Diminutive Bacteria Implications for Sterile FiltrationPage 1 of 7 Diminutive Bacteria Implications for Sterile Filtration P.T. Blosse*, E.M. Boulter* and S. Sundaram** Pall Scientific and Laboratory Services. Acknowledgements Isolation and Culture of Diminutive Bacteria Summary Introduction Evolution of Filtration Standards Penetration Studies on Filter Cartridges Penetration Studies on Filter Discs Penetration of 0.2µm and 0.22µm Rated Filters Short Term Penetration of 0.2µm and 0.22µm Filters Can 0.1µm Filters Reduce Validation Effort and Costs? References Acknowledgements The authors wish to acknowledge the conscientious experimental work performed by Vanessa G Carmen Chinnery of the Microbiological Laboratories, Pall Scientific and Laboratory Services, Po Top Isolation and Culture of Diminutive Bacteria Diminutive bacteria were obtained by filtering tap water through 0.45µm filter discs. The mixed po bacteria obtained in the filtrate was repeatedly grown and filtered through 0.45µm filter discs. One isolated was of special interest, as it was found to consistently penetrate 0.2µm filter discs. This b although not yet fully classified, has been identified as a Pseudomonas species and will be referr Pseudomonas sp. in this publication. A culture technique was developed that provided: A pure culture of the diminutive bacteria. No reversion to larger sizes. Sufficient quantities for bacterial challenge studies. Top Summary In the 1960s, sterilizing filters used for liquids were based on 0.45µm membranes, but when pene Brevundimonas (Pseudomonas) diminuta was reported, 0.2µm /0.22µm was adopted as the new now recognized that bacteria smaller than B. diminuta exist. Furthermore, occasional filter penetr reported for certain product and process conditions. Filter validation has therefore become more because of the need to simulate the process during challenge testing. A diminutive pseudomonas species has been isolated and cultured successfully to allow detailed studies to be performed. Consistent penetration of 0.2µm and 0.22µm filters has been shown in c of 60 - 90 minutes. Full retention was obtained with 0.1µm filters. The recent availability of high fl http://www.pall.com/34445_3813.asp 2/20/2007 Diminutive Bacteria Implications for Sterile Filtration Page 2 of 7 efficiency 0.1µm filters can provide enhanced sterility assurance and may help to simplify the pro validating sterile filtration processes. Top Introduction Membrane filters have been used widely to sterilize liquids, especially those which cannot be hea the final container. The definition of a sterilizing filter has been the subject of much discussion an the last 40 years. The initial definitions attempted to describe the physical structure of the membr that it was a simple thin screen which removed bacteria on its surface. The concept of ‘pore size’ introduced and the classification of filters by differences in pore size rating was developed. It is now known that 0.2µm sterilizing filters consist of a complex matrix of non-circular pores with pore sizes within the structure, with some at least 0.5µm in size. This was shown very effectively electron microscope studies of Osumi et al.1 An example is presented in Figure 1. Figure 1 Scanning electron micrograph of surface of 0.2µm rated filter challenged with B. diminuta at 5 x 1 Although the numerical ‘pore size’ definition helps us to classify filters into general groups such a 0.2µm, 0.1µm, etc., a more meaningful definition must include some reference to its microbiologi capability. In this way, the potential for specific bacterial types to be retained by the filter may be determined. This approach requires specification of the organism, the laboratory test method and efficiency level if the definition is to become a meaningful and appropriate industry standard. However, even a definition and specification based on removal efficiency does not necessarily gu in the process. The filter may not be physically or chemically compatible with the process. Also, b than B. diminuta may be present. These diminutive forms may be naturally occurring or induced conditions. Process validation may be able to establish the most suitable sterilizing filter type and difficult to predict or monitor during routine production the type and quantity of bioburden present batch. For this reason, there has been a major increase, especially over recent years, in the leve validation required to justify the use of the current 0.2µm or 0.22µm sterilizing filters. This publication reviews the evolution of sterile filtration with specific reference to penetration by