigorously to remove bubbles. Draw a calibration curve from the corrected absorbance for the five reference solutions and the corresponding content of acetylcholine chloride and read from the curve the content of acetylcholine chloride in the test solution for each volume tested. to a lesser extent.1) modified to the extent that 0.0 ml. Dissolve 0.0 per cent and not more than 120. Add 1.0 ml of alkaline hydroxylamine solution to each tube. 99 Typhoid Vaccine Typhoid Vaccine is a sterile suspension of inactivated Salmonella typhi containing not less than 5 x 108 and not more than 1 x 109 bacteria per human dose. The label states (1) the method used to inactivate the bacteria. When injected into susceptible laboratory animals. Ty 2. Identification It is identified by specific agglutination. Free formaldehyde (2. The final vaccine represents not more than 3 subcultures from the strain on which were made the laboratory and clinical tests that showed it to be suitable.5 ml of a mixture of 1 volume of water and 2 volumes of dilute hydrochloric acid to each of the reaction tubes. (2) the number of bacteria per human dose.2 g/l. 1 mol of acetylcholine chloride (181. Production The vaccine is prepared using a seed-lot system from a suitable strain of S. Calculate the mean of the two values. determine the amount of antimicrobial preservative by a suitable chemical method. The human dose does not exceed 1.11).0 per cent of the content stated on the label. Allow the reaction to proceed for exactly 2 min and add 0. anti-H and. Immediately before use. 0.5 per cent w/v. would comply with the test for abnormal toxicity (2. Measure the absorbance of each solution at 540 nm using the blank as the compensation liquid.5 ml of a mixture of 1 volume of water and 2 volumes of dilute hydrochloric acid to each of the correction tubes and to the blank. such as Ty 2.0 ml into each guineapig.2. Phenol (2. Antigenic power.0 ml and 1. The content is not less than 85. by phenol or by heating or by a combination of the last two methods.05 g). Maximum 0. dilute 0. the content is not more than 2. The fiducial limits of error (P = 0.20). place in duplicate (reaction and correction solutions) 0. (2) the total quantity of polysaccharide in the container.5 ml of the stock solution to 50 ml with water (working dilution containing 150 µg/ml of acetylcholine chloride). 0. The estimated amount of polysaccharide per dose is 80.IP 2007 TYPHOID VACCINE (FREEZE DRIED) Reference solutions.7 g) is equivalent to 1 mol of O-acetyl (43.2 ml.3. Labelling.5 ml of the vaccine is injected subcutaneously or intramuscularly or intraperitoneally into each mouse and 1. it elicits anti-O. The final vaccine represents not more than 3 subcultures from the strain on which were made the laboratory and clinical tests that showed it to be suitable. The production method is validated to demonstrate that the product. Production The vaccine is prepared from a seed-lot system from a suitable strain of S.0 ml of the reconstituted vaccine. Where applicable. Typhoid Vaccine (Freeze Dried) Freeze Dried Typhoid Vaccine is a freeze-dried preparation of inactivated Salmonella typhi. the concentration is not more than 0. Assay Determine Vi polysaccharide content by a suitable immunochemical method (2.95) of the estimated amount of polysaccharide are not less than 80.5 g/l.36). Add 0.1 ml. Sterility (2. typhi. Complies with the test for sterility. In ten tubes. The cell suspension is disrupted by sonication.0 per cent. SEED LOT The strain of varicella virus shall be identified as being suitable by historical records which shall include information on the Identification The virus harvest contains virus that is identified as varicella virus by serum neutralisation in cell culture. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. Only a virus seed lot that complies with the following requirements may be used for virus propagation.5 ml of the vaccine is injected subcutaneously or intramuscularly or intraperitoneally into each mouse and 1. Antigenic power. virus seed lots are prepared in large quantities and stored at temperatures below -20°. The vaccine is distributed into sterile containers and freezedried to a moisture content favourable to the stability of the vaccine. The virus in the final vaccine shall not have been passaged in cell cultures beyond the 38th passage from the original isolated virus. if tested. Extraneous agents (2. The production method is validated to demonstrate that the product. would comply with the test for safety and efficacy. if freeze-dried. released from the support surface and pooled. Approved animal (but not human) serum may be used in the media. would comply with test for abnormal toxicity (2.0 ml into each guineapig. Neurovirulence (2. Identification The vaccine reconstituted as stated on the label is identified by specific agglutination. Only a virus harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine.2. Virus concentration. of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells). anti-H and. 5.2. Seed lots are prepared in the same kind of cells as those used for the production of the final vaccine. anti-Vi agglutinins. origin of the strain and its subsequent manipulation. Substrate for virus propagation The virus is propagated in human diploid cells (2. Complies with the test for neurovirulence of live virus vaccines. using specific antibodies. Phenol is not used in the preparation. Live Varicella Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of Herpesvirus varicellae.7. LIVE IP 2007 formaldehyde or by heat.5). The production method shall have been shown to yield consistently live varicella vaccines of adequate immunogenicity and safety in man. Identification The master and working seed lots are identified as varicella virus by serum neutralisation in cell culture. (2) the number of bacteria per human dose.1).11). (3) that the vaccine should be used within 8 hours of reconstitution. Varicella Vaccine. Labelling. Virus concentration. Not more than 5.0 percent.VARICELLA VACCINE.2). To avoid the unnecessary use of monkeys in the test for neurovirulence. The concentration of infective virus in virus harvests is determined as prescribed under assay to monitor consistency of production and to determine the 100 . The reconstituted vaccine complies with the tests for sterility. Water. using specific antibodies. modified to the extent that 0.3). The production method is validated to demonstrate that the product.7. the reconstituted vaccine elicits anti-O. if not freeze-dried. The virus concentration of the master and working seed lots is determined as prescribed under Assay to monitor consistency of production. The cell culture medium may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. Sterility (2. The label states (1) the method used to inactivate the bacteria.7. but not less than 50. Complies with the requirements for seed lots for live virus vaccines. Production General provisions The production of vaccine is based on a virus seed-lot system and a cell-bank system. The virus shall at no time have been passaged in continuous cell lines. The infected cells constituting a single harvest are washed. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from extraneous agents. if tested. to a lesser extent. It is preferable to have a substrate free from antibiotics during production. a sample of 50 ml is taken for the test in cell cultures. When injected into susceptible laboratory animals.0 ml. or below -60°. Complies with the test for abnormal toxicity. Inject 0.2. it is no longer able to infect susceptible cell cultures.6 mg with 1 ml of polyvalent antisnake venom serum and incubate the mixture at 37o for 30 minutes. Bovine serum albumin.2. These solutions are then distributed in single dose sterile glass containers. The containers are then closed so as to prevent contamination and the introduction of moisture.5 ml of the mixture intravenously into a group of mice weighing between 18 and 20 g. Inject 0. Description. mixed. Production Immediately after extraction. Further dilutions of the stock solution are made with water for injection under aseptic conditions to give solutions with the required number of Mouse Units per ml. Labelling.0 per cent. The virus concentration is not less than the minimum stated on the label. dissolved in ice-cold water for injection and then filtered through a bacteria-proof filter to give a stock solution. Use a suitable virus reference preparation to validate each assay. Tests and Assay may be released for use. Viper Venom contains not less than 50 Mouse Units per mg.75 kPa. Not more than 3. Identification A.3). Assay Titrate for infective virus. Abnormal toxicity (2. Observe the animals for 24 hours. Assay. dry powder which when mixed with water yields a clear solution with some insoluble residue. (3) that contact with disinfectants is to be avoided.11) Complies with the test for sterility. Viperidae).7. tamper-proof containers and freeze-dried to a moisture content shown to be favourable to the stability of the vaccine.2. The control cells of the production cell culture from which the single harvest is derived comply with a test for identity and with the requirements for extraneous agents (2. (5) that the vaccine is not to be administered to pregnant women. Water (2.11). Only a final lot that is satisfactory with respect to each of the requirements given below under Identification. Complies with tests for sterility. FINAL BULK VACCINE Virus harvests that comply with the above