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Meltzer Introduction When microporous membranes were first manufactured in the United States they were prepared in two porosity grades, namely, 1.2 um and 0.45 um. The latter type was intended for filtrative sterilizations. Early success with these filters led to their expanded usage. Novel situations became disclosed. Developments in our understanding of filtration phenomena followed. Particle arrests by filters had been seen as expressions of size exclusion, or sieve retentions situations wherein the particle was too large to enter, let alone pass through, the filter pore. However, Bowman et al (1969) found that an organism, later identified as Brevundimonas diminuta, normally retained by 0.45 um rated membranes, was not removed from its suspensions when a protein,penicillinase, was present. Clearly , something more than sieve retention was involved. These findings were later ascribed to adsorptive influences wherein protein molecules preemptively occupied the adsorptive sites, denying them access by the organisms. The filtration sterilization problem was, however, solved by reducing the rating of the sterilizing filter. Its porosity grade was halved to create the 0.2 um or 0.22 um rated membrane (0.2/0.22), as it was variously labeled by its different manufacturers. It came to be considered the sterilizing grade filter. Brevundimonas diminuta As just explained, the sterilizing grade filter was defined in terms of its retention of B. diminuta. To the extent that the mechanism of capture relied upon size exclusion, the size of the microbe relative to the pore size, organisms of smaller size could be expected to escape capture by penetrating the 0.2um rated “sterilizing filter”. Organisms smaller than B. diminuta were known. Indeed, Howard and Duberstein (1980) reported that smaller organisms present in Long Island well waters penetrated such filters. This situation was known to the FDA when in 1987 it defined the sterilizing filter as one that retained 1x107 B. diminuta per square centimeter of filter surface. Yet so successful was the application of the 0.2/0 .22 um rated membrane to filtrative sterilizations that occasional failures were often regarded as aberrations. A companion assumption was that the B. diminuta were perhaps among the smallest organisms commonly encountered in pharmaceutical contexts. Otherwise, the sterilizing actions of the 0.2 um rated filters it would seem, could not prove so successful. However, B. diminuta was selected to be the test organism for the validation of the 0.2/0.22 sterilizing filters not because it was presumed to be especially small, but because its size so closely matched the supposed dimension of the 0.2/0.22 um rating. It served, therefore, as the model organism for assessing filtrative sterilizations. A the time it was believed that the pore size rating described the diameter or openness of the pore and that this dimension also defined the minimum size of the particles the filter could retain. Both these concepts were later understood to be incorrect. Underlying these views was the belief that filtrative particle removal was solely the product of sieve retention. The adsorptive sequestration mechanism was not yet sufficiently appreciated.
Adsorptive Effects It became evident that adsorptive retention mechanisms operated to remove organisms by fixing them to filter surfaces. Sieve retention is not the only particle trapping mechanism. Elford (1933) showed that the degree of organism arrests was a function of the challenge density. Leahy and Sullivan (1978) demonstrated that it was influenced by the applied differential pressure. Tanny et al (1979) proved the same and recognized that it reflected adsorptive phenomena. The adsorptive influences may derive from charge interactions or from the hydrophobic effect of lowering the free surface energy of the system. In either case, the dimensions involved in the electric double layer are important (Jornitz and Meltzer 2001). These may be modified by the properties of the organismsuspending medium, e.g. by its ionic strength, pH, etc. (Levy et al 1990,Mittelman et al 1997 , Meltzer et al 1998). It, therefore, follows that achieving the filtrative sterilization of a drug preparation may be dependent on the peculiarities of the drug solution as well as on the filter (PDA Report # 26). Restated, a given filter used to sterilize a given drug may or may not be effective, depending upon the filtration conditions and the nature of the pharmaceutical preparation. It is for this reason that filtrative sterilization operations require being product validated, as well as process validated; the latter under worst case conditions; those that are least conducive to adsorptive organism captures. Shrinkage In Organism Size One of the reasons requiring that filter validations involve the very pharmaceutical preparations of interest, is that the drug formulation may cause shrinkage of the organism; frustrating efforts to filter it as based on its normal size. Geesey (1987), Gould et al (1993) and others have demonstrated this occurrence, as triggered by conditions of poor nutrition. As, expressed by Gould (1993), “Bacteria invoke a number of survival mechanisms in the transition of feast to famine. They minimize cell size in order to maximize surface-to-volume ratio. By becoming small round cells or very thin long cells, they minimize the amount of cellular material they need to synthesize, while maximizing their contact with scarce nutrients”. Bergey (1986) stated,”sizes in culture are not the same as under industrial process conditions”. In particular, Leo et al (1997) demonstrate by way of scanning electron micrography that Burkholderia (formerly Pseudimonas) pickettii diminish in size as a result of exposure to