Grow-Through and Penetration of the 0.2/0.

22 "Sterilizing" Membranes
Mar 2, 2006 By: Maik W. Jornitz, Theodore H. Meltzer Pharmaceutical Technology

In the 1960s, researchers discovered that organisms retained on the upstream side of a microporous membrane could penetrate it after a period of time and appear on the downside. Investigations showed this occurred both when the liquid flow was ongoing, as in dynamic aqueous systems, and during static periods, when there was no flow, but the filter was still in place in a wet condition. Not all organisms exhibited this action, nor was the time interval the same for those that did. Although grow-through was still a present concern, its imminence was assuaged by the US Food and Drug Administration. In 1976, in its proposed good manufacturing practices (GMPs) for large-volume parenterals (LVPs), FDA addressed the problem by requiring that the mixing and filtration of a batch be completed within the eight-hour period of a single shift, an interval too brief for grow-through to occur (1). Although this proposal was never finalized, in the actual event it became the rule. (Indeed, its application discouraged the long-term use of membrane filters at points-of-use in water systems before the validation of sanitizing practices enabled its management.) Wallhusser described a companion phenomenon, "blow-through," as resulting from the imposition of pressure upon a wet membrane within whose pores organisms had already partly grown or penetrated (2). For example, instead of a "normal" release of 1 or 2 bacteria per liter through a membrane, 29 penetrated after an overnight shutdown and restart under pressure the next day. Following a flush of some liters, the bacterial passage declined to a "normal" number. The grow-through phenomenon was rationalized as being a result of a binary fission whereby an organism cell divides into two during its reproduction. Presumably, in this reduced cell size, the organism can penetrate an otherwise impermeable membrane (3, 4). Another explanation recognized the existence of pore-size distributions in membranes. Researchers postulated that the continuing growth of organisms on the wet membrane eventually yields numbers sufficient to encounter even the occasional large pore, thereby leading to penetration. (Interestingly, this explanation was strongly advanced by a filter manufacturer who had the good fortune to recognize the potential shortcoming in competitive filters that he asserted was absent from his.) The grow-through experience proved difficult to investigate because not all organisms showed the effect, and those that did differed in their response times. The total contact time between the organism and the drug is seldom known, as also whether the same organism types have been involved in each of the exposures, and whether under roughly the same conditions such as temperature. Indeed, some investigators dispute the "growthrough" and "blow-through" episodes. Carter and Levy discuss that view, stating, "Both phenomena are hypotheses and have never been rigorously demonstrated to be real events in pharmaceutical processes" (5). There is the belief that such events, culminating in organism passage through 0.2/0.22 m-rated membranes, are overstated as they pertain to pharmaceutical processing contexts. Grow-through versus reverse osmosis membranes

