Diminutive Bacteria Implications for Sterile Filtration Page 1 of 7

Diminutive Bacteria Implications for Sterile Filtration

P.T. Blosse*, E.M. Boulter* and S. Sundaram**

Pall Scientific and Laboratory Services.

z Acknowledgements
z Isolation and Culture of Diminutive Bacteria
z Summary
z Introduction
z Evolution of Filtration Standards
z Penetration Studies on Filter Cartridges
z Penetration Studies on Filter Discs
z Penetration of 0.2µm and 0.22µm Rated Filters
z Short Term Penetration of 0.2µm and 0.22µm Filters
z Can 0.1µm Filters Reduce Validation Effort and Costs?
z References

Acknowledgements

The authors wish to acknowledge the conscientious experimental work performed by Vanessa G
Carmen Chinnery of the Microbiological Laboratories, Pall Scientific and Laboratory Services, Po

Top

Isolation and Culture of Diminutive Bacteria

Diminutive bacteria were obtained by filtering tap water through 0.45µm filter discs. The mixed po
bacteria obtained in the filtrate was repeatedly grown and filtered through 0.45µm filter discs. One
isolated was of special interest, as it was found to consistently penetrate 0.2µm filter discs. This b
although not yet fully classified, has been identified as a Pseudomonas species and will be referr
Pseudomonas sp. in this publication. A culture technique was developed that provided:

z A pure culture of the diminutive bacteria.

z No reversion to larger sizes.
z Sufficient quantities for bacterial challenge studies.

Top

Summary

In the 1960s, sterilizing filters used for liquids were based on 0.45µm membranes, but when pene
Brevundimonas (Pseudomonas) diminuta was reported, 0.2µm /0.22µm was adopted as the new
now recognized that bacteria smaller than B. diminuta exist. Furthermore, occasional filter penetr
reported for certain product and process conditions. Filter validation has therefore become more
because of the need to simulate the process during challenge testing.

A diminutive pseudomonas species has been isolated and cultured successfully to allow detailed
studies to be performed. Consistent penetration of 0.2µm and 0.22µm filters has been shown in c
of 60 - 90 minutes. Full retention was obtained with 0.1µm filters. The recent availability of high fl

http://www.pall.com/34445_3813.asp 2/20/2007
Diminutive Bacteria Implications for Sterile Filtration Page 2 of 7

efficiency 0.1µm filters can provide enhanced sterility assurance and may help to simplify the pro
validating sterile filtration processes.

Top

Introduction

Membrane filters have been used widely to sterilize liquids, especially those which cannot be hea
the final container. The definition of a sterilizing filter has been the subject of much discussion an
the last 40 years. The initial definitions attempted to describe the physical structure of the membr
that it was a simple thin screen which removed bacteria on its surface. The concept of ‘pore size’
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
pore sizes within the structure, with some at least 0.5µm in size. This was shown very effectively
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 1

Although the numerical ‘pore size’ definition helps us to classify filters into general groups such a
0.2µm, 0.1µm, etc., a more meaningful definition must include some reference to its microbiologi
capability. In this way, the potential for specific bacterial types to be retained by the filter may be
determined. This approach requires specification of the organism, the laboratory test method and
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 gu
in the process. The filter may not be physically or chemically compatible with the process. Also, b
than B. diminuta may be present. These diminutive forms may be naturally occurring or induced
conditions. Process validation may be able to establish the most suitable sterilizing filter type and
difficult to predict or monitor during routine production the type and quantity of bioburden present
batch. For this reason, there has been a major increase, especially over recent years, in the leve
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
organisms. New data are also presented on a diminutive organism, isolated from water, which ca
quantity under laboratory conditions without losing its diminutive form. This organism has been s
penetrate various commercially available 0.2µm or 0.22µm sterilizing filters, but not 0.1µm filters.
implications for filter validation are discussed and the future role for 0.1µm filtration assessed.

Top

Evolution of Filtration Standards

0.45µm filtration standard.

http://www.pall.com/34445_3813.asp 2/20/2007
Diminutive Bacteria Implications for Sterile Filtration Page 3 of 7

In the 1960s, both sterilizing sheet filters and membrane filters were used in pharmaceutical prod
Membrane filters were available mostly in flat disc form and used singly or in multi-plate configura
membranes were successfully used for sterile production at that time as they enabled reasonable
achieved through the relatively low area disc systems. The membranes were qualified using Serr
marcescens, with a typical size of 0.6µm x 1µm. However, the safe use of 0.45µm filters was que
Bowman2 established that an organism, Pseudomonas diminuta, could consistently penetrate 0.4
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) s

the industry standard organism for 0.2µm filters. In 1987, the FDA ‘Guidelines on sterile drug pro
by aseptic processing’3 incorporated P. diminuta as the standard challenge organism for a steriliz
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 i
have been available commercially for almost twenty years and are being increasingly used in pro
processes. The primary area of application was initially in the processing of serum and tissue cul
where removal of mycoplasma is required. These deformable bacteria are known to penetrate 0.
filters. However, there has been an increasing use of 0.1µm filters in other applications where dim
organisms have been identified, or are of potential concern. They are also being used for enhanc
assurance in certain types of products or processes.