organisms. New data are also presented on a diminutive organism, isolated from water, which ca quantity under laboratory conditions without losing its diminutive form. This organism has been s penetrate various commercially available 0.2µm or 0.22µm sterilizing filters, but not 0.1µm filters. implications for filter validation are discussed and the future role for 0.1µm filtration assessed. Top Evolution of Filtration Standards 0.45µm filtration standard. http://www.pall.com/34445_3813.asp 2/20/2007 Diminutive Bacteria Implications for Sterile Filtration Page 3 of 7 In the 1960s, both sterilizing sheet filters and membrane filters were used in pharmaceutical prod Membrane filters were available mostly in flat disc form and used singly or in multi-plate configura membranes were successfully used for sterile production at that time as they enabled reasonable achieved through the relatively low area disc systems. The membranes were qualified using Serr marcescens, with a typical size of 0.6µm x 1µm. However, the safe use of 0.45µm filters was que Bowman2 established that an organism, Pseudomonas diminuta, could consistently penetrate 0.4 filters, but could be retained by the next finer grade commercially available - 0.22µm. 0.2µm/0.22µm filter standard. Bowman2 proposed in 1967 that P. diminuta (recently reclassified as Brevundimonas diminuta) s the industry standard organism for 0.2µm filters. In 1987, the FDA ‘Guidelines on sterile drug pro by aseptic processing’3 incorporated P. diminuta as the standard challenge organism for a steriliz defined a minimum qualifying level of 107/cm2 of filter area. Since that time, no further standards have been developed, even though 0.1µm sterilizing filters i have been available commercially for almost twenty years and are being increasingly used in pro processes. The primary area of application was initially in the processing of serum and tissue cul where removal of mycoplasma is required. These deformable bacteria are known to penetrate 0. filters. However, there has been an increasing use of 0.1µm filters in other applications where dim organisms have been identified, or are of potential concern. They are also being used for enhanc assurance in certain types of products or processes. In the absence of a defined industry standard, filter manufacturers have qualified 0.1µm filters us standards. PALL uses Acholeplasma laidlawii, a mycoplasma type organism, for 0.1µm filter valid addition to B. diminuta. Top Penetration Studies on Filter Cartridges In a production environment, filter cartridges and not filter discs are normally used. It was therefo establish whether the more complex cartridge structure would give similar results to those using d In these studies, a mixed challenge of B. diminuta and Pseudomonas sp. was used to show that 0.22µm filters retained B. diminuta while allowing penetration of Pseudomonas sp. The results of studies are shown in Table III. The results showed: Substantial and rapid penetration of all 0.2µm and 0.22µm filters by Pseudomonas sp. giv counts of between 102 and 105 cfu. Full retention of B. diminuta. Total removal of both Pseudomonas sp. and B. diminuta by 0.1µm filters. Further work is in progress to identify the diminutive species and to assess its suitability for wider qualification testing. Table III Mixed bacterial challenge of sterilizing filter cartridges * Filter Type Rating B. diminuta Challenge PALL Ultipor® N66® Grade NF/NR PALL Fluorodyne® II Grade DFL 0.2µm 1 x 1011 1 x 1011 9 x 1010 1 x 1011 7 x 1010 1 x 1011 Recovery 0 0 0 0 0 0 Pseudomonas sp. Challenge 2 x 1010 6 x 109 2 x 109 1 x 1010 3 x 109 4 x 1010 Recovery 1 x 105 1 x 104 1 x 104 1 x 104 2 x 102 7 x 102 0.2µm http://www.pall.com/34445_3813.asp 2/20/2007 Diminutive Bacteria Implications for Sterile Filtration Page 4 of 7 Non-PALL PVDF 0.22µm 1 x 1011 8 x 1010 8 x 1010 1 x 1011 1 x 1011 6 x 1010 1 x 1011 1 x 1011 2 x 1011 0 0 0 0 0 0 0 0 0 1 x 109 9 x 109 8 x 108 2 x 109 3 x 109 4 x 1010 6 x 109 3 x 109 5 x 1010 1 x 104 1 x 104 1 x 104 0 0 0 0 0 0 PALL Ultipor® N66® Grade NT PALL Fluorodyne® II Grade DJL 0.1µm 0.1µm *All studies used 254mm (10 inch) cartridges. Flow rate 0.3 L/min. Top Penetration Studies on Filter Discs The first objective was to show that Pseudomonas sp. could consistently penetrate 0.2µm and 0. and be retained by 0.1µm membranes. Various 47mm discs were selected and challenged with P sp. at 106cfu/cm2. Filter penetration was identified by passing the filtrate through a 0.1µm analys downstream. The results are presented in Table II. Table II Removal of Pseudomonas sp. by sterilizing grade filter