Curiously, organisms may, on occasion, be found on the downside of reverse osmosis (RO) membranes, although these have smaller "pores" than 0.1 m-rated membranes and would seem far less likely to permit grow-through. Unlike the case of microporous membranes, the integrity testing of RO membranes does not involve correlations with organism retentions. It is, therefore, possible that "imperfect" RO membranes rather than grow-through is the cause of such occurrences. Carter hypothesized the presence of pinholes in RO filters (6). Manufacturing techniques have undoubtedly improved since 1976. Nonetheless, the intersegmental spaces among the convoluted polymer molecules that constitute the pores of the RO membranes are exceedingly small. Their castings are, therefore, much more liable to be compromised by the incorporation of miniscule particle-inclusions that are not significant in microporous membranes. Tellingly, perhaps, RO membranes are not relied upon to produce sterile effluent. Indeed, FDA is generally regarded as being wary even of the use of two-pass, product-staged RO, at least for the production of water for injection. Grow-through concerns Grow-throughalthough seemingly avoided by the time restriction set on filtrationsremains unresolved and an ongoing matter of concern, largely because its cause is still little understood and its control, therefore, is uncertain. Regardless, the possibilities of its occurrence ought never really pose a threat in pharmaceutical filtrations, given the requirement for validation. Whenever a terminal filter is used to sterilize a fluid, validation is required to demonstrate that the filter will perform satisfactorily, reliably, and repeatedly over the entire processing interval. Thereby, grow-through will be detected. Its occurrence being time dependent, its avoidance can be undertaken by expeditious filtrations, time restraints, larger effective filtration area (EFA), or filter change-outs. Likewise, other activities such as prolonged filling times will receive their needed documented experimental verification. Desired filter retentivity should thus be ensured by validation, regardless of the possibility of grow-through (7). Awareness of organism size alterations It was known, most likely among microbiologists, that the size of organisms could differ depending upon the stage of their development and upon how they were cultured. The classical work of Leahy and Sullivan on the cultivation of Brevundimonas diminuta should have made that evident to filtration practitioners (8). It seems, however, that those dealing with organisms in filtration processing contexts largely assumed that the cell sizes within a drug preparation essentially remained constant. It was partly for this reason that efforts were made to relate organism and pore sizes in the search for the "sterilizing" filter. Subsequent findings that the size of the organism did not necessarily remain constant during drug processing gave rise to the suggestion that 0.1 mrated membranes should be substituted for their 0.2/0.22 m-rated counterparts that were considered "sterilizing filters" at the time. It is now accepted that the physicochemistry of the suspending fluid may serve to alter the size of suspended organisms as a consequence of its limited nutritive power or as an expression of the Donnan equilibrium's response to ionic strengths. Commonality of organism shrinkage and grow-through What is of interest is that certain organisms undergo morphological diminution after exposure to particular drugs. This singular occurrence manifests itself in two different circumstances. One characterizes the "growthrough" phenomenon wherein after a filtration both the filter and its retained organisms are maintained wet by the drug preparation. In the second circumstance, the organisms are suspended for a time in a drug formulation awaiting the filtration step. The commonality is that the size alterations in each case seem consequent to contact with a drug. Unfortunately, the literature does not record the total contact time of the organism and drug in any of the reported cases. Also unknown is whether the same organism types have been involved in each of the exposures and whether under roughly the same conditions of temperature and so forth. The authors surmise that the same phenomenon is being expressed in the two different situations. It is a purpose of this writing to encourage further investigation of the matter. Pore size versus grow-through Recent data secured by Sundaram et al. reveal that with regard to certain organisms, 0.2- and 0.22-m-rated membranes provided sterile effluent and/or a high titer reduction only for various lengths of time before

penetration occurred (9). Five 0.2-m-rated nylon 66 filters were tested. Penetration times varied from 24 to 96 h, and the cumulative challenge at which penetration was first observed ranged from 1.2 X 107 to 1.1 X 108 cfu/cm2. Two 0.22-m modified poly(vinylidene difluoride) (PVDF) hydrophilized filters showed bacterial penetration after 72 h, corresponding to a cumulative challenge level of 6.58.7 X 107 cfu/cm2. Two 0.2-mrated nylon 66 filters in series were unable to fully retain Ralstonia pickettii at 72 h, corresponding to a cumulative challenge of 2.4 X 107 cfu/cm2. The more extensive penetration of the nylon 66 membranes compared with that of the PVDF filters is in keeping with their greater degree of openness (10). None of the 0.1-m-rated membranes that were examined showed evidence of grow-through. Five nylon 66 filters tested yielded sterile effluent over the entire duration of the test (120196 h), up to challenge levels from 5.7 X 107 to 2.0 X 108 cfu/cm2. Similar results were obtained with the PVDF filters tested: No R. pickettii were detected at challenge levels from 5.9 X 107 to 6.0 X 108 cfu/cm2. The organisms were sufficiently small to penetrate the corresponding 0.2-m-rated membranes, however, and all 0.1-m-rated filters tested provided consistent and complete retention of R. pickettii for the entire duration of the test (120192 h).These results demonstrate that 0.1-m-rated filter membranes provided sterile effluents under conditions that allowed bacterial penetration to occur through conventional 0.2- and 0.22-m-rated sterilizing grade filters (9). Choosing the pore-size rating On the basis of the above data, one can argue that 0.1-m-rated membranes would quell grow-through concerns and would permit longer-term formulation and filtration operations. Indeed, the authors believe that long-term filtrations should incline toward a reliance on the 0.1-m-rated filters. Nevertheless, it is recognized that significant penalties are incurred by the unnecessary use of tighter membranes (1113). Objections to the tighter filters are the consequences that derive from their lower rates of flow. It should also be noted that longer term operations would promote endotoxin production, usually a matter of some importance. A responsible choice requires that both the 0.1-m-rated membranes and the 0.2/0.22-m-rated membranes be validated. If both prove appropriate, the higher pore-size rating should be used to avoid the penalties of reduced flows. If, however, the validation data do not permit a clear resolution, the 0.1-m-rated membranes should be selected. Retention is more important than flow rate or flux. It has been stated that advances in filter manufacture have resulted in 0.1-m-rated membranes that are faster flowing than their 0.2/0.22-m-rated counterparts. Contrary to common experience, however, it is noteworthy, and substantiating data are awaited. Its publication would accord with Lord William Thompson Kelvin's famous dictum, long a guiding principle of the scientific approach, "When you can measure what you are speaking about and express it in numbers, you know something about it." This condition, necessary to the removal of objections to the use of the 0.1-m-rated membranes, remains unfulfilled. Summary It is hypothesized that the occurrence of grow-through and the diminution in the size of certain organisms when in contact with given liquids are the same phenomenon manifested under different circumstances. If this be so, the identification of liable organism types and the elucidation of the kinetics of their size diminution will be of considerable practical value. It will resolve the uncertainties of grow-through, will dissipate its concerns, and will contribute to a more reliable usage of the 0.1-m-rated membrane. Moreover, process validation requirements defined 10 years ago reduce the fear of grow-through to an unresolved phenomenon instead of a safety issue. Maik W. Jornitz is group vice-president for product management at Sartorius North America, 131 Heartland Blvd., Edgewood, NY 11717. Theodore H. Meltzer* is principal of Capitola Consulting Company, 8103 Hampden Lane, Bethesda, MD 20814-1244, tel. 301.986.8640. He also is a member of Pharmaceutical Technology's Editorial Advisory Board. *To whom all correspondence should be addressed. References 1. US Food and Drug Administration, FDA Guidance Document: Current Good Manufacturing Practice in the Manufacture, Processing, Packing, or Holding of Large Volume Parenterals for Human Use (FDA, Rockville, MD, 1976).