tests are pooled and clarified to remove cells. Identification When the vaccine reconstituted as stated on the label is mixed with specific Herpesvirus varicellae antibodies.43). Tests Sterility (2. A suitable stabiliser may be added and the pooled harvests diluted as appropriate. Carry out the biological assay of snake venom described below: Biological assay of snake venom Dissolve a quantity of the freeze-dried venom equivalent to 50 Mouse Units in 25 ml of saline solution.3). (2) the type and origin of the cells used for the preparation of the vaccine. Produces almost immediate coagulation of blood and citrated human plasma. Tests Sterility (2.2.5 µg per human dose. it may be omitted on the final lot. Viper Venom Daboia Venom Viper Venom is the dried secretion obtained from the poison glands of Viperae russelli and other species of Viperae (Fam. Carry out test for sterility using 10 ml of bulk for each sterility medium. (6) the time within which the vaccine must be used after reconstitution. using at least ten cell cultures for each fourfold dilution or by a technique of equal precision. Extraneous agents (2. The label states (1) the strain of virus used for the preparation of the vaccine. Sterility (2. dried from the frozen state and sealed at a pressure not exceeding 2. Mix the soluble fraction from at least 0. B.1).3. FINAL LOT The final bulk vaccine is distributed aseptically into sterile. An almost white or very light yellow. Use 50 ml for the test in cell cultures. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. determined by a suitable immunochemical method (2. determined by the semi-micro determination of water. Not more than 0. Control cells.IP 2007 VIPER VENOM dilution to be used for the final bulk vaccine. no animal dies. The dried venom is pooled.7. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine.5 ml 101 . the poisonous secretion is dried from the frozen state. (4) the minimum virus concentration.14).2.11). (2) the volume of water for injection to be used for reconstitution.3). Complies with the tests for extraneous agents (2. The production method shall have been shown to yield consistently the yellow fever vaccine (live) of acceptable immunogenicity and safety for man. If stabilisers are added. Control eggs. Extraneous agents (2. Master and working seed lots shall not contain any human protein or added serum. Express the result in terms of number of Mouse Units per mg. Storage. The label states (1) the number of Mouse Units per container. Reference preparation. Only a single harvest that complies with the following tests may be used in the preparation of the final bulk vaccine.7. Substrate for virus propagation Virus for the preparation of master and working seed lots and for all vaccine batches is grown in the tissues of chick embryos from a flock free from specified pathogens (2. A test for protein nitrogen 102 .3). light-resistant containers. In the test for neurotropism. the virus in the final vaccine shall be between passage levels 204 and 239 from the original isolate of strain 17D. using specific antibodies. Virus seed lots are prepared in large quantities and stored at a temperature below -60°. A working seed lot shall be used without intervening passage as the inoculum for infecting the tissues used in the production of a vaccine lot. Two per cent but not less than twenty and not more than fifty eggs are set aside as uninfected control eggs. The age of the embryo at the time of virus harvest is reckoned from the initial introduction of the egg into the incubator and shall not be more than 12 days. NOTE — The quantity in mg of the venom which will kill in 2 to 24 hours not less than 3 and not more than 8 mice represents one Mouse Unit. change the dilution of the venom suitably. the extract of embryonic pulp is tested as described below and kept at -70° or colder until further processing. FINAL BULK VACCINE Single harvests that comply with the tests prescribed above are pooled and clarified again.7. so that no vaccine virus is more than one passage from a seed lot that has passed all the safety tests. each single harvest is titrated as described under Assay. a suitable batch of vaccine known to have satisfactory properties in man is used as the reference preparation. if tested. Labelling. Unless otherwise justified and authorised. be only one passage from a master seed lot. using specific antibodies. Virus harvests that comply with the prescribed tests may be pooled. Extraneous agents (2. Identification The single harvest contains virus that is identified as yellow fever virus by serum neutralisation in cell culture. Virus concentration. A working seed lot shall All processing of the fertilised eggs is done under aseptic conditions in an area where no