Theodore H. Meltzer is principle of Capitola Consultancy.

Theodore H. Meltzer

There are advantages and disadvantages to using model microorganisms in the validation of sterilizing filtration. Although filtration validation studies may demonstrate the removal of the challenge organisms (whatever they may be), those studies can only imply that any particular filter used to filter a batch of drug product will yield a sterile filtrate. Successfully passing a bubble-point test (1) (or multipoint diffusive-flow integrity test), the results of which have been correlated to successful microbial challenge testing and validation studies, provides additional assurance that a sterile filtrate has been obtained. The filter manufacturer's quality-management system and production-control program further support the validity of the sterilizing filtration process. All three elements must be present to predict the successful outcome of a particular sterile filtration event (2). 

Perhaps the best method of determining whether a filter can yield sterile effluent is to subject the drug product to a proper microbial challenge and to analyze the filtrate for its microorganism content. This method of direct measurement would seem to be the most reliable way of characterizing a filter's sterilizing ability. Even though an individual filter can be characterized by a direct microbial challenge, however, the tested filter may not then be used in a processing operation because it has been contaminated by the test organisms. 

In practice, the measurement of a filter's sterilizing capability is derived from a correlation of its degree of bacterial retention with a characterization of its largest pores, essentially in terms of their width. The latter is obtained by means of the bubble-point integrity test method (3). It is necessary to confirm this correlative relationship because to assume it exists could prove a mistake. For instance, two qualities or properties of a filter may so constantly occur together as to be assumed to be correlated. Actually, they may in fact be two independent but parallel events that are not correlated (1). For example, single-point air diffusion rates of a filter measured at 5 psi had formerly been thought to correlate with a bubble point of some 50 psi for that filter, but it was discovered that it was necessary to determine the bubble point by measurements made at the actual bubble-point pressure. It did not necessarily correlate with the extension of the 5 psi single-point air diffusion test reading in all cases. So what had seemed to be a correlation turned out to be a case of parallel events. As a result, the use of single-point diffusive airflows measured at 5 psi was abandoned. In the present instance, extensive examination by many investigators has established the validity of correlating the bubble point with microbial retention (4). 

A filter can be characterized by direct microbial challenge as being capable of producing a sterile effluent. Extrapolating from the properties of the tested filter to other "identical" filters would, however, involve assumptions related to the uniformity of the filters' manufacture. To investigate this aspect of the filtration picture to a statistically meaningful level would involve a large number of correlated studies. Moreover, it might well necessitate the disclosure of proprietary information in a competitive market. This would prove highly impractical. From this one consideration alone, it becomes evident that a fundamental assumption must be made, namely, that the filter manufacturers can be depended upon to control their manufacturing processes so that "identical" filters achieve the same level of retention performance. 

Reliance upon the filter manufacturer is one necessary factor in this action. Identifying the requirements does not ensure the attainment of sterile effluent. 

Until rather recently, it was believed that the sterilization of liquids could be achieved by their filtration through a sterilizing membrane whose proper and pertinent identity was confirmed by its pore-size rating, which was itself determined by integrity testing. The pore-size rating, plotted against organism size, indicated an inverse correlation. However arranged, organism removal by a filter was seen as resulting from the mechanism of size exclusion, also known as sieve retention, wherein the organism is retained because it is larger in size and shape than the filter's pores. The sizes of various organisms are available from listings in the literature, and the size of a filter's largest pores derives from its bubble-point value. Therefore, it was possible to accommodate the size of the restraining pore to the size of the organism to be retained. In the interest of obtaining a maximum flow rate consonant with complete organism removal, the 0.2/0.22-μm-rated membranes were widely accepted as being sterilizing filters. 

The sterilizing filter was defined in 1987 by the US Food and Drug Administration on the basis of its retaining a minimum of 1 × 10

(at that time taxonomically identified as 

) per square centimeter of effective filtration area (5). 

The situation is complicated in its application by the absence of pore-size rating standards. The pore sizes may not be assumed to have been rated in the same fashion by the various filter manufacturers. Thus, while filters may bear the same pore-size designation, they may not be identical in this regard. Nevertheless, such filters may prove interchangeable in filtration processing operations. Manipulative adaptations often make it possible to substitute one such filter for another. 

Although the individual rating methods differ, bubble-point measurements performed in as similar a manner as possible enable comparisons to be made among the various types of filters. Given the importance of gauging the sizes of the largest pores, the integrity testing should be as accurate and reproducible as possible. Accordingly, using automated integrity test instruments is recommended, because their use eliminates the subjectivity inherent in manual integrity testing (6). For the same reason, the bubble point should be identified by the straight line plotted from multipoint diffusion measurements extended through and beyond it to a robust flow of air. Single-point diffusive airflow measurements are not sufficiently reliable for this purpose (7). 

There are numerous types and sizes of microorganisms. To explore the sterilizing capabilities of the available filters with each of these different organisms would not be practical and might not even be meaningful from the standpoint of assessing patient risk. Given the large number of organism types and their differences in size, it would be advantageous to select for testing an organism that could serve as a model for all the others, or at least for those commonly encountered in pharmaceutical operations. The smaller the test organism, the more its removal by a filter would ensure the sieve retention of larger organisms. Properties other than size, however, also are important in the selection of the model microbe. Ease and safety in cultivation and handling are real considerations. The likelihood of the selected organisms' being encountered in pharmaceutical operations is another influence on its selection. 