an actual drug product. The organism in its reduced size penetrated 0.2/0.22 um rated filters that restrain them when they are of normal sized. Their alterations in size and morphology are significant. The ability of an organism to penetrate a filter previously able to arrest it can manifest itself as a function of exposure time to the suspending solution. It is possible that this reflects the rate of size alteration. It is interesting to speculate regarding the relationship of this phenomenon to that of grow-through. Differences in rates of size-changes, depending upon the preparation and the organism, could explain the experimental difficulties that have beset grow-through investigations naware of the character of the particular organism / drug interaction. Organisms Smaller Than B. diminuta Regardless of the particle capture mechanism involved, it became increasingly evident that the 0.2/0.22 um rated filter was not universally effective in providing sterile effluent. Likewise, B. diminuta exhibited limitations as a model for some of the organisms whose removal from drug preparations was sought. Increasingly, smaller organisms seemed more often encountered or the failure of the 0.2/0.22 um rated membrane to yield sterile filtrate was better recognized. Sundaram et al (1999) present a list of such occurrences There was a heightened apprehension of organisms that could be present, including L-forms, nanobacteria, viable but nonculturables, and, by inference, Others yet unknown (Hargreaves 1993, Haycock et al 1994). The FDA (CDER 1996) states, ”Recently, several instances have come to the Agency’s attention, where passage of a drug product through a 0.2 micron rated filter was not sufficient to remove contaminating microorganisms”.
Suggested Substitution of 0.1 um for 0.22 um Some suggest, therefore, that 0.1 um rated membranes be substituted for their 0.2/0.22 um rated counterparts as the sterilizing filters. This is seen as a prudent action ensuring the likelihood of removing organisms smaller than B. diminuta, including the worrisome as-yet-unknown but possibly-present varieties, from pharmaceutical preparations. The situation is identical to that of the 1960's “when the pharmaceutical industry changed rapidly from sterilizing filters of 0.45 um to 0.2 um when the existence of B. diminuta was confirmed (Agalloco 1998). There is , it seems, an atavistic flight to tighter filters. However, since that time, (1987), FDA has advised consideration of the size of the bioburden in the product fluid as being essential in guiding the filter choice. Threats of smaller organisms will not be allayed by the use of 0.1 um rated filters. There will always be those who fear smaller or unknown organisms. If the correct general approach is to employ tighter than 0.2/0.22 um rated filters as a prophylactic safeguard, then perhaps the semiconductor industry usage of 0.04 um rated membranes , or of ultrafilters, to effect particle (organism) removal should be emulated. Bioburden studies and appropriate validations should be relied upon to indicate the proper filter and filter rating required for the sterilization of a particular drug preparation. As stated by Agalloco (1998), “The filter user must have knowledge of the characteristics of the material being filter sterilized. Screening of the bioburden in the product fluid is necessary and should be considered standard practice in the validation of any sterilizing grade filter.” The Filtration Sterilization Standard The FDA definition of a sterilizing filter is one that retains challenges of 1x107 B. diminuta per cm2 of filter surface. This capacity, while identified with the 0.2/0.22 um rated membranes, and assumed to derive from sieve retentions, would seem innately to apply to tighter filters as well, with the same assumption. Leahy and Sullivan (1978) demonstrated that a mixed esters of cellulose membrane did so perform under applied differential pressures of both 5 and 50 psi. Given the strong reciprocal influence of applied differential pressure on adsorptive sequestrations (Tanny et al 1979), it is reasonable to conclude that sieve retention is the operative capture mechanism in the Leahy and Sullivan case. Other cases may involve adsorptive captures as well. As Tanny et al (1979) also showed, 0.45 um rated membranes perform as sterilizing filters when the applied differential pressure is low enough to enable adsorptive arrests to augment the reduced sieving characteristic of the more generous 0.45 um pore dimension. It is evident, then, that filtration conditions can influence the attainment of sterile effluent. Therefore, process validation of the filter action is required. However, a sterilizing filter should be defined by its performance under the specified conditions of its operation, regardless of its pore size rating. The 0.1 um rated Filter There is no industry standard by which filter manufacturers rate any of their membranes. It is for this reason that the FDA defined the 0.2/0.22 um rated filter in terms of the 1 x 107 cfu/cm2 challenge. Otherwise these filters can differ, and do; as in flow rate characteristics, which, incidentally, also reflect pore sizes. Tolliver and Schroeder (1983) showed (Table1) that five different 0.2/0.22 um rated membranes all of which met the FDA definition of sterilizing grade filter differed in their retention o f 0.198 um latex particles; especially so when adsorptive influences were blocked by the presence of surfactant.. It follows that while the five membranes examined all qualify as sterilizing filters, they each have different pore dimensions. Small wonder that the various 0.2/0.22 um rated filters exhibit differences in retentions, particularly given the dissimilar organism adsorption propensities of their several polymeric compositions (Ridgway 1987).
