DATA AND REVIEWAseptic Production, Gowning Systems, and Airborne Contaminants Bengt Ljungqvist and Berit Reinmüller* B Using a modified dispersal chamber, the authors have studied the protective efficacy of cleanroom clothing systems. Study results show that the state of a cleanroom clothing system—new or much used—influences the protection efficacy of the system. Suitable combinations of cleanroom underwear and cleanroom garments also improve the protection of the clean environment against airborne contaminants from people. ecause the production of sterile products for global sale requ i res manu f actu ring conditions that comply with inc re a s i n gly stri ct nati onal and intern a ti onal requ i rem en t s of cleanliness and documen t a ti on , s terile dru gs today are manufactured in classified cleanrooms. In the In tern a ti onal Orga n i z a ti on for Standard s’ (ISO) stand a rd 14644-1, “Cl e a n rooms and As s oc i a ted Con tro ll ed Environments,” a cleanroom is defined as, “A room in wh i ch the con cen trati on of airborne parti cles is controlled, and which is constructed and used in a manner to m i n i m i ze the introdu cti on , gen era ti on , and reten ti on of particles inside the room and in wh i ch other rel evant para m eters , e.g., tem perature, hu m i d i ty, and pressure, are con tro ll ed as necessary.” (1) Bengt Ljungqvist, PhD, is a professor of safety ventilation and head of the Department of Civil and Architectural Engineering, and Berit Reinmüller, PhD, is a senior researcher in safety ventilation, Building Services Engineering, in the Department of Civil and Architectural Engineering, both at KTH, The Royal Institute of Technology, Brinellvägen 34, SE-100 44 Stockholm, Sweden, tel. 146 (0) 8 790 75 37, fax 146 (0) 8 411 84 32,
[email protected]. *To whom all correspondence should be addressed. As technical solutions have improved , c u rrent good manufactu ring practice (CGMP) requ i rem ents rega rding manu f acturing conditions (e.g., the total number and vi a ble number of a i rb e orn p a rti c u l a tes) for sterile dru gs have become more stri n gen t . Th e va rious internati onal GMP requ i rem en t s , however, have not yet been fully harmonized . For pharmaceutical cleanrooms, the most commonly used guidelines and requirem ents are: • The US Food and Drug Administration’s CGMPs , which apply to products sold in the United States; • The European Union’s GMP Annex 1, wh i ch applies to products sold in the European Union (EU). An n ex 1 of the Eu ropean Commission (EC) Guide to Good Ma nu f a cturing Practi ce provi des su pp l em en t a ry guidance rega rding the app l i c a ti onof the principles and guidance of GMPs to sterile produ cts (2). The guidance inclu des recom m en d ations for standards of envi ron m ental cleanliness for cleanrooms. Ta ble I lists, as of January 2005, the maximum perm i t ted or recommended number of vi a ble particles in cleanrooms according to EU An n ex I, FDA GMPs, and the US Pharmacopeia (USP 2 4–NF 19) (2, 4, 5). Nei t h er ISO 14644-1 nor 14698-1 def i n e s the nu m ber of vi a ble airborne parti cles permitted in a cl a s s i f i ed cleanroom or assoc i a ted con tro ll ed envi ron m ent (1, 3). To avoid contaminati on ori gi n a ting from the process, a ll materials (ingredients and products) transferred into clean aseptic areas must be sterile-filtered , a utoclaved , or cl e a n ed and steri l i zed in some other way. In ad d i ti on , pers on n el must en ter the cleanroom through appropriate changing rooms. w w w. p h a rm t e ch . c o m s30 Pharmaceutical Technology ASEPTIC PROCESSING 2005 Cleanroom clothing The primary purpose of cl e a n ro clothing is to proom tect produ cts and the processing environ m ent from airborne contamination. According to the requ i rem ents for current good manu f actu ring practi ces (CGMPs). 