In the mid-1960s, Frances Bowman of FDA identified an organism that would penetrate the 0.45-μm-rated membranes that typically were used for sterilizing filtration at that time (8). The organism, currently known as 

 American Type Culture Collection 19146, had the ability to distinguish between 0.22- and 0.45-μm-rated membranes (it readily penetrated the latter) and, at concentrations higher than about 1 × 10

, it penetrated 0.22-μm-rated membranes, demonstrating that because penetration was concentration-dependent, mechanisms other than sieve retention were active. Although not the smallest organism known (smaller organisms had been known for decades), 

generally was considered small enough to represent whatever smaller organisms were likely to be present in pharmaceutical preparations. ASTM standard F 838, "Standard Test Method for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration," is based on the use of 

 as the challenge organism. The standard was originally approved in 1983 and was designed to determine the bacterial retention characteristics of membrane filters for liquid filtration. 

Since then, however, some 25 cases have been noted wherein the 0.2/0.22-μm-rated membranes qualified by withstanding the requisite 

 challenge did not yield sterile effluent (9, 10). Obviously, 

 does not serve as a universal model for all organisms. This finding has led some to advocate that the choice of the model organism whereby a filter will be qualified for use as a sterilizing filter should be an organism native to the drug preparation. In this view, 

 suffers from the demerit of not being part of the drug preparation's bioburden. Be that as it may, selecting a component of the bioburden as the model organism is not enough. The test organism must be amenable to identification and cultivation. Its life stages must allow amply for its management in the necessary test manipulations. Above all, its susceptibility to size diminution by contact with a given drug preparation will have to be investigated, and the kinetics and direction of the morphological changes, if any, will require elucidation. 

As previously stated, it had been believed that a sterilizing filter could be defined by way of its pore-size designation as identified by integrity testing. Developments in filtration practices showed this belief to be too simplistic. What had once seemed simple now is recognized as being quite complex. It was discovered that the conclusions based on pore-size ratings were subject to modification by the physicochemical specificity of the organism-suspending fluid, the individuality of the organism type in its size-changing response to the fluid, the possible change in pore size induced by the fluid, and the adsorptive qualities of the filter resulting from its particular polymeric composition, all influenced by the filtration conditions in their numerous varieties, but especially by the transmembrane pressure (11, 12). 

				A filter may not sterilize the same preparation under different filtration conditions, especially under dissimilar differential pressures (13). A given membrane may or may not retain a particular organism type suspended in a different drug vehicle (8). The organism type need not remain constant in size, but may alter in response to its suspending fluid (14–16). The effect of the vehicle upon the polymeric membrane may cause a change in its pore size (17). 			

The certainty of obtaining sterile effluent requires far more than the identification of a sterilizing filter by a pore-size rating. The variety of influences governing the outcome of an intended sterilizing filtration necessitates a careful validation of the process, including the filter. The very drug preparation of interest, the exact membrane type, the precise filtration conditions, and the prefiltration bioburden, including specific organism type(s) and number, should be used in the necessary validation. 

Given the complexity of the organism removal operation, it is doubtful whether a universal sterilizing filter can be devised. Certainly, there is no known absolute filter, one that will retain all organisms under all conditions, especially if viruses are included. Therefore, the successful attainment of a sterile filtration with regard to specified organisms of interest must in every individual filtration sterilization be corroborated by the documented experimental evidence that constitutes validation. The specifics of such operations are discussed in the Parenteral Drug Association's "Technical Report No. 26." Adherence to the teachings of this report is a second factor that is essential to attaining sterile effluent. 

Obviously, the many organisms whose presence in pharmaceutical contexts deserves consideration may differ from one another so greatly as to compel individual techniques for their identification and cultivation. Conclusions cannot be made regarding the sterile filtration of microorganisms unless methods of quantifying them by culturing and counting are available. Organisms such as the L-forms, nanobacteria, and "viable but nonculturable" entities may not be amenable to such analyses. Concerns about their presence may be justified, but without the means to cultivate and count them, it is impossible to attest to their complete absence. It follows that a sterilizing filter can be judged only by its performance in the removal of identifiable and culturable organisms known to be present in the drug preparation (18). 

Model organisms, although perhaps necessitated at least as an opening gambit, will not truly serve their intended, more general use as surrogates for most other organisms. In the present circumstance, however, 

 can serve as a model as well as any bioburden organism. Albeit with surprise to some and consternation to others, 

 did fail to serve as a filtration model in some specific circumstances. Nevertheless, the failures were relatively few. Moreover, they were the result of organism size reductions occasioned for particular organisms by contact with particular drug preparations. Many involved in pharmaceutical filtration were unaware that an organism's morphology could reflect its manner of cultivation, as well as the nutritional value of its suspending fluid, but those facts are known to microbiologists. 

It was not, nor is it as yet, sufficiently recognized within the pharmaceutical industry that organisms penetrating the 0.2/0.22-μm-rated membranes were likely to have undergone size reductions occasioned by exposures to particular drug preparations (

Ralstonia pickettii, Burkholderia cepacia,

 in particular). The dependability of the exclusion mechanism was predicated on the stability of the organism and pore sizes as measured in certain specific situations. It does require modification when organisms or pore sizes do not remain constant. Each may undergo change upon contact with particular drugs. At present, little is known about the kinetics of these size alterations. 