The 0.1 um rated membranes lack even the modicum of standardization afforded by the FDA definition for their 0.2/0.22 opposites. A filter manufacturer can for any reason he considers proper and valid label any filter 0.1 um rated. This is not a situation that lends conviction to the application of 0.1 um rated filters for general use. Discrepancies between the pore size ratings of 0.1 um rated membranes and their latex trapping properties were probed by Wrasidlo et al (1983). These investigators examined three 0.1 um rated filters, namely, a track-etched polycarbonate, a nylon 66, and an asymmetrical polysulfone. The polysulfone and the tracketched filters retained essentially 100% of a challenge of 0.198 um latex particles, whether in the presence or absence of surfactant. The indifference to surfactant indicates an absence of adsorption. The particles, essentially 0.2 um in size, were completely sieve retained by the 0.1 um rated filters The nylon 66 retained the 0.198 um challenge 99.4% in the absence of surfactant, but slightly less than 89% in its presence, an indication of some adsorptive influence, as enabled by the more open construction of the nylon membrane. These same filter types in their 0.1 um manifestations were challenged by 0.085 um latex particles. The polysulfone filter retained 100% without surfactant and 99.7% with; an attestation of its appropriate retention rating. The polycarbonate membrane captured 99.4% of the latex particles in the absence of surfactant, and 47% when surfactant was present. The latter figure, indicative of the sieve retention level, attests that the filter is more open than its assigned rating indicates. Were it truly a 0.1 um membrane, the retention would have been (100%), completely by sieving. The latex retention by the nylon 66 filter was even more revealing. In the absence of surfactant its retention was 40%; in its presence 38%. Clearly, the nylon 0.1 um rated filter is not 0.1 um particle retentive. Yet, all three filters, in addition to others not tested, are described as being 0.1 um rated. Obviously, the 0.1 um rating is not a meaningful guide. At present, it cannot be depended upon to ensure retentions unequivocally based on particle size. Substituting 0.1 for 0.2/0.22 Membranes In support of the advocacy that 0.1 um rated filters should be substituted in place of their 0.2/0.22 um rated counterparts, Sundaram et al (1999) demonstrated that a number of organisms that are able to penetrate 0.2/0.22 um rated membranes were retained by 0.1 um rated filters. The organisms were identified as: Acidovorax (formerly Pseudomonas) avenae citrulli, Comamonas (formerly Pseudomonas) acidovorans, Hydrogenophaga (formerly Pseudomonas) pseudoflava and Janthinobacterium lividum. It has been suggested that H. pseudoflava, which like B. diminuta, is said to be found in pharmaceutical waters, be utilized as the test organism for 0.1 um rated membranes. It is not inappropriate, however, to ask how often it or the other of these organisms is encountered in drug products. “To sterilize a fluid we need only utilize a filter which will quantitatively retain the microorganisms that are normally present in the fluid” (Agalloco 1998). It is not necessary , however useful and desirable, that the sterilizing filter selected to accomplish its purpose be capable of universally retaining all types of microbes, whether present or not . Colwell and her associates (Shahamat et al 1999) found, as reported by Cooper (Apr. 2000), that B. diminuta, three strains of Ralstonia pickettii, Pseudomonas aeruginosa and two water isolates penetrated 0.2/0.22 um rated membranes as a function of time. These organisms were, however, restrained by 0.1 um rated filters that were validated using Acholeplasma laidlawii. Nevertheless it would be incorrect to assume that organisms escaping 0.2/0.22 um rated filters are necessarily restrained by their 0.1 um rated opposites. Sundaram et al (1999), in the above referenced work, state that the organism types that were found to penetrate the 0.2/0.22 um rated filters also penetrated three of the seven 0.1 um rated filters that were challenged !
