as far as filtes go here's anarticle that blows .22s out of the water. hell .1s are not 100% effective dur to viruses and mycroplasms. il let you read it.
Introduction
Membrane filters have been used widely to sterilize liquids, especially those which cannot be heat-sterilized in the final container. The definition of a sterilizing filter has been the subject of much discussion and changes over the last 40 years. The initial definitions attempted to describe the physical structure of the membrane, assuming that it was a simple thin screen which removed bacteria on its surface. The concept of ‘pore size’ was therefore introduced and the classification of filters by differences in pore size rating was developed.
It is now known that 0.2µm sterilizing filters consist of a complex matrix of non-circular pores with a distribution of pore sizes within the structure, with some at least 0.5µm in size. This was shown very effectively by the scanning electron microscope studies of Osumi et al.1 An example is presented in Figure 1.
Figure 1
Scanning electron micrograph of surface of 0.2µm rated filter challenged with B. diminuta at 5 x 108cfu/cm2
Although the numerical ‘pore size’ definition helps us to classify filters into general groups such as 0.45µm, 0.2µm, 0.1µm, etc., a more meaningful definition must include some reference to its microbiological removal capability. In this way, the potential for specific bacterial types to be retained by the filter may be more accurately determined. This approach requires specification of the organism, the laboratory test method and a removal efficiency level if the definition is to become a meaningful and appropriate industry standard.
However, even a definition and specification based on removal efficiency does not necessarily guarantee sterility in the process. The filter may not be physically or chemically compatible with the process. Also, bacteria smaller than B. diminuta may be present. These diminutive forms may be naturally occurring or induced by process conditions. Process validation may be able to establish the most suitable sterilizing filter type and rating, but it is difficult to predict or monitor during routine production the type and quantity of bioburden present in every product batch. For this reason, there has been a major increase, especially over recent years, in the level of process validation required to justify the use of the current 0.2µm or 0.22µm sterilizing filters.
This publication reviews the evolution of sterile filtration with specific reference to penetration by specific organisms. New data are also presented on a diminutive organism, isolated from water, which can be grown in quantity under laboratory conditions without losing its diminutive form. This organism has been shown to penetrate various commercially available 0.2µm or 0.22µm sterilizing filters, but not 0.1µm filters. The implications for filter validation are discussed and the future role for 0.1µm filtration assessed.
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Evolution of Filtration Standards
0.45µm filtration standard.
In the 1960s, both sterilizing sheet filters and membrane filters were used in pharmaceutical production. Membrane filters were available mostly in flat disc form and used singly or in multi-plate configuration. 0.45µm membranes were successfully used for sterile production at that time as they enabled reasonable flow rates to be achieved through the relatively low area disc systems. The membranes were qualified using Serratia marcescens, with a typical size of 0.6µm x 1µm. However, the safe use of 0.45µm filters was questioned when Bowman2 established that an organism, Pseudomonas diminuta, could consistently penetrate 0.45µm ‘sterilizing’ filters, but could be retained by the next finer grade commercially available - 0.22µm.
0.2µm/0.22µm filter standard.
Bowman2 proposed in 1967 that P. diminuta (recently reclassified as Brevundimonas diminuta) should become the industry standard organism for 0.2µm filters. In 1987, the FDA ‘Guidelines on sterile drug products produced by aseptic processing’3 incorporated P. diminuta as the standard challenge organism for a sterilizing filter and defined a minimum qualifying level of 107/cm2 of filter area.
Since that time, no further standards have been developed, even though 0.1µm sterilizing filters in cartridge form have been available commercially for almost twenty years and are being increasingly used in production processes. The primary area of application was initially in the processing of serum and tissue culture media, where removal of mycoplasma is required. These deformable bacteria are known to penetrate 0.2µm or 0.22µm filters. However, there has been an increasing use of 0.1µm filters in other applications where diminutive organisms have been identified, or are of potential concern. They are also being used for enhanced sterility assurance in certain types of products or processes.
In the absence of a defined industry standard, filter manufacturers have qualified 0.1µm filters using their own standards. PALL uses Acholeplasma laidlawii, a mycoplasma type organism, for 0.1µm filter validation4 in addition to B. diminuta.
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Penetration Studies on Filter Cartridges
In a production environment, filter cartridges and not filter discs are normally used. It was therefore important to establish whether the more complex cartridge structure would give similar results to those using discs.
In these studies, a mixed challenge of B. diminuta and Pseudomonas sp. was used to show that the 0.2µm and 0.22µm filters retained B. diminuta while allowing penetration of Pseudomonas sp. The results of these cartridge studies are shown in Table III.
The results showed:
* Substantial and rapid penetration of all 0.2µm and 0.22µm filters by Pseudomonas sp. giving downstream counts of between 102 and 105 cfu.
* Full retention of B. diminuta.