There is no universal sterilizing filter. Sterile effluent results from a combination of many influences, not solely from the use of filters of any particular pore size. Each filtration is an independent event that arises from a proper combination of several stated requirements.

Sterility must be defined in terms of specific organisms that are targeted in a given filtration. The ability to identify and cultivate the organism of interest is indispensable.

 as the model organism likely to represent the bioburden of the drug preparations presented for aseptic processing by sterilizing filtration is limited to those microbes that do not undergo shrinkage upon contact with the drug preparations. Also, it is clear from considerable experience that risks arising from the use of the 

 model are small enough in the vast majority of cases to be considered completely insignificant to public health.

Choosing an actual bioburden organism to replace 

 as a filtration model is unlikely to improve the situation. The substituting organism will replace the present problems with those peculiar to itself. It is questionable whether a universal model is at all possible.

 has served its model role adequately, except for those organisms like itself that undergo size diminution during their cultivation or that manifest size shrinkage through contact with drug preparations that are nutritionally poor for them. Thus far, these have been limited to relatively few, 

 These Gram-negative rods can be kept to extremely low numbers in prefiltration bioburden with reasonable and effective bioburden control measures.

Bacterial challenge studies should be conducted under process conditions by means of inoculating the product to be evaluated with 

  and evaluating the filtrate for the presence of the challenge organism. In the event that 

 is not viable in the product formulation, precondition the filter by recirculating product through it to simulate process conditions and follow this with a microbial challenge by modifying the process to ensure the viability of the challenge organism (

 change temperature), modifying the formulation to ensure the viability of the challenge organism (

 adjust pH, remove bactericidal component), reducing the exposure time to ensure the challenge organism remains viable, or change from 

 to a microorganism that has been isolated from the formulation. It is important to use the product formulation if possible, because there have been instances in which 

 has penetrated a sterilizing filter in contact with the product formulation but has been retained by the same filter when inoculated into a surrogate fluid. 

The ability to predict the successful outcome of a particular sterile filtration event requires a filter manufacturing enterprise that is highly capable and consistent, a dependable and accurate method to conduct integrity testing, and possession of the understanding and ability to implement effective filtration validation strategies. 

is president of the Williamsburg Group, LLC, 18907 Lindenhouse Rd., Gaithersburg, MD 20879, tel. 301.869. 5016, 

is president of Akers Kennedy and Associates. 

is group vice-president of global product management, bioprocess of Sartorius North America. 

is a consultant for Capitola Consulting. Madsen, Akers, and Meltzer also are members of 

*To whom all correspondence should be addressed.

 (DHI Publishing, River Grove, IL, 1st ed., 2003). 

 2. Parenteral Drug Association, "Technical Report No. 26: Sterilizing Filtration of Liquids," 

 3. P.R. Johnston and T.H. Meltzer, "Comments on Organism Challenge Levels in Sterilizing-Filter Efficiency Testing," 

 (Marcel Dekker, New York, NY, 1st ed., 2001), p. 623. 

Guideline on Sterile Drug Products Produced by Aseptic Processing

 6. M.W. Jornitz, "Integrity Testing," in 

M.W. Jornitz, Ed. (Springer-Verlag, Heidelberg, Germany, 2006), pp. 143–180. 

 "Considerations in Sterile Filtrations, Part I: The Changed Role of Filter Integrity Testing," 

 8. F. Bowman, M.P. Calhoun, and M. White, "Microbiological Methods for Quality Control of Membrane Filters," 

 "An Application of Membrane Filtration for Removal of Diminutive Bioburden Organisms in Pharma Products and Processes," 

 10. M.S. Cooper, "Microbial Retentive Filtration," 

 11. R.E. Madsen, "Filter Validation," in 

 M.W. Jornitz, Ed. (Springer-Verlag, Heidelberg, Germany, 2006), pp. 125–141. 

Articles

Model organisms are useful when validating sterile filtration, but successful retention of the model organism does not always guarantee that effluent is sterile. The authors explore the various factors that influence sterile filtration. 

The Use of Model Organisms in Sterilizing Filtration

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.) 

Wallhäusser 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 "grow-through" 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 μm-rated membranes, are overstated as they pertain to pharmaceutical processing contexts. 

Curiously, organisms may, on occasion, be found on the downside of reverse osmosis (RO) membranes, although these have smaller "pores" than 0.1 μm-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—although seemingly avoided by the time restriction set on filtrations—remains 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). 