8).only a minimal nu m ber of particles and be resistant to wear and era ting mach i n ery. and sterilizing. clean and Tod ay ’s aseptic cl e a n ro clothing consists of a com bi n aom sterile protective garments. the main ti cle reten ti on . should not work in an aseptic cleanroom. we a r. hood s . c o m .000 100 of the same model made by a single manu f acClass 100. including sterile gloves and masks. Thus. because of f actors su ch as agi n g. According to Akers. ing. US Customary 10 10. The two reusable cl e a nControlled area.tear. Com p a rom Background to critical ative studies have been performed in the disarea. parti cle filtra tion. and must be provided for each working session. a ll pers on n el en tering an aseptic producti on area must be qualified with rega rd to gowning tech n i ques. contaminants from being dispers ed into a cl e a n ro . p h a rm t e ch . during aseptic produ cti on . particle particle particle Grade Area customary At KTH (the Royal Insti tute of Tech n o l ogy ) count count count class (cfu/m3) (cfu/m3) (cfu/m3) in Sweden.000 tu rer. For reu s a ble parts. Sep a ra te laundry fac i l i ties for su ch cl o t h. a modified dispersal chamber has . Most tests are conducted on new material and stu dy particle EU GMP Annex 1 FDA 2004 (4) USP 24–NF 19 (5) gen era ti on . Within a cl e a n room. borne parti cles. Peop l e with skin probl em s . People within the asepti c produ ction and the cri tical areas are frequ en t ly mon itored by a com bi n a ti on of sampling methods. clean. and sterile (sterilized or ad. f ace masks. and goggles. opera tors should however. Cleanroom cloth. and pore size . cl e a n . process as well as the steri l i z a ti on process or the saniti z a ti on processes must be validated.000 The tests performed. om The properties of the fabrics used for cl e a n room cl o t hing can be assessed by measu ring air perm e a bi l i ty. ti on of dispo s a ble or reusable covera ll s . l ong boo t s .3 A Critical area 1 100 been used to stu dy the pro tective ef f i c acy of cl e a n ro clothing (see Figure 1). For every opera tor in a cri tical (grade A or B) are a . washing. room clothing systems studied were two sets C 100 US Customary 100 100. Det a i l ed re sults have been B 10 area. Class 10. people are the main source of su ch microbi o l ogical airborne contaminants. The airborne d i s pers i on of su ch particles va ries from pers on to pers on and from time to time.1 . most cases of microbi o l ogical produ ct contamination are caused by airborne contamination (6). The materials con s i s ted of con ti nuous D 200 f i l a m ent po lye s ter fabric with an el ectro s t a tic s32 Pharmaceutical Technology ASEPTIC PROCESSING 2005 w w w.must be of appropriate high qu a l i ty. All items ing must be of high quality. and re s i s t a n ce Maximum Maximum Maximum US to wear. and sterile (steri lequ a tely saniti zed). Clothing should cover a pers on as Figure 1: The arrangement of a modified dispersal chamber (body box) used to com p l etely as po s s i ble to prevent significant nu m bers of study the protective efficacy of clothing systems.000 20 reported by Ljungqvist and Reinmüll er (7. p a rBecause su pp ly air is filtered thro u gh HEPA-filters . People are the principal source of viable air. The fabric itself should dispers e sources of airborne parti cles in cl e a n rooms are people and op.i zed or adequ a tely saniti zed ) . the washing ing are desirable. The ef fectiveness of cl e a n room clothing wi ll deteriora te . or clothing manu f acturer. Te s ting the material properties for asepti c Table I: Maximum permitted or recommended airborne viable particles clothing is mainly performed by the material (colony-forming units/m3) in pharmaceutical cleanrooms. US Customary 7 persal ch a m ber on sel ected clothing sys tems Class 1000 of both new sys tems and sys tems washed and Background to critical steri l i zed 50 times.acce s s ories su ch as glove s . drywear specially designed cleanroom clothing. to protect the air qu a l i ty. who are referred to as shedders.DATA AND REVIEW People People disperse fragments from the skin. 5 mm Highitself and not by the activi ty of the test Particles quality 9 70 36 su bj ects. The number of co l ony-formcleanroom >5 mm ing units genera ted .com). incre a s ed Colonyclothing as the nu m ber of washing and steri l iforming 0.f i l tered warm air. More particles After 1 wash and After 25 wash and After 50 wash and were gen erated after 25 washing and Clothing Contaminant sterilization cycle sterilization cycles sterilization cycles s teri l i z a ti oncycles than after 50.. Discussion discharge (ESD) stripe.24 0. enhanced the fil*The 95% confidence intervals (t-distribution) for source strengths of tra ti on efficacy of both parti cles and co l ony .10 0.