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Validation of sterilizing grade filtration

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Pharmaceutical Bioprocess Filtration | Validating Sterile Filtration: Overcome the Fear of ... Page 1 of 9

Validating Sterile Filtration: Overcome the Fear of Failure

Common practices, including the selection of model organism, reflect lack of
process knowledge. Are they driven by science, irrational fear, or marketing?
BY MAIK W. JORNITZ, SARTORIUS STEDIM NORTH AMERICA, AND THEODORE H. MELTZER, CAPITOLA CONSULTANCY

For a process as critical and sensitive as sterile filtration, its not surprising that many filter
end-users experience some fear of failure. As risk management consultant Peter Sandman
has said, people become fearful when they feel they are not in control [1]. As a result, some may
see established sterile filtration practices as inadequate. Compelled by fear to do more than
necessary, they may overdesign their filtration systems, adding wasteful and costly procedures.

By embracing this fear, rather than studying the root causes of filter penetration and the
specifics of each process, practitioners of sterile filtration impede the advancement of filtration
science and its understanding within the industry and global regulatory agencies.

This article will examine several unnecessary practices that are burdened upon the industry
today, using process validation principles to explain why they are inadequate. It will also touch
on problems that result when model organisms used to evaluate sterile filters are seen as
universal archetypes that can characterize all process situations and filter types.

First, lets consider the concept of validation itself, which is designed to establish documented
evidence which provides a high degree of assurance that a specific process will consistently
produce a product meeting its pre-determined specifications and quality attributes [2].

Validation is a global regulatory requirement for drug manufacturing, particularly for aseptic
processes. As industry consultant Jim Agalloco has written [3], there is already a substantial
body of knowledge on best validation practices for sterile injectable biotech products,
including:

Fluid influences on the organisms, membrane polymer and retention mechanism

Process parameter influences on the membrane polymer, filter construction and retention
mechanism

Validated processes are under control, yet their reliability continues to be questioned by
regulators and those in the field, suggesting that many practitioners do not fully understand
the validation concepts. This can often be traced to subjective fears rather than valid technical

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concerns.

Here are some examples of current concerns, requirements or practices, pulled directly from
regulatory or company documents, that illustrate that fear:

0.2-m filters can be penetrated by microorganisms, so the industry must

switch to 0.1-m-rated filters.

Increasingly, there are detectable but non-culturable organisms or L-forms or

nano-bacteria in our processes.

Redundant 0.2-m filtration is necessary and should be used.

I need an absolute 0.1- or 0.2-m filter.

Diffusive flow integrity testing is better than bubble point testing. (Currently,
there is a difference in preference between U.S. and European regulators)

Flawed filters will not be detected by a post-use test, as the pore will be plugged
during filtration.

The maximum bioburden in front of a sterilizing filter should be

10cfu/100mL.

We will address many of these statements individually, later in this article.

First, however, lets consider the industrys current understanding of the role of the model
organism in filter testing and validation practices. Obviously, the industry needs a model,
since it would be impractical to test each and every microorganism, given the number and
diversity of microbes in pharmaceutical settings. Furthermore, most of the organisms are not
of concern, since their size offers no challenge to modern sterilizing grade filters.

The question is: does the most widely used model

organism always reflect specific process and filter
characteristics?

Beyond Size Exclusion: There Is No

Universal Model Organism
Brevundimonas diminuta is typically used as the
model for organisms that are expected to be found
in pharmaceutical manufacturing environments. It
was selected, based on its presence in
pharmaceutical operations, and within native bioburden [4].

The organism has been found particularly suitable for validating sterilizing grade filters due to

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its size and ease of cultivation. However, it should only be used to model situations where its
dimensions closely match the organisms of interest in a given application, relative to the filter
pore size and shape.

Furthermore, in some cases, sieve retention may not be the mode of organism removal. For
instance, in some cases, it may be adsorption, as the organism forms hydrogen bonds to the
filters polymeric surface. This could account for the observation, 26 years ago, that
Pseudomonas aeruginosa organisms are more strongly retained by polyamide membranes than
by cellulose triacetate filters [5]. It also explains the removal of latex particles from aqueous
suspensions by polyamide membranes in the presence of surfactant, but not in its absence
(Table 1) [6].