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 

 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 μm-rated membranes should be substituted for their 0.2/0.22 μm-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 "grow-through" 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. 

 reveal that with regard to certain organisms, 0.2- and 0.22-μm-rated membranes provided sterile effluent and/or a high titer reduction only for various lengths of time before penetration occurred (9). Five 0.2-μm-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 10

. Two 0.22-μm modified poly(vinylidene difluoride) (PVDF) hydrophilized filters showed bacterial penetration after 72 h, corresponding to a cumulative challenge level of 6.5–8.7 X 10

. Two 0.2-μm-rated nylon 66 filters in series were unable to fully retain 

 at 72 h, corresponding to a cumulative challenge of 2.4 X 10

. 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-μm-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 (120–196 h), up to challenge levels from 5.7 X 10

. Similar results were obtained with the PVDF filters tested: No 

 were detected at challenge levels from 5.9 X 10

. The organisms were sufficiently small to penetrate the corresponding 0.2-μm-rated membranes, however, and all 0.1-μm-rated filters tested provided consistent and complete retention of 

for the entire duration of the test (120–192 h).These results demonstrate that 0.1-μm-rated filter membranes provided sterile effluents under conditions that allowed bacterial penetration to occur through conventional 0.2- and 0.22-μm-rated sterilizing grade filters (9). 

On the basis of the above data, one can argue that 0.1-μm-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-μm-rated filters. Nevertheless, it is recognized that significant penalties are incurred by the unnecessary use of tighter membranes (11–13). 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-μm-rated membranes and the 0.2/0.22-μm-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-μm-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-μm-rated membranes that are faster flowing than their 0.2/0.22-μm-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-μm-rated membranes, remains unfulfilled. 

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-μm-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. 

 is group vice-president for product management at Sartorius North America, 131 Heartland Blvd., Edgewood, NY 11717. 

 is principal of Capitola Consulting Company, 8103 Hampden Lane, Bethesda, MD 20814-1244, tel. 301.986.8640. He also is a member of 

*To whom all correspondence should be addressed. 

FDA Guidance Document: Current Good Manufacturing Practice in the Manufacture, Processing, Packing, or Holding of Large Volume Parenterals for Human Use

2.  K.H. Wallhäusser, "Grow-Through and Blow-Through Effects in Long-Term Sterilization Processes," 

3.  J.A. Simonetti and H.G. Schroeder, "Evaluation of Bacterial Grow-Through," 

4.  D.A. Christian and T.H. Meltzer, "The Penetration of Membranes by Organism Grow-Through and Its Related Problems," 

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. 103–114.  

7.  PDA–FDA Special Scientific Forum, "Validation of Microbial Retention of Sterilizing Filters," Bethesda, Maryland, July 12–13, 1995. 

8.  T.J. Leahy and M.J. Sullivan, "Validation of Bacterial Retention Capabilities of Membrane Filters," 

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. 232–251. 

11.  K. Kawamura, M.W. Jornitz, and T.H. Meltzer, "Absolute or Sterilizing Grade Filtration—What is Required?" 

12.  J. Lindenblatt, M.W. Jornitz, and T.H. Meltzer, "Filter Pore Size 

 Process Validation—A Necessary Debate?" 

13.  M.W. Jornitz and T.H. Meltzer, "Sterilizing Filtrations with Microporous Membranes," 

The authors encourage the investigation into whether 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.

Grow-Through and Penetration of the 0.2/0.22 "Sterilizing" Membranes

The myriad of successful sterile drug preparations by way of filtrations attests to the pharmaceutical industry's confident application of this purification technique. The constant search for improvements in purification has resulted in a growing interest in multifilter arrangements, which offer greater assurances regarding organism retentions. Multifilter arrangements are often over-designed and the most successful are based on solid scientific principles.  

Committee for Proprietary Medicinal Products (CPMP) guidelines from the EU and FDA, and FDA's Aseptic Guide (2004) recommend the use of redundant or serial 0.2/0.22 µm-rated membranes. FDA's new aseptic guideline states: "Use of redundant filtration should be considered in many cases." This guidance is unspecific and reveals the level of uncertainty surrounding the subject.

This article will review and critique multifilter arrangements, and examine filter combinations in terms of flow rates and throughputs. Flow rates are important for their very practical significance with respect to filtration time, filling speed and filter contact time. Additionally, they connect to other factors that relate to time and durations, for example, organism proliferation and an increase in endotoxin. The utilitarian value of throughput is self-evident in terms of yield. It will also measure what has so far been attained (i.e., a 50–75% increase in throughput of filters in the last 15 years) within the operational limits set by the industry's unswerving drive for sterility. 

				Two (or more) filters can be arranged to  				

ensure against an imperfect final filter by using repetitive filters.

The latter is more common in Europe than in the US. However, there is a distinct difference between two 0.2 μm filters in series and redundant 0.2 μm filtration, meaning the two filters are directly connected to each other (Figure 1). No other equipment is in-between the two filters.  

Figure 1 Schematic of redundant and serial filtration.			

Two types of double-layered filters are available. The heterogeneous form consists of layers of dissimilar pore-size ratings. The homogeneous type is composed of two membranes of the same pore-size rating. The two different constructions are intended for different purposes.  

An example of a heterogeneous construction would be a 0.45 µm-rated membrane upstream from a 0.2/0.22 µm-rated final filter. The more open filter, in its position upstream of the tighter version, serves in the protective role of prefilter; accepting a portion of the particulate load, which prolongs the service life of the final downstream membrane (Figure 2). The flow rate is slowed by the longer overall flow path of the double construction. The slowing effect is greater than that of either single membrane.  

Figure 2 Schematic of a heterogeneous filter layer construction.			

When serial filtration is used, each filter is in its own housing and separated so can be tested independently. When the filter arrangement is redundant, neither filter can be tested without invading the space separating them unless some very unusual arrangements are devised. This is a disadvantage of redundant filter system designs.  