other infectious agents or cells are handled at the same time. Complies with the tests for extraneous agents. Production General provisions The production of vaccine is based on a virus seed-lot system. Store in single dose. After inoculation and incubation at a controlled temperature.YELLOW FEVER VACCINE (LIVE) IP 2007 intravenously into each of 10 mice weighing between 18 and 20 g and observe the animals for 24 hours. After homogenisation and clarification by centrifugation.3). Each working seed lot complies with the test for extraneous agents. SEED LOT The 17D strain shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. only living and typical chick embryos are harvested. Identification The master and working seed lots are identified as containing yellow fever virus by serum neutralisation in cell culture. PROPAGATION AND HARVEST Yellow Fever Vaccine (Live) Yellow Fever Vaccine (Live) is a freeze-dried preparation of the 17D strain of yellow fever virus grown in fertilised hen eggs. Not less than 3 and not more than 8 of the mice die in 2 to 24 hours. The production method is validated to demonstrate that the product. Only a virus seed lot that complies with the following requirements may be used for virus propagation.7. they shall have been shown to have no antigenic or sensitising properties for man. If the number of deaths is not within this range. would comply with the test for safety and efficacy.7). In order to calculate the dilution for formulation of the final bulk.7. No human protein is added to the virus suspension at any stage during production. or any other diluent that has been shown to be equivalent for maintaining the infectivity of the virus).000 mouse LD50. Bacterial endotoxins (2. Tests Sterility (2.3. The method shown below. Viraemia (Viscerotropism). Protein nitrogen content (2. Thermal stability. The relationship between mouse LD50 and TCID50 or PFU is established by each laboratory and approved by the competent authority. Mice paralysed on the twenty-first day of observation are counted as survivors.0 per cent. they shall not have been inoculated by other routes with neurotropic viruses or with antigens related to yellow fever virus. Complies with the test for sterility. The virus concentration is not less than the equivalent in TCID50 or PFU of 1 × 103 mouse LD50 per human dose.43). Only a final bulk vaccine that complies with the following tests may be used in the preparation of the final lot. Inject the test dose into one frontal lobe of each monkey under anaesthesia and observe the monkeys for not less than 30 days.11). are injected intracerebrally under anaesthesia with 0. Take blood from each of the test monkeys on the second. Mouse LD50. Sterility (2.IP 2007 YELLOW FEVER VACCINE (LIVE) content is carried out.1).2.3. The statistically calculated quantity of virus suspension that is expected to produce fatal specific encephalitis in 50 per cent of mice of a highly susceptible strain. fourth and sixth days after Identification When the vaccine reconstituted as stated on the label is mixed with specific yellow fever virus antibodies. Assay Titrate for infective virus in cell cultures. The monkeys shall be Macaca spp. there is a significant reduction in its ability to infect susceptible cell cultures. determined by a suitable immunochemical method (2. the series of dilutions is chosen so as to cover the range 0 to 100. Groups of not less than eight mice are used for each dilution. immunogenicity and neurotropism. Tests and Assay may be released for use. Only a final lot that is satisfactory with respect to thermal stability and each of the tests given under Identification. Carry out test for sterility using 10 ml of bulk for each sterility medium. Provided that the test for ovalbumin has been performed with satisfactory results on the final bulk vaccine.0 per cent mortality of the mice. Furthermore.30). Complies with the test for abnormal toxicity. before the addition of any stabiliser. or another suitable technique. They shall be healthy and shall not have received previously intracerebral or intraspinal inoculation.03 ml of the vaccine dilution.25 ml containing the equivalent of not less than 5000 mouse LD50 and not more than 50. determined by a titration for infectious virus and using the established equivalence between virus concentration and mouse LD50 (see under Assay). A suitable stabiliser may be added and the pooled harvests diluted as appropriate.2.11). Abnormal toxicity (2. Viscerotropism is indicated by the amount of virus present in serum. 