discs* Filter Type PALL Ultipor® N66® Grade NR PALL Fluorodyne® II Grade DFL Non-PALL PVDF PALL Ultipor® N66® Grade NT PALL Fluorodyne® II Grade DJL Non-PALL PVDF * 47mm discs challenged at 106cfu/cm2. Filter Rating 0.2µm 0.2µm 0.22µm 0.1µm 0.1µm 0.1µm Sterile Filtrate? No No No Yes Yes Yes Bacteri >100cfu >100cfu >100cfu 0 0 0 Flow - 20ml/min. Filtration Time - 25min. Penetration of all 0.2µm and 0.22µm disc filters was observed with high bacterial counts of >100c The three 0.1µm filters tested were fully retentive. Of equal importance was the short challenge t minutes, which would restrict any significant time-dependent penetration or bacterial growth. The bacterial ‘growthrough’, or other time related effects, cannot therefore explain the extensive pene in these studies. The results suggest that the number and size of the bacteria challenging the filte to exceed the removal capability (titer reduction) of the filters. Top Penetration of 0.2µm and 0.22µm Rated Filters The European GMP Guide5 defines a sterilizing filter as having “a nominal pore size of 0.22 micr with at least equivalent micro-organism retaining properties”, but then states that “Such filters can bacteria and molds, but not all viruses or mycoplasmas”. The FDA3 state that “Validation should include microbiological challenges to simulate ‘worst case conditions particularly regarding the size of micro-organisms in the material to be filtered.” The F accept B. diminuta as a sound basis for such assessment, but also state that “It is important to as influent bioburden does not contain micro-organisms of a size and/or concentration that would re targeted high level of filtrate sterility assurance”. http://www.pall.com/34445_3813.asp 2/20/2007 Diminutive Bacteria Implications for Sterile Filtration Page 5 of 7 These statements indicate that filter users must establish the possible risk of filter penetration an product. Since the 1960s, there have been occasional reports of bacteria other than mycoplasma species 0.2µm and 0.22µm sterilizing filters. Representative examples are given below: • In 1967, Braun et al.6 reported penetration of waterborne bacteria (Spirillaceae) through 0.22µm during development of methods for isolating Leptospires. • In 1980, Howard and Duberstein7 demonstrated penetration of a range of commercially-availab 0.22µm filters with naturally-occurring waterborne bacteria, including Leptospira species shown in Figure 2 Leptospira species isolated downstream of 0.2µm sterilizing filters7 The same organisms were fully retained by a PALL Ultipor® N66® (grade NT) 0.1µm rated filter, a Table I. Table I Removal of diminutive bacteria by 0.1µm filters7 Filter Number 1 2 3 Filter Size 142mm Disc 293mm Disc 254mm Cartridge Total Challenge 1.2 x 107 8.9 x 107 1.7 x 1010 Bacteria Re 0 0 0 In 1985, Andersen et al.8 reported penetration of 0.2µm filters by Burkholderia (Pseudom saline solution. In 1993, the FDA reported presence of Pseudomonas cepacia in deionized water downst filters9. In the same document, they reported a product recall in the USA for a solution of which was also suspected to involve penetration of the 0.2µm filter by P. cepacia. In a recent publication10, Leo et al. reported penetration of 0.2µm and 0.22µm filters by B (Pseudomonas) pickettii when suspended in product, but not when suspended in saline la (SLB). The penetration was associated with a 40% reduction in bacterial size, as shown i retention was achieved using a PALL 0.1µm Ultipor® N66® filter. Further work is in progre the mechanism of filter penetration. Figure 3 Size distribution of B. pickettii suspended in product10 http://www.pall.com/34445_3813.asp 2/20/2007 Diminutive Bacteria Implications for Sterile Filtration Page 6 of 7 Events of this type have resulted in an increasing requirement by regulatory authorities for produ specific filter validation. Such additional validation is required to be performed in a way that simul as possible the actual production conditions and also represents ‘worst case’3. More recently, the need to challenge test in the actual drug product being filtered has been emph FDA11, who have stated: “Since there is the possibility that the drug product may cause a reducti the micro-organism, it is best to test the microbial retentivity of the filter with the microbial challen drug product.” and “…There are products that fall within Millipore’s matrix which are not rendered filtered through a 0.22 micron filter.” Despite increased validation for 0.2µm and 0.22µm filters, the FDA12 in 1996 have commented th 0.1µm filter as the final filter may be “desirable to ensure sterility of