Homogeneous double constructions contain two membranes of similar pore size ratings, for example, both 0.2 µm-rated (Figure 3). This double-layer construction reduces the probability of particle passage. The intention is to ensure that even if an inappropriate or flawed membrane was somehow included, the second filter would serve as a safeguard; likewise, when single membranes cannot be cast to thicknesses sufficient to retain the target organism. All too often, the situation is that of a filter of an unknown pore size distribution being confronted by organisms of an unknown particle size distribution. The possibility of selecting an improper filter is conceivable.  

Figure 3 Schematic of a homogeneous filter layer construction.			

Unfortunately, a homogeneous double-layer configuration does not allow the detection of an integrity test flaw of only one membrane when the bubble point test is used. Therefore, there could be retention risks attached to the homogeneous double-layer design, when both membranes are required to be integral to ensure a sterile effluent. 

The flow rate through these double-filter constructions is less than through single membrane layers because the overall pore lengths are longer. The flow rate through homogeneous arrangements is less than through heterogeneous double filters because in the former combination both membranes have the tighter restrictions of the final filter. 

Repetitive filtration — in close contact design

In this case, the pore is conceived as being a convoluted, irregular capillary. It is, therefore, necessarily longer than the depth of the filter because of its tortuosity. Its restrictive dimensions may occur anywhere along its length. This assumption explains certain observations regarding repetitive filters. Thus, it would be expected that lengthening a pore — as by layering membranes — might proportionally increase the number of its restrictive regions. This should promote particle trapping. In effect, prolonging the pore length is the technological achievement that results when membranes are superimposed on one another in intimate contact. A second consequence is a homogenization of the pore size distribution. A pore with larger openings will likely juxtapose one with smaller openings because there are many more smaller pores. The result is a narrower — but longer — overall pore passageway. This occurrence assists sieve retentions by decreasing the number of larger (wider) pores. Moreover, the pore path length is doubled. This encourages adsorptive sequestrations. 

Repetitive filtration — space-separated layers

When two membranes separated from one another are used, a different result is obtained. Between the two membranes there exists a space where the fluid exiting the first membrane forms a pool from which it is hydrodynamically directed preferentially to the larger pores of the second membrane.  

Higher differential pressures can have the same effect as the direct and intimate superimposition of one membrane on the other. At the higher differential pressures, the effluent is more immediately directed perpendicularly from the pores of the top filter to those, of whatever sizes, of the second filter. There may not be enough time to permit the hydrodynamic flow to assert itself. This limits the influence of the hydrodynamic preference. 

This situation favours sieve retentions because the larger particles are denied time within the between-the-filters pool to orient towards the scarcer, larger pores of the second filter. Adsorptive retentions would seem to be marginally favoured given the preponderance of smaller pores (Figure 4).

  In these cases, filter efficiency is increased. Where the total suspended solids (TSS) content is large enough, albeit an unlikely condition, the system would incline towards lower throughputs.  

Figure 4 Adsorptive sequestration and bridging of particles.			

The common term used to describe the organism-reducing power of a filter is to state its log reduction value (LRV). In effect, FDA defines a sterilizing filter as one that can cause a seven log reduction in the titre of a suspension of unspecified magnitude. The LRV is log to the base 10 of the ratio of the organisms in the influent stream to those that emerge in the filtrate.

The LRVs of a redundant filter combination are not necessarily multiples of the single filter. The exact sum depends upon the probabilities of a given size organism encountering an appropriately sized pore within each membrane. In any case, the likelihood is that the organisms penetrating the upstream filter will be smaller. This shifts the organism size distribution confronting the second filter in the direction of smaller sizes. The smaller organisms are more elusive, making a lower LRV for the downstream filter more likely. In a case study the LRV of the second filters has been found to be 2–4 logs lower than the first filter.

  The situation where particles of an unknown size distribution challenge pores of an unknown size distribution is complex enough to permit numerous (but profitless!) hypothetical combinations to be considered. Nevertheless, perhaps the following question should be asked: If a given filter cannot completely retain the organism challenge, why should an identical second filter be expected to do so? Once again, the filter industry's neglect of the important area of quantifying membrane pore size distributions is evident. In any case, the literature attests to the nonadditivity of the LRVs of individual filters.

This term is often used to designate a repetitive or double-filter arrangement where the two filters are housed separately, but connected to each other. The term 'redundant' is usually associated with being wasteful or unnecessary. Its intended meaning in filtration is to imply 'enough and to spare'. Redundant filtration incurs the cost of two housings, but the separate housings permit the integrity testing of each filter, although the risks inherent to asepsis in prefiltration integrity testing still apply. Within a single housing the upstream testing of the downstream filter is manageable, but will require the isolation of the upstream filter. The housing modifications necessary to this action is rarely undertaken. 

The first filter is expected to fulfil the retention requirement.

  In the event of its failure, the second filter ensures this function. This arrangement is regarded as prudent by its practitioners. However, given the dependable performance of stipulated integrity testing, the endorsement of the use of backup (repetitive) membranes may also reflect some degree of personal insecurity. While understandable, this subjective reaction would be an intrusion into what ought to be an entirely technical decision.  

A filter's integrity can undergo damage during use; most likely (but not only) from in-line steaming. Hence, the requirement for final integrity testing.