* i zed only on ce ) . Bad Liebenzen. Pacific Scientific Instruments/Hach Ultra Analytics.51 0. Mi c robiological The stu dy re sults show that the source s tren g t h—nu m ber of parti cles inclu ding the number of colony-forming units generated from peop l e—i n c reases with the nu m ber of washing and steri l i z a ti on cycles even wh en high . Aerobic cfu/s Microbiological results. No significant perform a n ce difMean value Confidence interval ference was seen between the disposable clothing and the two sets of reu s a ble clothing that were on ly washed and Part (number of test subjects) Lower Upper sterilized once. All instruments were opera ted according to the manu f actu rers’ instructi ons. the on ly microbi o l ogical con t a m i n a ti onsource is the opera tors. In a 90-m3 aseptic filling room with 20 air changes per hour. units Table II: The effect of using special cleanroom underwear on the filtration efficacy of particles and colony-forming units (cfu)*. The current co l ony-forming unit limit for grade B areas (under EU GMPs) and for the back ground to cri tical areas ( u n der FDA rules) is 10 cfu/m3. Ta ble III summari zes the re sults of testing three sets of cl e a n room cl o t hTable III: Performance of cleanroom clothing systems after various numbers of ing: n ew clothing (washed and steri lwashing and sterilization cycles. clothing washed and steri l i zed 25 times. The TSA plate s were incubated for at least 3 days at 32 8C fo llowed by at least 2 days at room temperature. Some estimates are given in the following example.s a m p l er (Andersen 6-Stage Sampler. Wen dell . *Comparison of data (mean values) of the source strength (generated particles and colonyforming units [cfu] per second). A dispo s a ble sys tem also was tested for comparison. how many operators can be allowed in an aseptic filling room if the limit of 10 cfu/m3 cannot be exceeded during operating conditions? This can be determ i n ed by calculating the cfu concen tra tion in the filling room by taking the source strength for one person (from Table III) and dividing it by the total air volume flow. Parts 1 and 2 comprised all 10 test subjects.39 special cleanroom underwear. or 50 times. and long-legged cleanroom underpants.49 1. People were dressed in various clothing systems washed and sterilized once.fa-klotz.24 0. An derson Sa m p l ers Inc. The weave.pacsciinst. 25 times. respectively. There were five test subjects in each group: clothing systems. Th eoreti c a lly.de) and in some cases also with a sieve . The con t a i n m ent tests in the dispersal chamber eva lu a ted the pro tecti on ef f i c ac of the clothing sys tems by measu ring the y concentration of total airborne particulates and viable particulates (as aerobic colony-forming units. rinsing steps with filtered dei on i zed water. Example. the five test subjects in part 2 wore a cleanroom undershirt room unders h i rt and lon g . Viable particles were collected with a slit-sampler (Impactor FH3/FH5. The theoretical maximum number of people allowed in the aseptic filling room wi ll be the limit va lue divi ded by the calculated con cen trati on for one pers on.forming units airborne aerobic cfu/s from test subjects dressed in new cleanroom (see Table II). Th i s re sult su ggests that the parti cles may Particles 585 3950 2860 have been gen era ted from the material >0.qu a l i ty cleanroom clothing is used . GA). and clothing washed Particles/s and colony-forming unit (cfu)/s and sterilized 50 times. Markus Klotz GmbH.78 gre a ter than the va ri a ti on bet ween the sys tems. The five the test su bj ects in part 1 wore a cleanroom unders h i rt and test subjects in part 1 wore a cleanroom undershirt and short short underpants and the subjects in part 2 wore a cleanunderpants. OR. or cfu) in the exhaust air du ct .l egged cleanroom underpants.growth med ium for all tests was standard med ium tryptic soy agar (TSA) in 9-cm Petri dishes. The use of Part 2 (n 5 5) 0. and surface treatment. www. www. The sterilizati on cycle was carried out in a steam autoclave at 121 8C for at least 20 minutes. The variation among the test subjects was Part 1 (n 5 5) 0.38 0. h owever. vari ed . and dry-tu m bling with HEPA. Grants Pass. The washing process and sterilizing cycle used in the stu dy con s i s ted of a washing process at a tem pera ture of 73 6 2 8C for 10 minute s . Table IV shows the source strengths (cfu/s) for high-quality cl e a n room clothing and the esti m a ted maximum nu m ber of operators allowed in an aseptic filling room with a maximum level of 10 cfu/m3. weight (g/m2). however. Several types of u n derwear were te s ted in combination with the cleanroom coveralls. Calculations based on the reported results and the dilution ef fect of a ventilati on sys tem with at least 20 air ch a n ges per Pharmaceutical Technology ASEPTIC PROCESSING 2005 s33 . The total number of airborne parti cles was determ i n ed using a discrete parti cle co u n ter (Hiac / Royco model 245. however. Germany.14 zation cycles increased. Manufacture of Sterile Medicinal Products (European Commission. . .34 Baxter BioPharma Solutions . . . . . . . . . . . . . . . . . 