103 . The difference in the virus concentration between unheated and heated vaccine does not exceed 1. Maintain samples of the final lot of freezedried vaccine in the dry state at 37° for 14 days. susceptible to yellow fever virus and shall have been shown to be non-immune to yellow fever at the time of injecting the seed virus.14).2. and the virus concentration of the heated vaccine is not less than the number of TCID50 or plaque-forming units (PFU) equivalent to 1 × 103 mouse LD50 per human dose. Not more than 5 IU of bacterial endotoxin per human dose. determined by the semi-micro determination of water. Not fewer than ten monkeys are used for each test. Water (2. Mice of a highly susceptible strain.75 per cent solution of bovine albumin in phosphate-buffered saline pH 7. is not more than 0. 4 to 6 weeks of age.2. it may be omitted on the final lot. 4 to 6 weeks of age. Not more than 5 µg of ovalbumin per human dose. Use an appropriate virus reference preparation to validate each assay. Not more than 3. may be used to determine the mouse LD50.4. The protein nitrogen content. Tests in monkeys for Yellow Fever Vaccine Each master and working seed lot complies with the following tests in monkeys for viraemia (viscerotropism).0 log10.2. tamper-proof containers and freeze-dried to a moisture content shown to be favourable to the stability of the vaccine. The containers are then closed so as to prevent contamination and the introduction of moisture. Only survivors and deaths caused by typical yellow fever infections are counted in the computations. Ovalbumin. Appropriate serial dilutions of the reconstituted vaccine are made in diluent for yellow fever virus (0. Injection of the mice is performed immediately after the dilutions have been made. FINAL LOT The final bulk vaccine is distributed aseptically into sterile. The mice are observed for 21 days and all deaths are recorded. Use a test dose of 0.3). Determine the virus concentration as described under Assay in parallel for the heated vaccine and for unheated vaccine.25 mg per human dose. after intracerebral inoculation. 03 ml and at most one serum contains more than the equivalent of 100 mouse LD50 in 0.YELLOW FEVER VACCINE (LIVE) IP 2007 inoculation and prepare serum from each sample. Moderate: 4 or more focal inflammatory infiltrates. Histological evaluation. Degeneration or loss of neurons affecting not more than one third of cells.0 per cent at the 1:10 dilution.2 ml of each serum-virus mixture to each of four cell-culture plates and proceed as for the determination of virus concentration. Cervical and lumbar enlargements of the spinal cord are each divided equally into six blocks. Severe: moderate focal or diffuse inflammatory infiltration. unco-ordinated. slow moving. Block IV — the pons and cerebellum at the level of the superior olives. tremors. shaky. high-pitched voice. such as paralysis or inability to stand when stimulated. Clinical evaluation. The seed lot complies with the test if at least 90. Minimal: 1 to 3 small focal inflammatory infiltrates. — the thalamus at the level of the mamillary bodies. Inoculate similarly ten plates with the same amount of virus plus an equal volume of a 1:10 dilution of monkey serum known to contain no neutralising antibodies to yellow fever virus. 1:100 and 1:1000 dilutions from each serum and inoculate each dilution into a group of at least six cell culture vessels used for the determination of the virus concentration. such as paresis. compare the mean number of plaques in the plates receiving virus plus non-immune serum with the mean number of plaques in the plates receiving virus plus dilutions of each monkey serum. 1:40 and 1: 160 of serum from each monkey with an equal volume of 17D vaccine virus at a dilution that will yield an optimum number of plaques with the titration method used. the monkeys are removed from their cage and examined for signs of motor weakness or spasticity). Take blood from each monkey 30 days after the injection of the test dose and prepare serum from each sample.95) than the mean for the group of monkeys injected with the reference preparation. Grade 2 — Grade 3 — Grade 4 — A clinical score for a particular monkey is the average of its daily scores. 15 µm sections are cut from the tissue blocks embedded in paraffin wax and stained with gallocyanin. The seed lot is not acceptable if in the monkeys injected with it the incidence of severe signs of encephalitis. lethargy. Block III — the mesencephalon at the level of the superior colliculi.03 ml. Overwhelming: variable but often severe inflammatory reaction. inability to stand. limb weakness. It has been found that inoculation of yellow fever vaccine into the monkey brain causes histological lesions in different 104 . inactive. Grade 4. Neurotropism. The seed lot is not acceptable if either the onset and duration of the febrile reaction or the clinical signs of encephalitis and pathological findings are such as to indicate a change in the properties of the virus. 