  However, in the actual event, both filters would likely be affected similarly given their mutually shared exposures. Even where employed for reasons of prudence, the use of the repetitive filters is not necessarily justified economically.  

Calculations can be made to approximate the economics of enlisting repetitive filtrations to safeguard the quality (sterility) of an effluent. The question is: In the event of a failed filter, what would be the value of the drug product that had to be discarded, and what would be the cost of the additional filters needed to prevent the loss? Hypothetically, if a filter failure happens every 10000 batches, and the filtered batch has a value of $100000, the expense of saving the batch by using repetitive filters for every batch would be: 10000 batches×filter unit cost of $150=$1.5 million. The total gained by the saved batch is its value of $100000 plus $50000 in downtime and overheads that replacing the batch would have cost; a total of $150000. 

The expense of saving the batch by using repetitive filters for every batch would be the cost of $1.5 million less savings of $150000: a total loss of $1.35 million. The costs involved from a risk management standpoint do not substantiate a redundant filtration. However, some biologic drug products easily reach a value of $1.5 million a batch. In these cases an 'insurance' filter might be useful. However, a second filter adds to unspecific adsorption and consequently yield losses and possibly higher hold-up volumes. These costs need to be evaluated, before a decision can be made. The question that needs to be addressed is why an insurance filter might be required? When the process is appropriately validated, retentivity and performance of the filter should be ensured.  

Regarding the EU and FDA, both the European Agency for the Evaluation of Medicinal Products' (EMEA's) guideline (CPMP, April 1996)

  and the EC Guide to Good Manufacturing Practice Annex 1,

  recommend the use of redundant 0.2/0.22 µm-rated membranes. FDA's new aseptic guideline states: "Use of redundant filtration should be considered in many cases." The placement of the filters is to be "as close as possible" (or practical) to the filling needles. This will reduce the likelihood of contamination resulting from random biofilm shedding. The usual location is just before the filling needles or before the reservoir that feeds them. In practice, the 'recommendations' are enforced as if they were law. Thus, in Europe they are becoming common practice. 

The recommendation is not based on any known survey or experimental data, nor have the regulatory authorities explained their reasoning. As stated, overdesign is a normal response to uncertainties. The technically oriented individual will resist making unsupported decisions. He/she may insist on waiting for data. Those responsible for public safety may be obliged to make decisions when adequate supporting data are not yet available. 

So what is required regarding the integrity testing of the repetitive filters? If the downstream filter is intended as the final filter, then it alone needs testing. If the filter pair is being relied on to ensure against the likelihood of filter failure, both membranes should be tested in the hope that one will remain unflawed. From an engineering point of view this is manageable if the redundant membranes are housed separately. 

The employment of automated integrity test machines is especially advantageous as it avoids invading the system downstream of the filters that are housed separately. The threat of asepsis is, therefore, eliminated from the integrity testing activity. As previously said, placing both filters in the same housing makes it difficult to perform upstream integrity testing of the downstream filter. It is manageable, but would require the isolation of the upstream filter. 

The regulating authorities have, it seems, a preference for the application of multimembrane combinations to maximize organism retentions.

A review and critique of multifilter arrangements

Sterilizing grade membrane filters are widely used for the intermediate and final filtration of liquid drug products. Their function is either to reduce the bioburden level within intermediate production steps or to sterilize the final filled product. In 1918, Nobel Prize winner Richard Zsigmondy invented membrane filters that found commercial use in the pharmaceutical industry during the 1930s as flat filter devices (mainly 293 mm discs) and during the 1960s as pleated filter cartridges. When Pseudomonas diminuta (now Brevundimonas diminuta) penetrated 0.45 Âµm rated filters, the standard for sterilizing grade filters became 0.2 Âµm. In 1982, the Health Industry Manufacturers Association (HIMA) stipulated that these filters must retain 107 per cm2 B. diminuta; this is now (since 1988) required by the American Society for Testing and Materials (ASTM).1,2 Since this time, appropriate process validation has received greater priority than pore size ratings; under process conditions, such validation has provided a catalyst for improvement. This article evaluates performance developments of sterilizing grade filters during the last decade and the unfulfilled optimization potential of these systems compared with revalidation costs.	

Materials. Flat 293 mm disc holders (Figure 1) or manifolds were used in process filtration before pleated filter cartridge devices (Figure 2) were developed. Such manifolds had disadvantages including a large footprint, possible leaks, cross-contamination and difficult assembly. The first pleated filter cartridge devices were designed and built to provide a larger filtration area within a smaller filter. These devices had a filtration area of approximately 4000 cm2 and a cylindrical pleat pack that was resin-bonded to the end caps.

Figure 1:  A 293 mm flat filter holder.				

Polyester materials were commonly used in the prefilter and support fleeces in the filters. Both the polyester and the resin (used to bond the membrane to the end cap) gave the filters low chemical and thermal resistance because of oxidative degradation during use. Furthermore, the fleece and resin components added to increasing extractable levels, which would be unacceptable under current standards. Cellulose acetate, cellulose nitrate, mixed cellulose esters, polyamide and polyvinylidenefluoride were the most commonly used filter membrane materials. These materials usually included surfactant or membrane surface treatments to achieve pleatability, wetability and membrane stability. Large water flush volumes were required before the filter could be used to wash out any residual surfactant or treatment.		