In the absence of a defined industry standard, filter manufacturers have qualified 0.1µm filters us
standards. PALL uses Acholeplasma laidlawii, a mycoplasma type organism, for 0.1µm filter valid
addition to B. diminuta.

Top

Penetration Studies on Filter Cartridges

In a production environment, filter cartridges and not filter discs are normally used. It was therefo
establish whether the more complex cartridge structure would give similar results to those using d

In these studies, a mixed challenge of B. diminuta and Pseudomonas sp. was used to show that
0.22µm filters retained B. diminuta while allowing penetration of Pseudomonas sp. The results of
studies are shown in Table III.

The results showed:

z Substantial and rapid penetration of all 0.2µm and 0.22µm filters by Pseudomonas sp. giv
counts of between 102 and 105 cfu.
z Full retention of B. diminuta.
z 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
qualification testing.

Table III

Mixed bacterial challenge of sterilizing filter cartridges *

Filter Type Rating B. diminuta Pseudomonas sp.
Challenge Recovery Challenge Recovery
PALL 0.2µm 1 x 1011 0 2 x 1010 1 x 105
Ultipor® N66® 1 x 1011 0 6 x 109 1 x 104
Grade NF/NR 0
9 x 1010 2 x 109 1 x 104
PALL 0.2µm 1 x 1011 0 1 x 1010 1 x 104
Fluorodyne® II 7 x 1010 0 3 x 109 2 x 102
Grade DFL 0
1 x 1011 4 x 1010 7 x 102

http://www.pall.com/34445_3813.asp 2/20/2007
Diminutive Bacteria Implications for Sterile Filtration Page 4 of 7

Non-PALL 0.22µm 1 x 1011 0 1 x 109 1 x 104

PVDF 8 x 1010 0 9 x 109 1 x 104
0
8 x 1010 8 x 108 1 x 104
PALL 0.1µm 1 x 1011 0 2 x 109 0
Ultipor® N66® 1 x 1011 0 3 x 109 0
Grade NT 0 0
6 x 1010 4 x 1010
PALL 0.1µm 1 x 1011 0 6 x 109 0
Fluorodyne® II 1 x 1011 0 3 x 109 0
Grade DJL 0 0
2 x 1011 5 x 1010

*All studies used 254mm (10 inch) cartridges. Flow rate 0.3 L/min.

Top

Penetration Studies on Filter Discs

The first objective was to show that Pseudomonas sp. could consistently penetrate 0.2µm and 0.
and be retained by 0.1µm membranes. Various 47mm discs were selected and challenged with P
sp. at 106cfu/cm2. Filter penetration was identified by passing the filtrate through a 0.1µm analys
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? Bacteri

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 >100c
The three 0.1µm filters tested were fully retentive. Of equal importance was the short challenge t
minutes, which would restrict any significant time-dependent penetration or bacterial growth. The
bacterial ‘growthrough’, or other time related effects, cannot therefore explain the extensive pene
in these studies. The results suggest that the number and size of the bacteria challenging the filte
to exceed the removal capability (titer reduction) of the filters.

Top

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 micr
with at least equivalent micro-organism retaining properties”, but then states that “Such filters can
bacteria and molds, but not all viruses or mycoplasmas”.

The FDA3 state that “Validation should include microbiological challenges to simulate ‘worst case
conditions particularly regarding the size of micro-organisms in the material to be filtered.” The F
accept B. diminuta as a sound basis for such assessment, but also state that “It is important to as
influent bioburden does not contain micro-organisms of a size and/or concentration that would re
targeted high level of filtrate sterility assurance”.

http://www.pall.com/34445_3813.asp 2/20/2007
Diminutive Bacteria Implications for Sterile Filtration Page 5 of 7

These statements indicate that filter users must establish the possible risk of filter penetration an
product.

Since the 1960s, there have been occasional reports of bacteria other than mycoplasma species
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
during development of methods for isolating Leptospires.

• In 1980, Howard and Duberstein7 demonstrated penetration of a range of commercially-availab

0.22µm filters with naturally-occurring waterborne bacteria, including Leptospira species shown in

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, a
Table I.

Table I

Removal of diminutive bacteria by 0.1µm filters7

Filter Number Filter Size Total Challenge Bacteria Re
1 142mm Disc 1.2 x 107 0

2 293mm Disc 8.9 x 107 0

3 254mm Cartridge 1.7 x 1010 0

z In 1985, Andersen et al.8 reported penetration of 0.2µm filters by Burkholderia (Pseudom

saline solution.
z In 1993, the FDA reported presence of Pseudomonas cepacia in deionized water downst
filters9. In the same document, they reported a product recall in the USA for a solution of
which was also suspected to involve penetration of the 0.2µm filter by P. cepacia.
z In a recent publication10, Leo et al. reported penetration of 0.2µm and 0.22µm filters by B
(Pseudomonas) pickettii when suspended in product, but not when suspended in saline la
(SLB). The penetration was associated with a 40% reduction in bacterial size, as shown i
retention was achieved using a PALL 0.1µm Ultipor® N66® filter. Further work is in progre
the mechanism of filter penetration.