* Total removal of both Pseudomonas sp. and B. diminuta by 0.1µm filters.
Further work is in progress to identify the diminutive species and to assess its suitability for wider use in filter qualification testing.
Table III
Mixed bacterial challenge of sterilizing filter cartridges *
Filter Type Rating
B. diminuta
Pseudomonas sp.
Sterile
Challenge Recovery Challenge Recovery
PALL
Ultipor® N66®
Grade NF/NR 0.2µm 1 x 1011
1 x 1011
9 x 1010 0
0
0 2 x 1010
6 x 109
2 x 109 1 x 105
1 x 104
1 x 104 No
No
No
PALL
Fluorodyne® II
Grade DFL 0.2µm 1 x 1011
7 x 1010
1 x 1011 0
0
0 1 x 1010
3 x 109
4 x 1010 1 x 104
2 x 102
7 x 102 No
No
No
Non-PALL
PVDF 0.22µm 1 x 1011
8 x 1010
8 x 1010 0
0
0 1 x 109
9 x 109
8 x 108 1 x 104
1 x 104
1 x 104 No
No
No
PALL
Ultipor® N66®
Grade NT 0.1µm 1 x 1011
1 x 1011
6 x 1010 0
0
0 2 x 109
3 x 109
4 x 1010 0
0
0 Yes
Yes
Yes
PALL
Fluorodyne® II
Grade DJL 0.1µm 1 x 1011
1 x 1011
2 x 1011 0
0
0 6 x 109
3 x 109
5 x 1010 0
0
0 Yes
Yes
Yes
*All studies used 254mm (10 inch) cartridges. Flow rate 0.3 L/min.
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Penetration Studies on Filter Discs
The first objective was to show that Pseudomonas sp. could consistently penetrate 0.2µm and 0.22µm filter discs and be retained by 0.1µm membranes. Various 47mm discs were selected and challenged with Pseudomonas sp. at 106cfu/cm2. Filter penetration was identified by passing the filtrate through a 0.1µm analysis disc downstream. The results are presented in Table II.
Table II
Removal of Pseudomonas sp. by sterilizing grade filter discs*
Filter Type Filter Rating Sterile Filtrate? Bacteria Recovery
PALL Ultipor® N66® Grade NR 0.2µm No >100cfu
PALL Fluorodyne® II Grade DFL 0.2µm No >100cfu
Non-PALL PVDF 0.22µm No >100cfu
PALL Ultipor® N66® Grade NT 0.1µm Yes 0
PALL Fluorodyne® II Grade DJL 0.1µm Yes 0
Non-PALL PVDF 0.1µm Yes 0
* 47mm discs challenged at 106cfu/cm2. Flow - 20ml/min. Filtration Time - 25min.
Penetration of all 0.2µm and 0.22µm disc filters was observed with high bacterial counts of >100cfu downstream. The three 0.1µm filters tested were fully retentive. Of equal importance was the short challenge time of 25 minutes, which would restrict any significant time-dependent penetration or bacterial growth. The concept of bacterial ‘growthrough’, or other time related effects, cannot therefore explain the extensive penetration obtained in these studies. The results suggest that the number and size of the bacteria challenging the filters are sufficient to exceed the removal capability (titer reduction) of the filters.
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Penetration of 0.2µm and 0.22µm Rated Filters
The European GMP Guide5 defines a sterilizing filter as having “a nominal pore size of 0.22 microns (or less), or with at least equivalent micro-organism retaining properties”, but then states that “Such filters can remove most bacteria and molds, but not all viruses or mycoplasmas”.
The FDA3 state that “Validation should include microbiological challenges to simulate ‘worst case’ production conditions particularly regarding the size of micro-organisms in the material to be filtered.” The FDA Guidelines accept B. diminuta as a sound basis for such assessment, but also state that “It is important to assure that actual influent bioburden does not contain micro-organisms of a size and/or concentration that would reduce the targeted high level of filtrate sterility assurance”.
These statements indicate that filter users must establish the possible risk of filter penetration and non-sterile product.
Since the 1960s, there have been occasional reports of bacteria other than mycoplasma species penetrating 0.2µm and 0.22µm sterilizing filters. Representative examples are given below:
• In 1967, Braun et al.6 reported penetration of waterborne bacteria (Spirillaceae) through 0.22µm membranes, during development of methods for isolating Leptospires.
• In 1980, Howard and Duberstein7 demonstrated penetration of a range of commercially-available 0.2µm and 0.22µm filters with naturally-occurring waterborne bacteria, including Leptospira species shown in Figure 2.
Figure 2
Leptospira species isolated downstream of 0.2µm sterilizing filters7
The same organisms were fully retained by a PALL Ultipor® N66® (grade NT) 0.1µm rated filter, as shown in Table I.