Figure 2:  A resin-bonded filter cartridge.			

Optimization. The next development phase to optimize sterilizing grade filter products was replacing the resin bonding with heat welded thermoplastic materials used in the end caps, core, outer support and bonding materials (Figure 3). These filters demonstrated higher chemical, thermal and mechanical resistance, but still generated membrane weaknesses or potential membrane damage if an excess of welding material (commonly polypropylene melt) was forced into the tight pleats of the membrane. This could create hydrophobic spots that showed false negative integrity test results. Pleating of the membranes and the support layers developed further, and a higher pleat density was achieved resulting in significantly increased effective filtration areas from 0.4 m2 to up to 0.75 m2. To avoid inefficient use of the entire pleat pack, new fleece structures were developed to gain a wide enough gap between the pleatings. 	

Configurations. In addition to optimized prefilter fleeces, different membrane filter configurations were utilized as a single layer (for example, 0.2 Âµm only), a homogeneous double layer (for example, 0.2/0.2 Âµm) or a heterogeneous double layer filter (for example, 0.45/0.2 Âµm). Depending on the filter configuration, these filters displayed an effective filtration area of up to 7500 cm2. This represented a 15 fold increase compared with the 293 mm disc filter area and a 1.9 fold increase compared with the first pleated filter. The development of an effective filtration area resulted in a tremendous increase in total throughput for the industry; heterogeneous double layer membrane configurations increased the total throughput furthermore because of a fractionate retention of any contaminant. The contaminant load no longer restricted itself to the 0.2 Âµm final filter membrane.		

Figure 3:  A high pleat density filter cartridge.			

Ultrasonic technology. To avoid heat welding weaknesses, ultrasonic welding was utilized and is still currently used. This allowed the mechanical and thermal stability to increase to an extraordinary level, considering the fragility of the membrane material. With polymer evolution, additional treatment steps and new membrane refinements eliminated the use of wetting agents and surface treatments that previously caused increased extractable levels and high flush volumes. 	

Surface treatments were included within the membrane production process, leading to chemical integration in the membrane matrix instead of having an individual membrane modification step. This also meant that the hydrophilicity of the membrane was retained even under stringent conditions such as steam sterilization or sodium hydroxide contact. The leachable level of such membranes was greatly reduced because of bonding between the surface treatments and the membrane. End caps. Eventually, polyester end caps became obsolete in multiple use applications such as air filtration because of the oxidization and degradation of the polymer. The most common end cap and support material is now polypropylene, which complies with regulations; some new poly-propylenes are also gamma irradiation stable. Safety was also enhanced by improving the designs of filter cartridge adapters, which are now tightly installed into the recess of filter housings. Instead of a double step adapter (where one O-ring is smaller than the other [also known as 2216 and 2218]), adapters with double O-ring seals of similar size (also known as 2226) and with bayonet locks are commonly used.3,4 This type of adapter is more secure because of the firm seat of the filter cartridge housing, particularly during variable pressures, back pressures and steam sterilization conditions. The increase in effective filtration areas and the new membrane configurations allowed higher flow rates and total throughputs, which still incorporated the same geometric dimensions as the first filter cartridges (Figure 4). 	

Today, the design and construction of sterilizing grade membrane filters are optimized to fulfil the ever changing and increasingly stringent requirements of regulatory authorities and the biopharmaceutical industry. Optimization is continually evolving and has led to the following features of modern sterilizing grade filter elements:				

steam sterilization resistance up to 134 ÂºC and gamma irradiation resistance

mechanical resistance of 5000 pulsations at 5 bar differential pressure

optimal total throughputs to significantly reduce costs per litre

optimized flow rates to reduce the processing times or effective filtration area

integrity testing of common non-destructive tests, such as diffusive flow, pressure decay, water intrusion or bubble point

full validation documentation and individual process validation under process conditions with the actual drug product.

Major filter manufacturers not only improved their production processes, but also asked the biopharmaceutical industry to help find reliable solutions. Research and development (R&D) investment in filter elements enhanced aseptic process security and also improved production economics. Today, filter production is undertaken to the highest pharmaceutical quality standards. By receiving input from the industry and regulatory authorities, filter manufacturers are able to produce a wide range of optimized filter products.	

Table I:  Total cost calculation of a historic and modern optimized filter system.			

Savings. The total throughput of commercially available filters today is double compared with filters supplied 10 years ago (Figures 5 and 6). Improvements to reduce the effective filter area have resulted in a decrease in the running costs per litre and associated capital costs. Expensive multi-round stainless steel filter housings can now be replaced by single-round housings; a particular application that required a 3320 in. housing a decade ago, today only requires a single-round 30 in. housing. This, potentially, means a capital cost reduction of up to 70%. A similar cost calculation for filter elements can be demonstrated; assuming that a 10 in. filter element costs $150 and the filtration system is used for 200 days, then the consumable expenditure of multi-round versus single-round would be $900 and $450, respectively. This would give annual savings for consumables of up to $90000 (Table I). 	