Table 1: Retention (%) of 0.198-m spheres by various 0.2-m-rated membranes

Filter Type In Water (% ) In 0.05% Triton X-100 (%)

Polycarbonate 100.0 100.0
Asymmetric polysulfone 100.0 100.0
Polyvinylidene fluoride 74.8 19.2
Nylon 66 82.1 1.0
Cellulose esters 89.4 25.1

From Tolliver and Schroeder (1983) courtesy of Microcontamination

B. diminuta should not be viewed as a universal model organism, as some native bioburden
may be a better alternative as challenge organism, being close to the actual process settings.
Unfortunately, rare penetrations of sterilizing grade filters have caused an exaggerated doubt
in the reliability of filtration. Appropriate process validation though should render such doubts
and be trusted by even the most critical reviewer of sterile filtration..

Sources of Variability: Size and Shape

B. diminuta varies in size and shape, depending on how it is cultivated. Back in 1967, Bowman
and colleagues described the B. diminuta size as 0.3 1.0 m [7]. However, in 1978, Leahy and
Sullivan found [8] that the organism grown at 30 C and incubated for 24 hours in saline
lactose broth, a minimally nutritional medium for that microbe, yielded cocci-like cells

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approximately 0.3 1.0 m (Figure 1). Similar considerations have to be accounted for when
challenge tests are performed with native bioburden forms.

B. diminuta are typically cultivated to develop as spherical a form as possible, since spheres are
least amenable to retention. Thus, Leahy and Sullivan proposed, back in 1978, that it be used as
the model organism for 0.2/0.22-m-rated membranes, partly because of its size relative to the
0.2-m dimension [8].

Subsequently, the FDA designated it for that very purpose [9], defining a sterilizing filter as
one that retains a minimum of 1 x 107 cfu of Brevundimonas diminuta ATCC 19146 / per cm2
of effective filtration area (EFA).

Although it isnt the smallest organism known, B. diminuta was considered diminutive enough
to represent whatever smaller organisms were likely to be present in pharmaceutical
preparations. The smaller the test organism, goes the logic, the more likely that its removal by
a filter would assure the sieve retention of larger organisms.

However, ease and safety of cultivation and handling are also important considerations. In
2001, Sundaram and colleagues found an increasing number of cases where filtration in
0.2/0.22-m-rated membranes failed to yield sterile effluent [10]. Experimental studies
showed that penetrating organisms had shrunken because they had been cultivated in broths
that were nutritionally inadequate. In such cases, the physicochemistry of the suspending fluid
may serve to alter the size of the suspended organisms as expressed by the Donnan equilibrium
consequent to ionic strengths.

Organism Shrinkage During Processing

Sundarams team [11] also found that organisms
underwent size changes after exposure to certain
drugs. In cases with 0.2-m-rated membranes, the
researchers found, the larger pore size could only
provide sterile effluent and/or a high titer
reduction with regard to certain organisms for
various lengths of time, before penetration
occurred.

Penetration times varied from 24 to 96 hours, and the cumulative challenge at which
penetration was first observed ranged from 1.2 x 107 to 1.1 x 108 cfu/cm2. Two 0.2-m rated
Nylon-66 filters in series were unable to fully retain Ralstonia pickettii (now Burkholderia
pickettii) with penetration observed at 72 hours, corresponding to a cumulative challenge of
2.4 x 107 cfu/cm2. The more extensive penetration of the Nylon-66 membranes, compared
with the PVDFs, is in keeping with their greater degree of openness, as Krygier and colleagues
showed in 1986 [12].

As a result, it has been suggested that 0.1-m-rated membranes be substituted for their 0.2-

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m-rated counterparts.

Sundarams team evaluated five 0.1-m-rated membranes and found that they yielded sterile
effluent over the entire duration of the test (120-196 hours), up to challenge levels of 5.7 x 107
to 2.0 x 108 cfu/cm2. Similar results were obtained with the PVDF filters tested; no B. pickettii
were detected at challenge levels of 5.9 x 107-6.0 x 108 cfu/cm2.

In addition, all 0.1-m-rated filters tested provided consistent and complete retention of B.
pickettii for the entire duration of the test (120-192 hours), suggesting that the smaller pores
would ensure sterile product at conditions where penetration could occur through
conventional 0.2- and 0.22-m-rated sterilizing grade filters. Proponents argue that using 0.1-
m-rated membranes would permit longer term formulation and filtration operations. In fact,
0.1 m-rated filters may be the best choice for long-term filtrations.