What significance does the 0.1 rating label have when the very organisms that pass through the 0.2/0.22 um rated filters also escape so large a proportion of the 0.1 um rated membrane that are tested? The present status of the 0.1 um rated filter does not invite confidence. Sundaram and associates point out that the 0.1 um rated membranes that do remove the organisms that penetrated their 0.2/0.22 counterparts are validated to retain Acholeplasma laidlawii, a mycoplasma type, as well as B. diminuta.. The retention of this mycoplasma seemingly being key to the 0.1 um rating process, these investigators logically urge its use as a standard for defining the membrane. Other manufacturers of such filters agree, but there is no standard testing protocol. The lack of such a standard impugns the reliability of the A. laidlawii challenge results (Lindenblatt 1999). The reported findings translate to an endorsement of a single manufacturer’s 0.1 um rated filters; hardly a situation upon which to establish a new sterilizing grade filter for general use. Universal Sterilizing Filter It may not be possible to designate a single filter rating to serve as a sterilizing grade for all the possible organisms that could be encountered. This would require the singular condition of absolute filtration wherein the smallest organism would be larger than the largest pore of that filter’s pore size distribution. Only in this circumstance would influences associated with adsorptive captures be eliminated, obviating the need for process validation. Even so, to ensure that the organism and /or pore sizes are not altered by the liquid media, product validation would be required.. If devising such a single rating were possible, the pores would be so small as to seriously restrict flow rates and throughputs. In that situation, practicality might well compel the use of tangential flow arrangements, higher differential pressures and /or prefiltration. The present status of filtrative sterilizations does not seem to make possible a particular filter rating suitable for the removal of all organisms regardless of size. In the not unlikely situation that a universal sterilizing grade filter cannot be found, the present 0.2/0.22 um rated filters w ill do as well as can presently be managed. Their performance has largely been positive, and the pharmaceutical industry’s experience with them has been quite favorable since their inception. When necessary their usefulness can be modified, as directed by bioburden studies, to filters having higher or lower ratings, to secure sterile effluent without unnecessarily compromising flow rates and throughputs. The sterilizing filter’s identity is not confined to any particular pore size rating. It is recognized by its performance under stipulated conditions and with regard to the specific drug preparation filtered. Although not universally applicable, the 0.2/0.22 um rated membranes should be continued to be regarded as the sterilizing grade filters for the majority of pharmaceutical applications.
References -Agalloco, J. (1998), “Guest Editorial - It Just Doesn’t Matter”, PDA J. Pharm. Sci. Tech 52 (4): 149 - 150 -Bergey’s (1986), Manual of Systematic Bacterio logy, Vols 1 & 2, pg. 24, Williams and Wilkins, Balto, MD. -Bowman, F.W., Calhoun, M.P. and White, M. (1967), “Microbiological Methods for Quality Control of Membrane Filters”, J. Pharm. Sci. 56: 222 - 225. -Cooper, M.S. (Apr. 2000), “Microbial Retentive Filtration”, Vo l. 18 (1): 1-2. -Elford, W.J. (1933), “The Principles of Ultrafiltration as Applied in Biological Studies”, Proceedings of the Royal Society, 112-B, pp 384 - 406, London, U.K . -Food and Drug Administration (1987), “Guideline on Sterile Products Produced by Aseptic Processing”, Division of Manufacturing and Product Quality, Office of Compliance, Center for Drugs and Biologics, Rockville, MD. -Food and Drug Administration (1996), “Perspectives