Additional savings can be made but are often overlooked; for example, the integrity testing of multi-round housings (large volumes) is difficult and can result in longer downtimes, particularly in high water flush volumes. Assuming the net volume (housing including filter elements) of a 3320 in. multi-round housing is 6 L and that of a 1330 in. housing is 2 L, an additional 4 L are required just to fill the multi-round housing. Also, the assembly of 3320 in. filter elements requires a minimum of double the flush volume compared with a 1330 in. element. This would be 30 L more if it is assumed that there is only a 10 L flush volume per 10 in. filter cartridge. Therefore, the total amount of higher flush volume of water for injection (WFI) would be 34 L more at every filtration run, which would result in additional WFI usage of 6800 L.		

Figure 4:  A flow rate comparison of different sterilizing grade filter developments. Membrane material is 0.2 ÃÂµm cellulose acetate and Figure 5:  Total throughput comparison of 0.2 ÃÂµm sterilizing grade filter cartidges with polysulfon (PS) membrane materials.			

Savings differ greatly from application to application. In some, the throughput does not have much importance; however, processing time (flow rate through the filter element) is important. Some applications require a high batch volume filtration; therefore, an improved filter cartridge with enhanced flow rates allows high batch volumes to be processed faster without changing the equipment dimensions.	

Although membrane filter performance has greatly improved, some aseptic processes that utilize the filters have not advanced significantly. Filters resembling those used more than a decade ago are still used today - possibly because of regulatory implications or a reluctance to change.				

Obsolete filter products still seem to find a use, despite revalidation becoming easier5,6 and the savings that could be made by upgrading. All major filter manufacturers offer validation services and some provide a range of additional support services. Previously, such services and guidance were not available. Now, however, process validation and revalidation are necessary requirements that are well documented.	

Support services are additionally available when conditions change during stability tests. Filter manufacturers have also established thorough testing procedures, such as monitoring adsorption levels of individual membrane polymers.			

Figure 6:  Total throughput comparison of 0.2 ÃÂµm sterilizing grade filter cartidges composed of polyvinylidenefluoride (PVDF) membrane materials and Figure 7:  Throughput trials with different prefilters (47 mm discs).			

Because of economic pressures and the need for increased efficiency, the biopharmaceutical industry is looking to use the most up-to-date equipment whenever possible. This has meant improved quality systems being installed within the filter manufac-turing process. Although the performance of individual equipment can be improved, this does not imply that such improvements can only be achieved by exchanging types of filters. Improvements made to sterilizing grade membrane filters has meant that prefilters have also been thoroughly reviewed by manufacturers (Figure 7).3,7 Performance and economic improvements can be enhanced by finding the optimal solution or combination of pre- and final filter. To find this optimum, filter manufacturers have established software-driven, highly effective filterability test systems and methodologies.				

Initial trials are normally performed with 47 mm filter disc composites to establish the optimal retention rate/pore size combination. Once this optimal combination has been found, filter trials are performed with small-scale pleated filter devices. Filterability trials are useful in identifying the correct filtration combination and size; moreover, the filtrate of these trials can be used to test for any yield or activity losses resulting from unspecific adsorption. Yield losses, because of unspecific adsorption and undetermined dead volumes, must be avoided, particularly with low volume/high value products; for example, monoclonal antibodies or blood fractions. Any yield loss reduces the available production capability and product profitability. Evaluating filter size and material is a priority during the development and scale-up phases, and both should be evaluated within an existing filtration process.	

In addition to improvements in filter equipment, filter systems have developed to become disposable, such as capsule filters. These are available in larger sizes with up to 1.8 m2 of effective filtration area. The benefit of such systems is the reduction in cleaning and downtime, resulting in reduced costs. Operator protection is enhanced because the user is not exposed to the filter and separated contaminant during filter exchange. Installing disposable filter systems is easier and avoids the risk of possible O-ring damage, which can occur in classical filter systems.	

Sterilizing grade filters have experienced remarkable improvements during the last decade (such as thermal and mechanical stability), resulting in reliable, cost-effective filter units. Previous concerns regarding leachables from resins and surface treatments have almost been eliminated because of raw material and production process improvements. Additionally, the performance of sterilizing grade membrane filters (throughput and flow rates) has doubled, equating to lower costs per litre, and reduced downtimes and production times. Filter manufacturers are also providing a range of support services, and validation and revalidation support. 				

1.	ASTM Committee, "Standard Test Methods for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration," Annual Book of ASTM Standards (American Society for Testing and Materials, Philadelphia, Pennsylvania, USA, 1988) pp 790-795.				

2.	"Microbiological Evaluation of Filters for Sterilizing Liquids," HIMA Document 3, Volume 4 (Health Industry Manufacturers Association, Washington DC, USA, 1982).	

3.	M.W. Jornitz and T.H. Meltzer, Sterile Filtration - A Practical Approach (Marcel Dekker, New York, New York, USA, 2001).	

4.	P. Soelkner and J. Rupp, "Cartridge Filters" in T.H. Meltzer and M.W. Jornitz, Eds., Filtration in the Biopharmaceutical Industry (Marcel Dekker, New York, New York, USA, 1998).	

Sterilizing grade filters are widely used in the biopharmaceutical industry and were once thought of as being perfect. However, these filters have experienced rapid developments and improvements during the last decade, which have resulted in enhanced thermal and mechanical resistance. Moreover, their performance levels have been raised, which has led to significant cost savings within production processes.