However, penetration has also been found in 0.1 m-rated filters. In 1999, Sundarams team
found that B. pickettii, when its size was so affected, could be retained by certain 0.1-m-rated
filters. But, in a similar situation, they found that only four of seven commercially available 0.1-
m-rated membranes could remove a particular organism. Just because one type of membrane
so classified may provide proper retention, does not mean that any other 0.1-m-rated
membrane can also be depended upon for a like result.

It is important to remember that today, there are no industry standards by which 0.1 filters can
be judged. In addition, more research clearly needs to be done into the kinetics behind the
organisms size changes, evaluating different organisms in different fluids.

Based on available data, long term filtrations may best be handled by 0.1-m-rated filters,
subject to validations being performed. However, in other cases, substituting 0.1-m-rated for
0.2-m-rated membranes may be unnecessary, and could result in significant penalties,
including:

Slower flow and processing rates, resulting in longer term operations.

Higher costs for larger EFAs
More leaching and extractables
Higher product losses, due to adsorptive bonding to the ultimately greater filter area used.

A responsible choice requires that both the 0.1 m-rated membranes and the 0.2 m-rated
membranes be validated.

If both types of filter 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 m-rated membranes should be used, since retention is more critical than flow rate or flux.

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Below, we address some of the common sterile filtration concerns, requirements or practices
that appear to be motivated by fear and can best be resolved by careful process validation.

0.2-m filters are penetrated by organisms. The industry is, therefore, required
to switch to 0.1-m-rated filters.

In certain specific processes, 0.2-mrated filter can be penetrated by organisms, or by

organisms which would normally be retained by such filters. In such cases, the flltrative
removal of the organisms may well require the use of 0.1 mrated filters. Such instances are
not new. Their occurrences have been considered by regulators for years, at least since the PDA
and FDA held a special forum on this topic in 1995 [13].

Certain organisms, such as Burkholderia pickettii, Burkholderia cepacia, and Pseudomonas

aerugenosa. shrink as a result of their immersion in fluid media that are only minimally
nutritious for them [14]. Their reduction in size renders as invalid validations that use B.
diminuta as a model. Brevundimonas diminuta can undergo shape alterations in minimally
nutritious media but is not listed as undergoing size alterations occasioned by contacts with
process fluids.

The fact that some microbes require 0.1-mrated filters to arrest them does not signify that
all organisms are so disposed. The necessitated switch from 0.2-m-rated to 0.1-mrated
happens in only roughly 0.005% 0.01 % of sterilizing grade filtration applications.

A mandated switch is therefore scientifically and statistically unfounded. Its promulgation may
be shunned and process validation activities and data used as performance verification. Sole
reliance on pore size ratings have been found obsolete anyway.

Increasingly there are detectable but non-culturable organisms or L-forms or

nano-bacteria in our processes.

Conclusions cannot be made regarding the sterile filtration of microorganisms unless the
methods of quantifying them by culturing and counting are available. Organisms such as the L-
forms, nanobacteria, and viable but non-culturable 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 [15]. The
complex of influences governing the outcome of an intended sterilizing filtration necessitates a
careful validation of the process, including that of the filter [4]. The very drug preparation of
interest, the exact membrane type, the precise filtration conditions, and the specific organism
type(s) of concern must be employed in the necessary validation.

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Redundant 0.2-m filtration is necessary and should be used.

Not necessarily. Again, proper process validation will disclose whether a single filter will do the
job or not. However, there are some specific applications which traditionally, for whatever
reason, utilize a second (redundant) filter as an insurance filter, i.e. if the first filter fails, the
second may compensate. This holds, however, only when each filter has been validated to show
specified retentivity.

Even so, the wisdom of the exercise deserves careful evaluation, as it assumes added costs for
membrane EFA, increased leachables and extractables. The loss of drug product may
needlessly be incurred by the filters heightened product hold-up, and unspecified adsorption.

The maximum bioburden in front of a sterilizing filter should be 10 cfu per 100
mL of fluid.

This is true if one wishes to accord with EMA regulations, and especially if one wishes to export
product to Europe. The FDA makes no such stipulation, but bases its approval on process
validation.

Seemingly in conflict, the two views arise from the same premise. The EMA regulation tries to
establish the same sterility assurance level (SAL) for filtration as for thermal sterilization. EMA
recognizes that, the greater the number of challenges, the more likely that at least one will
succeed.