Table I
Removal of diminutive bacteria by 0.1µm filters7
Filter Number Filter Size Total Challenge Bacteria Recovered
1 142mm Disc 1.2 x 107 0
2 293mm Disc 8.9 x 107 0
3 254mm Cartridge 1.7 x 1010 0
*
In 1985, Andersen et al.8 reported penetration of 0.2µm filters by Burkholderia (Pseudomonas) pickettii in saline solution.
*
In 1993, the FDA reported presence of Pseudomonas cepacia in deionized water downstream of 0.2µm filters9. In the same document, they reported a product recall in the USA for a solution of povidone iodine which was also suspected to involve penetration of the 0.2µm filter by P. cepacia.
*
In a recent publication10, Leo et al. reported penetration of 0.2µm and 0.22µm filters by Burkholderia (Pseudomonas) pickettii when suspended in product, but not when suspended in saline lactose broth (SLB). The penetration was associated with a 40% reduction in bacterial size, as shown in Figure 3. Full retention was achieved using a PALL 0.1µm Ultipor® N66® filter. Further work is in progress to determine the mechanism of filter penetration.
Figure 3
Size distribution of B. pickettii suspended in product10
Events of this type have resulted in an increasing requirement by regulatory authorities for product and process specific filter validation. Such additional validation is required to be performed in a way that simulates as closely as possible the actual production conditions and also represents ‘worst case’3.
More recently, the need to challenge test in the actual drug product being filtered has been emphasised by the FDA11, who have stated: “Since there is the possibility that the drug product may cause a reduction in the size of the micro-organism, it is best to test the microbial retentivity of the filter with the microbial challenge in the actual drug product.” and “…There are products that fall within Millipore’s matrix which are not rendered sterile when filtered through a 0.22 micron filter.”
Despite increased validation for 0.2µm and 0.22µm filters, the FDA12 in 1996 have commented that the use of a 0.1µm filter as the final filter may be “desirable to ensure sterility of the product, especially when any contaminants are exposed to the drug product over a long period of time.”
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Short Term Penetration of 0.2µm and 0.22µm Filters
Filter penetration is often interpreted as a time-dependent and not size dependent occurrence. This can be explained by the difficulties in producing sufficient quantities of diminutive bacteria of constant morphology to assess the true removal performance of filters over short time periods. In addition, the diminutive species may be a very small sub-population of the natural bioburden. These factors have made it difficult to obtain reproducible results on filter penetration. Most studies have, therefore, been performed over extended time periods of several days, or weeks, in order to challenge the filters with significantly high levels of bacteria.
To overcome these limitations, an experiment was designed by Pall Scientific and Laboratory Services to try to recover and culture a single diminutive bacterial species from mains water so that extensive and controlled penetration studies could be performed over short time periods, ideally less than 1 hour.
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Isolation and culture of Diminutive Bacteria
Diminutive bacteria were obtained by filtering tap water through 0.45µm filter discs. The mixed population of bacteria obtained in the filtrate was repeatedly grown and filtered through 0.45µm filter discs. One of the species isolated was of special interest, as it was found to consistently penetrate 0.2µm filter discs. This bacterium, although not yet fully classified, has been identified as a Pseudomonas species and will be referred to a Pseudomonas sp. in this publication. A culture technique was developed that provided:
* A pure culture of the diminutive bacteria.
* No reversion to larger sizes.
* Sufficient quantities for bacterial challenge studies.
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Can 0.1µm Filters Reduce Validation Effort and Costs?
There is an increasing awareness that the current filtration standard for sterilizing filters does not necessarily guarantee sterility for all bacteria, under all conditions. Furthermore, the ability of bacteria to reduce in size due to interactions with the drug product, or the presence of low nutrient conditions, is further supported by the latest studies.
There is also the possibility that the bacteria present in the product may change from batch to batch as a function of production conditions, seasonal variations and other factors, which may be poorly understood and could make routine monitoring of bioburden type and quantity necessary . For these reasons, validation protocols for 0.2µm filters using B. diminuta are becoming increasingly complex.
One possible way to reduce the validation effort and cost is to enhance the sterility assurance provided by the filter. This can be achieved by using a filter with higher removal efficiency - a properly validated 0.1µm filter.
Although this option has been commercially available for almost twenty years, the limitations on flow capacity and filter life have restricted these filters to special applications. However, recent developments in filter technology have produced 0.1µm filters with flow rates comparable to some 0.2µm and 0.22µm filters and these are now being used successfully in a range of applications.
The enhanced sterility assurance provided by a properly validated 0.1µm filter may be the simple answer to the problem of validating the security of sterile filtration processes.
Although many filter suppliers offer 0.1µm grades, there is no international qualification standard for such filters. Therefore, it cannot be assumed that all filter cartridges rated as ‘0.1µm’ would provide the same removal efficiency as those tested in this study.