Figure 3
Size distribution of B. pickettii suspended in product10

http://www.pall.com/34445_3813.asp 2/20/2007
Diminutive Bacteria Implications for Sterile Filtration Page 6 of 7

Events of this type have resulted in an increasing requirement by regulatory authorities for produ
specific filter validation. Such additional validation is required to be performed in a way that simul
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 emph
FDA11, who have stated: “Since there is the possibility that the drug product may cause a reducti
the micro-organism, it is best to test the microbial retentivity of the filter with the microbial challen
drug product.” and “…There are products that fall within Millipore’s matrix which are not rendered
filtered through a 0.22 micron filter.”

Despite increased validation for 0.2µm and 0.22µm filters, the FDA12 in 1996 have commented th
0.1µm filter as the final filter may be “desirable to ensure sterility of the product, especially when
contaminants are exposed to the drug product over a long period of time.”

Top

Short Term Penetration of 0.2µm and 0.22µm Filters

Filter penetration is often interpreted as a time-dependent and not size dependent occurrence. T
explained by the difficulties in producing sufficient quantities of diminutive bacteria of constant mo
assess the true removal performance of filters over short time periods. In addition, the diminutive
a very small sub-population of the natural bioburden. These factors have made it difficult to obtai
results on filter penetration. Most studies have, therefore, been performed over extended time pe
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 Se
recover and culture a single diminutive bacterial species from mains water so that extensive and
penetration studies could be performed over short time periods, ideally less than 1 hour.

Top

Can 0.1µm Filters Reduce Validation Effort and Costs?

There is an increasing awareness that the current filtration standard for sterilizing filters does not
guarantee sterility for all bacteria, under all conditions. Furthermore, the ability of bacteria to redu
interactions with the drug product, or the presence of low nutrient conditions, is further supported
studies.

There is also the possibility that the bacteria present in the product may change from batch to ba
of production conditions, seasonal variations and other factors, which may be poorly understood
routine monitoring of bioburden type and quantity necessary . For these reasons, validation proto
filters using B. diminuta are becoming increasingly complex.

http://www.pall.com/34445_3813.asp 2/20/2007
Diminutive Bacteria Implications for Sterile Filtration Page 7 of 7

One possible way to reduce the validation effort and cost is to enhance the sterility assurance pro
filter. This can be achieved by using a filter with higher removal efficiency - a properly validated 0

Although this option has been commercially available for almost twenty years, the limitations on f
filter life have restricted these filters to special applications. However, recent developments in filte
have produced 0.1µm filters with flow rates comparable to some 0.2µm and 0.22µm filters and th
being used successfully in a range of applications.

The enhanced sterility assurance provided by a properly validated 0.1µm filter may be the simple
problem of validating the security of sterile filtration processes.

Although many filter suppliers offer 0.1µm grades, there is no international qualification standard
Therefore, it cannot be assumed that all filter cartridges rated as ‘0.1µm’ would provide the same
efficiency as those tested in this study.

Top

References

1. M.Osumi, N. Yamada and M. Toya. “Bacterial retention mechanisms of membrane filters”

Pharmaceutical Science and Technology 50: 30-34, (1996)
2. F.Bowman, M.P. Calhoun and M. White, “Microbiological methods for quality control of m
J. Pharm. Sci., 55, 818 (1967)
3. FDA, “Guidelines on sterile drug products produced by aseptic processing”. Centre for Dr
Biologics, Rockville. MD, (1987)
4. “Validation guide for Pall NT grade 0.1µm microbially-rated nylon 66 membrane cartridge
Corporation, East Hills, NY
5. “Rules governing medicinal products in the European Community Vol IV - Good manufac
for medicinal products”. ISBN 92-826-3180-X. Office for official publications of the Europe
Communities, Luxembourg, (1992)
6. J.L. Braun, S.L. Diesch and W.F McCulloch “A method for isolating leptospires from natur
waters”. Canadian Journal of Microbiology 14: 1011-1012 (1968).
7. G. Howard and R. Duberstein. “A case of penetration of 0.2µm rated membrane filters by
Journal of the Parenteral Drug Association.,34: p95 (1980)
8. R.L. Anderson, L.A.Bland, M.S. Favero, M.M. McNeil, B.J. Davis, D.C. Mackel and C.R. G
“Factors associated with Pseudomonas pickettii intrinsic contamination of commercial res
solutions marketed as sterile”. Applied and Environmental Microbiology 58: 1343-1348. (1
9. D.L. Michels “Validation and control of de-ionised water systems”. FDA, Rockville MD208
10. F. Leo, M. Auriemma, P. Ball and S. Sundaram, “Application of 0.1µm filtration for enhanc
assurance in pharmaceutical filling operations”. BFS News, August (1997) (in press)
11. Human Drug CGMP Notes. Vol 2, No. 4, p35. FDA CDER Office of Compliance. (1995)
12. CDER Perspective on Isolator Technology ISPE Barrier Isolation Technology Conference
(1996)

Top

http://www.pall.com/34445_3813.asp 2/20/2007