You have also bacteria penetration through 0.2µm filters but it's if you compared them to the 0.45 it's big differences bacause you have more a heavy particules who pass thru these .45 filters
Even filtering through a 0.20 micron filter doesn't make a filtered solution sterile as you can see here:
Diminutive Bacteria Implications for Sterile Filtration
P.T. Blosse*, E.M. Boulter* and S. Sundaram**
Acknowledgements
The authors wish to acknowledge the conscientious experimental work performed by Vanessa Gover
and Carmen Chinnery of the Microbiological Laboratories, Pall Scientific and Laboratory Services,
Portsmouth, UK.
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.
Summary
In the 1960s, sterilizing filters used for liquids were based on 0.45µm membranes, but when
penetration by Brevundimonas (Pseudomonas) diminuta was reported, 0.2µm /0.22µm was adopted
as the new standard. It is now recognized that bacteria smaller than B. diminuta exist. Furthermore,
occasional filter penetration has been reported for certain product and process conditions. Filter
validation has therefore become more complex because of the need to simulate the process during
challenge testing.
A diminutive pseudomonas species has been isolated and cultured successfully to allow detailed filter
penetration studies to be performed. Consistent penetration of 0.2µm and 0.22µm filters has been
shown in challenge tests of 60 - 90 minutes. Full retention was obtained with 0.1µm filters. The recent
availability of high flow and high efficiency 0.1µm filters can provide enhanced sterility assurance and
may help to simplify the problem of validating sterile filtration processes.
Introduction
Membrane filters have been used widely to sterilize liquids, especially those which cannot be heatsterilized
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.
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.
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
*All studies used 254mm (10 inch) cartridges. Flow rate 0.3 L/min.
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
* 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.
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 nonsterile
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 0.1µm rated filter, as
shown in Table I.
Table I
• 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
filter. Further work is in progress to determine the mechanism of filter penetration.
Figure 3
Size distribution of B. pickettii suspended in product10
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.”
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.
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.
Please Scroll Down to See Forums Below 