on Isolator Technology”, Center for Drug Evaluation and Research, (CDER), Rockville, MD. - Geesey, G.G. (1987), Chapter1, “Survival of Microorganisms in Low Nutrient Waters” in Biological Fouling of Industrial Water Systems - A Problem Solving Approach, Eds. Geesey, G.G. and Mittelman, M.W., Water Micro Associates, Long Beach, CA. -Gould, M.J., Dawson. M.A. and Novitsky, T .J. (1993), “Evaluation of Microbial / Endotoxin Contamination Using the LAL Test “, Ultrapure Water 10 (6): 43 - 47. -Hargreaves, P. (1993), “Paul Hargreaves Speaks Out On Pharmaceutical Manufacturing”, PDA Letter 29: 10 13. -Haycock, J.W., Joho, K.E., Gaylon, J.C., and Moses, R.L. (1994), “Bacterial L-forms Are Omnipresent in ‘Sterile’ Tissue Culture Sera”, Tissue Culture Conf. Assoc., Regulation of Cell & Tissue Differentiation, Research Triangle Park, NC. -(HIMA) Health Industries Manufacturing Association (1982), “Micribiological Evaluation Of Sterilizing Liquids”, Document 3, Volume 4, Wash., D.C. Filters for
-Howard, Jr., G., and Duberstein, R. (1980), “A Case of Penetration of 0.2 um-rated Membrane Filters by Bacteria”, J. Parenteral Drug Assoc. 34 (2): 95 -102. -Johnston, P.R. (1998), Chapter 1, “Liquid Flow Through Filter Media”, in Fluid Sterilization by Filtration”, 2nd Ed., Interpharm Press, Buffalo Grove, IL. -Jornitz, M.W. and Meltzer, T.H. (2001), “Mechanisms of Particle Removal from Liquids “, Chapter 5 in Sterile Filtration - A Practical Approach”, Eds. Jornitz, M.W. and Meltzer, T.H., Marcel Dekker, New York, NY. -Kawamura, K., Jornitz, M.W. and Meltzer , T .H. (2000), “Absolute or Sterilizing Grade Filtration - What is Required ? ”, PDA J. Pharm Sci Tech (In Press).
-Leahy , T.J. and Sullivan, M.J. (1978), “Validation of Bacterial Retention Capabilities of Membrane Filters”, Pharm Tech 2 (11): 64 - 75. -Leo, F., Auriemma, M., Ball,P., and Sundaram, S. (1997), “Application of 0.1 um Filtration for Enhanced Sterility Assurance in Pharmaceutical Filling Operations”, Pall’s Scientific & Technical Report, PPB - STR 30. -Levy, R.V.., Souza, K.S., Hyde, D.L., Gambale, S.M. and Neville, C.B. (1991), “The Matrix Approach: Microbial Retention Testing o f Sterilizing Grade Filters with Final Parenteral Products, Part II “, Pharm Tech 15: 58 - 68. -Lindenblatt, J. (1999), “0.1 um Versus 0.2 um Final Filtration in BFS Operations” Minutes From AGM, BFS News, Autumn Edition, pp 44 - 48. -Meltzer, T.H., Jornitz, M.W. and Mittelman, M.W., (1997), “ Surrogate Solution Attributes and Use Conditions: Effects on Bacterial Cell Size and Surface Charge Relevant to Filter Validation Studies”,PDA Second Asia Symposium, Feb 17 - 19, Osaka, Japan. -Mittelman, M.W, Jornitz, M.W,and Meltzer,T.H.(1998),“Bacterial Cell Size and Surface Charge Characteristics Relevant to Filter Validation Studies”, PDA J. Pharm Sci Tech 52(1):37 - 42. -PDA Technical Report #26 (1998), “Sterilizing Filtration of Liquids”, PDA J. Pharm Sci Tech Supplement Vol. 52, Number S1 -Shahamat, M.., Levin,M., Green, B., Felkner, C. Maugel,T. and Colwell, R. 1999), “Bacterial Retention by Filters”, Presentation at American Society of Microbiology, 99 th General Meeting, Chicago, IL. -Sundaram,S., Eisenhuth, J., Auriemma, M., Howard Jr., G. and Brantwein, H. (1999), Retention of ‘Diminutive’ Water-borne Bacteria by Membrane Filtration”, PDA Annual Meeting, December. -Tanny, G.B., Strong, D.K., Presswood, W.G., and Meltzer,T.H. (1999), “The Adsorptive Retention of Pseudomonas diminuta by Membrane Filters”, J. Parenteral Drug Assoc. 33 (1): 43 - 51. -Tolliver, D.L and Schroeder, H.G. (1983),“Particle Control in Semiconductor Process Streams”, Microcontamination 1(1):34 - 43 and 78. -Wrasidlo, W., Hofmann, F., Simonetti, J.A. and Schroeder, H.G. (1983), “Effect of Vehicle Properties on the Retention Characteristics of Various Membrane Filters”, PDA Spring Meeting, San Juan, Puerto R ico.
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