The FDA seems to agree, in that if the filter can sustain the removal of organism burdens far
above those liable to be encountered in real life situations, it can assuredly withstand lesser
insults. If, as the authors see it, the FDAs massive challenge fails to breach the filters pores, it
is needless to compel bioburden assessment in front of the filter. Filter validation would
gainfully serve the intended purpose. Process validation, effectively conducted, would reliably
demonstrate the filter action.

I need an absolute 0.1- or 0.2-m-rated filter.

As a former FDA authority, since retired, once observed, The word absolute should be used
only in conjunction with vodka. Absoluteness implies a complete independence from
conditions, an inherent ability to retain particles larger that than the filters pore size rating,
regardless of any other considerations. Without a complete knowledge of the properties of the
particles and filter pores at our disposal, the statement is devoid of technical significance or
guidance. It may, perhaps, be used in ignorance (although cynics may suspect that its utility
derives from marketing efforts, a practice not unknown in the competitive world of sales.)

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Control vs. Fear

As Sandman elucidated, human beings like to be in control, and, if this status cannot be
achieved, may move rapidly to fear. Unfortunately, when sterile filtration is concerned, fear can
result in the installation of wasteful, unnecessary safety nets that can create more problems
than they solve.

Being in control is the desired state, and such control can only come from process validation
studies. Their authority is at least as old as Lord Kelvins basic scientific principle, When you
can measure what you are speaking about, and can express it in numbers, you know something
about it.

It speaks to validation. In sterile filtration, as in most areas of pharmaceutical manufacturing,

science-based validation is the best cure for fear.

References

1. Hessler, A., Sandman, P.M. Squeaky Clean? Not Even Close.

http://www.nytimes.com/2004/01/28/dining/squeaky-clean-not-even-close.html?
sec=health?pagewanted=1
2. FDA. Guideline on General Principles of Process Validation, FDA CDER, 1987.
3. Agalloco, J.P. Compliance Risk Management Using a Top-Down Validation Approach,
Pharmaceutical Technology, July 2008.
4. PDA Technical Report 26 (2008), Sterilizing Filtration of Liquids, Parenteral Drug
Association, Bethesda, MD.
5. Ridgway, H.F., Rigby, M.G., and Argo, D.G. Adhesion of a Mycobacterium to Cellulose
Diacetate Membranes Used in Reverse Osmosis. Applied and Environmental Microbiology
47, 1984, pp. 61-67.
6. Tolliver, D.L. and Schroeder, H.G. Particle Control in Semiconductor Process Streams.
Microcontamination (l), 1983, pp. 34-43 and 78.
7. Bowman, F.W, Calhoun, M.P. and White, M. Microbiological Methods for Quality Control
of Membrane Filters. J. Pharm. Sci., 56/2, 1967, pp. 453-459.
8. Leahy, T.J., Sullivan, M.J. Validation of Bacterial Retention Capabilities of Membrane
Filters. Pharmaceutical Technology 2(11), 1978, pp. 64-75.
9. FDA. Guideline on Sterile Drug Products Produced by Aseptic Processing, FDA CDER,
1987.
10. Sundaram, S., Eisenhuth, J., Howard Jr., G.H., and Brandwein, H. Part 1: Bacterial
Challenge Tests on 0.2 and 0.22 Micron Rated Filters. PDA Journal of Pharmaceutical
Science and Technology, 55 (2), 1984, pp. 65-86.
11. Sundaram, S., Auriemma, M., Howard Jr., G.H., Brandwein, H., and Leo, F. An
Application of Membrane Filtration for Removal of Diminutive Bioburden Organisms in
Pharmaceutical Products and Processes, PDA Jour. Pharm. Sci. and Technol. 53 (4), 1999,

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pp. 186-201.
12. Krygier, V. Rating of Fine Membrane Filters Used in the Semiconductor Industry,
Transcripts of Fifth Annual Semiconductor Pure Water Conference, (1986), pp. 232-251, San
Francisco, CA
13. PDA/FDA Special Scientific Forum, Bethesda, MD; Validation of Microbial Retention of
Sterilizing Filters, July 12-13, 1995.
14. Mittleman, M.W., Jornitz, M.W., Meltzer, T.H., Bacterial Cell Size and Surface Charge
Characteristics Relevant to Filter Validation Studies, PDA Jour. of Pharm. Sci. and Technol.
52 (1), 1998, pp. 37-42.
15. Agalloco, J., Letter to the Editorre: It just doesnt matter, It just doesnt matter, It just
doesnt matter. PDA Journal of Science and Technology. Vol 52, No. 3, pp. 149-150.

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