1:10) may contain non-specific inhibitors that influence this test. These and other signs of encephalitis. as determined by examining their sera in the test for neutralisation of yellow fever virus described below. Block V — the medulla oblongata and cerebellum at the level of the mid-inferior olivary nuclei. The monkeys are examined daily for 30 days by personnel familiar with clinical signs of encephalitis in primates (if necessary. It has been shown that a low dilution of serum (for example. The seed lot complies with the test if none of the sera contains more than the equivalent of 500 mouse LD50 in 0. Lesions are scored as follows: Grade 1. in comparison with ten monkeys injected with the reference preparation. tremors or spasticity are assigned numerical values for the severity of symptoms by a grading method. Each day each monkey in the test is given a score based on the scale: Grade 1 — rough coat. or mortality is greater than for the reference vaccine. limb paralysis or death (a dead monkey receives a daily score of 4 from the day of death until day 30). Grade 2.0 per cent of neurons. Degeneration or loss of up to two third of the neurons. special consideration is given to any animal showing unusually severe signs when deciding on the acceptability of the seed lot. The seed lot is not acceptable if the mean of the clinical severity scores for the group of monkeys inoculated with it is significantly greater (P = 0. Five levels of the brain are examined including : Block 1 Block II — the corpus striatum at the level of the optic chiasma. Neurotropism is assessed from clinical evidence of encephalitis. not eating. the clinical score for the seed lot is the mean of the individual monkey scores. In addition. such serum shall be treated to remove inhibitors. At the end of the observation period. Incubate the serum-virus mixtures in a waterbath at 37° for 1 h and then cool in iced water. from incidence of clinical manifestations and by evaluation of histological lesions. Not more than 10 per cent of the test monkeys have serum that fails to reduce the number of plaques by 50. Mix dilutions of at least 1: 10. Degeneration or loss of more than 90. in-coordination.0 per cent of the test monkeys are shown to be immune. add 0. Degeneration or loss of a few neurons. Numerical scores are given to each hemisection of the cord and to structures in each hemisection of the brain as listed below. Prepare 1:10. Immunogenicity. Grade 3. Type of monkey Discriminator areas Target areas substantia nigra Macaca cynomolgus globus pallidus putamen anterior/ median thalamic nucleus lateral thalamic nucleus Macaca rhesus caudate nucleus substantia nigra globus pallidus cervical putamen anterior/ lumbar median thalamic enlargement nucleus lateral thalamic nucleus cervical enlargement lumbar enlargement enlargement Scores for discriminator and target areas are used for the final evaluation of the seed lot. S. (4) that contact 105 .IP 2007 YELLOW FEVER VACCINE (LIVE) anatomical formations of the central nervous system with varying frequency and severity (I.95) than for the reference preparation. The discriminator and target areas for monkey. 15. 305-313).. Journal of Biological Standardization. spared and discriminator areas. Labelling. The individual monkey score is calculated from the sum of individual target area scores in each hemisection divided by the number of areas examined. 1987. The seed lot is not acceptable if either of the mean lesion scores is significantly greater (P = 0. Discriminator areas are those where there is a significant increase in the frequency of more severe specific lesions with seed lots having a higher degree of neurovirulence. Spared areas are those which show only minimal specific lesions and in a minority of monkeys. Based on these two indicators. (3) the minimum virus concentration. Discriminator and target areas for Macaca cynomolgus and Macaca rhesus monkeys are shown in the table below: Table 1. (2) that the vaccine has been prepared in chick embryos. These two mean scores are taken into account when deciding on the acceptability of the seed lot. Levenbook et al. A separate score is calculated similarly for the discriminator areas. Target areas are those which show more severe specific lesions in a majority of monkeys irrespective of the degree of neurovirulence of the seed lot. Mean scores for the test group are calculated in two ways: (1) by dividing the sum of the individual monkey discriminator scores by the number of monkeys and (2) by dividing the sum of the individual monkey target and discriminator scores by the number of monkeys. the anatomical structures can be divided into target. The label states (1) the strain of virus used in preparation.