the product, especially when contaminants are exposed to the drug product over a long period of time.” Top Short Term Penetration of 0.2µm and 0.22µm Filters Filter penetration is often interpreted as a time-dependent and not size dependent occurrence. T explained by the difficulties in producing sufficient quantities of diminutive bacteria of constant mo assess the true removal performance of filters over short time periods. In addition, the diminutive a very small sub-population of the natural bioburden. These factors have made it difficult to obtai results on filter penetration. Most studies have, therefore, been performed over extended time pe days, or weeks, in order to challenge the filters with significantly high levels of bacteria. To overcome these limitations, an experiment was designed by Pall Scientific and Laboratory Se recover and culture a single diminutive bacterial species from mains water so that extensive and penetration studies could be performed over short time periods, ideally less than 1 hour. Top Can 0.1µm Filters Reduce Validation Effort and Costs? There is an increasing awareness that the current filtration standard for sterilizing filters does not guarantee sterility for all bacteria, under all conditions. Furthermore, the ability of bacteria to redu interactions with the drug product, or the presence of low nutrient conditions, is further supported studies. There is also the possibility that the bacteria present in the product may change from batch to ba of production conditions, seasonal variations and other factors, which may be poorly understood routine monitoring of bioburden type and quantity necessary . For these reasons, validation proto filters using B. diminuta are becoming increasingly complex. http://www.pall.com/34445_3813.asp 2/20/2007 Diminutive Bacteria Implications for Sterile Filtration Page 7 of 7 One possible way to reduce the validation effort and cost is to enhance the sterility assurance pro filter. This can be achieved by using a filter with higher removal efficiency - a properly validated 0 Although this option has been commercially available for almost twenty years, the limitations on f filter life have restricted these filters to special applications. However, recent developments in filte have produced 0.1µm filters with flow rates comparable to some 0.2µm and 0.22µm filters and th being used successfully in a range of applications. The enhanced sterility assurance provided by a properly validated 0.1µm filter may be the simple problem of validating the security of sterile filtration processes. Although many filter suppliers offer 0.1µm grades, there is no international qualification standard Therefore, it cannot be assumed that all filter cartridges rated as ‘0.1µm’ would provide the same efficiency as those tested in this study. Top References 1. 2. 3. 4. 5. M.Osumi, N. Yamada and M. Toya. “Bacterial retention mechanisms of membrane filters” Pharmaceutical Science and Technology 50: 30-34, (1996) F.Bowman, M.P. Calhoun and M. White, “Microbiological methods for quality control of m J. Pharm. Sci., 55, 818 (1967) FDA, “Guidelines on sterile drug products produced by aseptic processing”. Centre for Dr Biologics, Rockville. MD, (1987) “Validation guide for Pall NT grade 0.1µm microbially-rated nylon 66 membrane cartridge Corporation, East Hills, NY “Rules governing medicinal products in the European Community Vol IV - Good manufac for medicinal products”. ISBN 92-826-3180-X. Office for official publications of the Europe Communities, Luxembourg, (1992) J.L. Braun, S.L. Diesch and W.F McCulloch “A method for isolating leptospires from natur waters”. Canadian Journal of Microbiology 14: 1011-1012 (1968). G. Howard and R. Duberstein. “A case of penetration of 0.2µm rated membrane filters by Journal of the Parenteral Drug Association.,34: p95 (1980) R.L. Anderson, L.A.Bland, M.S. Favero, M.M. McNeil, B.J. Davis, D.C. Mackel and C.R. G “Factors associated with Pseudomonas pickettii intrinsic contamination of commercial res solutions marketed as sterile”. Applied and Environmental Microbiology 58: 1343-1348. (1 D.L. Michels “Validation and control of de-ionised water systems”. FDA, Rockville MD208 F. Leo, M. Auriemma, P. Ball and S. Sundaram, “Application of 0.1µm filtration for enhanc assurance in pharmaceutical filling operations”. BFS News, August (1997) (in press) Human Drug CGMP Notes. Vol 2, No. 4, p35. FDA CDER Office of Compliance. (1995) CDER Perspective on Isolator Technology ISPE Barrier Isolation Technology Conference (1996) 6. 7. 8. 9. 10. 11. 12. Top http://www.pall.com/34445_3813.asp 2/20/2007