8. 55–58 (2000). . . . . .7 Fres-Co System USA. . . . . .15 s34 Pharmaceutical Technology ASEPTIC PROCESSING 2005 w w w. . . . Number of washing and sterilizing cycles 1 25 50 Source strength (cfu/s) 0. . . .25 BD Medical-Pharmaceutical Systems . . 2004).3 Veltek Associates. The re sults of the stu dy show that the state of a cl e a n ro om clothing system—new or much used—is important because it influences the protection efficacy of the system. Cleanroom Clothing Systems. . . 7. . . . . . revised 2003). . Inc . . . . . . . . So far. .21 Cardinal Health. . US Food and Drug Ad m i n i s tration. Bi a rritz. . . . . . Vol. ISO 14698-1. 1997.76 0. . . . . . . J Paren. . . . . . . . . . . . .” p a per pre s ented at the A3P con gre s s . . . IL. (PDA/DHI Publishing. . . . Oct. . . .28 Maximum number of operators 13 10 3 References 1. . . . . 19–21. . . . . .27 Weiler Engineering. . . . . . . . . “ Devel opm ent of a High ly Autom a ted PAT. . Geneva. . . P T hour and HEPA-filters show that the limit of 10 cfu/m3 can be maintained with three people pre s ent in a com m only sized cleanroom for aseptic production (9). . France. Sci.49 1. . . . . . . 3. . p h a rm t e ch . MD.17 Ben Venue Laboratories. . 2004). IV. . . . . MD. Switzerland. .”Un i ted St a tes Pharmacopeia 27–Na tional Fo rmulary 22.” Eur. .DATA AND REVIEW Table IV: Source strengths (cfu/s) for high-quality cleanroom clothing and the estimated maximum number of operators allowed in an aseptic filling room with a maximum level of 10 cfu/m3. B. Sterile Dru g Products Produced by Aseptic Processing—Current Good Manufacturing Practice (FDA. . . . . . . .5 Vital Pharmaceuticals . . . River Grove. . . . Pa rt 1: Gen eral Principles and Methods ( International Organization for Standards. . . .19 Watson-Marlow Bredel Pumps . 4. . . . . Ljungqvist and B. .14 Concentration (cfu/m3) 0. . . . . . . .“Eva lu a ti onof Cl e a n room Garm en t s in a Di s persal Ch a m ber—Some Observa ti on s . . . . Rockville. 2. . . Geneva. . . . Inc . . Cleanrooms and As so ciated Co n troll ed Environments.31 Lyophilizaton Technology. . .13 Boston Analytical . . (United States Pharm acopeial Conven ti on . . . . . . . . .35 Sartorius Corp .38 0. . . . . . Belgium. .2. . . . . . . . Reinmüller. . . . . . 1999). Gu i d a n ce for Indu s try. . . . . . . J. . . . L ju n g q vist and B. . . B. . . . . . . . . . .. . . Sci. . . Inc . . Rockville. People as a Contamination Sou rce . . Ljungqvist. 2004. Akers et al. . 2004). .9 rommelag USA. . . Inc .6 Hospira One 2 One . . . B. . . . . Switzerland. LLC. . . C l e a n rooms and As so ci a ted Controll ed Envi ro n m en t s— Bioco n t a m i n a tion Control. .“Microbi o l ogical Eva lu a ti on of Clean Room s and Ot h er Contro lled Envi ron m en t s . . ISO 14644-1.36 Pall Corp . . . 5 (3). 25. . . . . . . Ad Index CO M PA N Y PAG E Advanced Scientifics. . . . Guide to Good Manufacturing Practice. .Com p a ti bl e As eptic Processing Sys tem. . . . . . . Suitable combi n a ti ons of cl e a n room underwear with the cl e a n room garm ents also improve the pro tecti on of the clean environ m ent a gainst airborne contaminants from peop l e . . . . . . . .29 ASEPCO . . . . Inc . . . . . . . . . . . . . . . . . . .11 Alconox. . . . . . . .. . . . . . Inc . . Pa rt 1: Classification of Air Cleanliness (Intern a ti onal Orga n i z a ti onfor Standards. . European Com m i s s i on. . . . Inc . . . “People as a Con t a m i n a ti onSo u rce: Cleanroom Clothing after 1. . . . . . . . Inc . J Paren. . . . Inc . Enterprise Directorate General. . . Rei n m ü ll er and B. 2004). . Inc .98 2. . . 6. . . . . . c o m . . . no other test method than measu rem ents in a dispersal ch a m ber have been reported to give information about cleanroom clothing’s protection efficacy during use. 9. G en eral Ch a pter ^1 1 1 6&. . . . . . . . Brussels. . 5. 75–80 (2003). . . .4 AES Clean Technology. . . . . . . . . Rei n m ü ll er. . . . and 50 Wa s h i n g / S terilizing Cycles. . 8 (3). Annex 1. Pharm. . . . . . The Rules Governing Medicinal Produ cts in the European Community.” Eur.