2. K.H. Wallhusser, "Grow-Through and Blow-Through Effects in Long-Term Sterilization Processes," Die Pharmazeutische Industrie 45 (5), 527531 (1983). 3. J.A. Simonetti and H.G. Schroeder, "Evaluation of Bacterial Grow-Through," J. Environ. Sci. 27 (6), 2732 (1984). 4. D.A. Christian and T.H. Meltzer, "The Penetration of Membranes by Organism Grow-Through and Its Related Problems," Ultrapure Water 3 (3), 3944 (1986). 5. J.R. Carter and R.V. Levy, "Microbial Retention Testing in the Validation of Sterilizing Filtration," in Filtration in the Biopharmaceutical Industry, T.H. Meltzer and M.W. Jornitz, Eds. (Marcel Dekker, New York, NY, 1998). 6. J.W. Carter, "Membrane Techniques (Reverse Osmosis and UltraFiltration)," in Towards Absolute Water, A Survey of Current Water Purification, Proceedings of International Symposium (Lane End, High Wycombe, Bucks, England, May 1976), pp. 103114. 7. PDAFDA Special Scientific Forum, "Validation of Microbial Retention of Sterilizing Filters," Bethesda, Maryland, July 1213, 1995. 8. T.J. Leahy and M.J. Sullivan, "Validation of Bacterial Retention Capabilities of Membrane Filters," Pharm. Technol. 2 (11), 6475 (1978). 9. S. Sundaram, "Retention of Diminutive Water-Borne Bacteria by Microporous Membrane Filters," presented at the PDA National Meeting, Washington, DC, Dec. 7, 1999. 10. V. Krygier, "Rating of Fine Membrane Filters Used in the Semiconductor Industry," in Transcripts of the Fifth Annual Semiconductor Pure Water Conference (San Francisco, CA, Jan. 1986), pp. 232251. 11. K. Kawamura, M.W. Jornitz, and T.H. Meltzer, "Absolute or Sterilizing Grade FiltrationWhat is Required?" PDA J. Paren. Sci. Technol. 54 (6), 485492 (2000). 12. J. Lindenblatt, M.W. Jornitz, and T.H. Meltzer, "Filter Pore Size versus Process ValidationA Necessary Debate?" Eur. J. Paren. Sci. 7 (3), 6771 (2002). 13. M.W. Jornitz and T.H. Meltzer, "Sterilizing Filtrations with Microporous Membranes," Pharm. Forum 30 (5), 29 (2004).