# Sterile Filtration: Process Optimization Through Real Compliance

by Hans G. Schroeder, Ph.D.

International Consultants Association

Encinitas, CA

As a general rule, the regulations imposed on the pharmaceutical
industry offer clear advantages to the patients who depend on their
products. Enforcement of the regulations and associated guidelines
provides assurance to the consumer that approved and acceptable quality
levels are met, and that similar products from different manufacturers
are, in essence, equivalent in safety and efficacy. The main benefit of
compliance to the industry is the corresponding license to stay in
business with a minimum of citations or regulatory actions by the
enforcing agency.

Over time, industry has learned to stay in compliance by following good
manufacturing practice regulations to the letter. It is, in effect,
impossible to run a pharmaceutical operation without a sound set of
written standard operating procedures and programs that assure important
contributions to the expected product quality, such as operator
training, internal audits, validation of critical processes, and change
control. Compliance is obviously a vital component of our industry, so
important that an entire journal is dedicated to this topic (1). Many
articles reflect the fact that current regulations are subject to
changes as well as interpretation, and suggest their readers update
their compliance programs by reviewing current FDA 483 observations,
Warning Letters and guidance provided by publications like the Pink
Sheet and the GMP Monitor (2,3). While such "Regulatory Compliance" may
keep inspectors happy, it unfortunately also leads to constant and
costly knee-jerk reactions to avoid similar observations in the next
inspection of their own facility.

A viable, and, on the long run more cost effective, alternative was
recently presented by Madsen (3). He proposes that a state of process
control well above regulatory compliance can be achieved by applying
fundamental scientific principles and technically sound operating,
control and documentation procedures. Such an approach must, of course,
keep current regulations in mind, and, if properly implemented,
regulatory compliance becomes, for practical purposes, a beneficial side
effect such "Real Compliance."

According to Madsen, key elements required to achieve real compliance
include an adequate number of trained operators, a technically sound set
of instructions and operating procedures, dependable redundancy,
appropriate verification and documentation and an immune system that
corrects process variations and discrepancies before they result in
defective product. Once a real compliance state of control is reached,
the process can be monitored by meaningful feedback loops, including
warning indicators and alarms, but in-process checks become confirmatory
rather than controlling. Unfortunately, the direct outcome of some
processes cannot be monitored on line and in real time. Good examples
are sterilization processes, for which the incubation time associated
with commonly practiced microbial monitoring represents an unavoidable
delay. In addition, many quality attribute tests, sterility included,
are destructive in nature. Thus, particularly in such cases, a real
state of control depends heavily on sound characterization and
understanding of the process, which, in turn, makes the success of such
processes most dependent on a sound validation program.

# Process Validation

Many approaches to validation have been presented in the technical
literature over the years, but, once the glitter is removed, a sound
process validation program can be reduced to responsibly pondering,
substantiating and documenting the following four questions:

-   What is the desired outcome or what is the objective of this process
    step?

-   Which of the process variables will affect the successful outcome of
    the step?

-   How are these process variables monitored and controlled?

-   Will the control limits imposed assure that the objective is met
    consistently?

By analyzing these questions it is possible to determine if a process is
in real compliance, even if no regulatory citations are issued against
the manner in which the process is currently run. Sterile filtration
will be discussed to illustrate this approach.

# Objective of the Sterile Filtration Process

By implication, the obvious objective of a sterile filtration step is
the removal of any viable organisms that may be present in the bulk
product solution as manufactured. This is typically accomplished by the
use of "Sterilizing Grade" membrane filters, defined in the FDA "Aseptic
Guideline" (4) as those capable of totally retaining a challenge level
of 10\^7 cfu / sq cm at a differential pressure of 30 psi. The FDA
currently accepts B. diminuta at this challenge level as a worst case
model, but it is also acceptable to use natural flora.

# Analysis of Variables

The variables recognized to have an impact on the outcome of a sterile
filtration exercise have been extensively discussed in the technical
literature and were recently reviewed by Jornitz et al (5,6). These
variables include the physical-chemical nature of the carrier liquid,
the hydrodynamic conditions and the duration of the process, qualitative
and quantitative aspects of the bioburden, and last, but certainly not
least, the list of variables must include the very filter chosen for the
process. Variables directly associated with the filter itself include
the type, its membrane material and any surface treatment, the actual
pore size (as opposed to an arbitrarily assigned pore size rating), pore
morphology, thickness and tortuosity, and the degree of "integrity" of
the filter, as determined by the actual size of the largest pores and
defects, if any, present on the filter used.

Retention has long been recognized to depend on complex interactions
between these variables. The membrane and its surface treatment, in
combination with the contaminant and the carrier liquid, typically
define the retention mechanism, while the integrity determines the
extent of the retention capability. In recognition of the impact of such
variables, the FDA Aseptic Guideline expects the validation to be run
simulating the actual processing conditions, using the actual product to
be filtered or a suitably similar surrogate as a carrier vehicle. In a
series of validation runs, the bioburden is also defined and kept
relatively constant, and, of course, the filter type is fixed by the
user's choice according to whatever filter selection criteria were
followed. This reduces the long list of variables that have an impact on
the retention to one, namely, the integrity of the filter. Hence, the
long-standing emphasis on establishing a sound correlation between a
non-destructive integrity test and bacterial challenge tests as a basis
for validating the retention capability of membrane filters.

Note that in an actual filtration process involving the filter type
validated, most of the variables discussed above will become fixed
parameters. This is true even for the integrity of the filter, since a
minimum acceptable integrity parameter will have to be met before it is
placed into service. On the other hand, the cell count and nature of the
bioburden will likely be different in actual filtration settings,
particularly if a model such as B. diminuta was used in the validation
runs. In light of recent publications that document the passage of
presumably smaller organisms through B. diminuta retentive membranes
(7,8,9), this could adversely affect the expected retention in an actual
filtration setting. For this reason, validations using naturally
occurring flora will likely eventually be preferred over any model
organism testing, even if natural flora may also vary from time to time.
To reach concentrations of 10\^7 / sq cm using natural flora it may also
be necessary to increase the inherent bioburden of the actual product by
additional steps, such as allowing incubation beyond the regular
processing time or the concentration of cells by cross-flow filtration.

# Monitoring the Integrity of Sterilizing Grade Membrane Filters

As has been extensively discussed in the technical literature, currently
practiced membrane filter integrity tests are based on the observation
that membrane samples wetted out with a suitable liquid are essentially
impermeable to viscous flow of gases at low pressures. As the applied
pressure is increased, the liquid within the largest pores present in
the sample will eventually start to dislodge, allowing the test gas to
flow through them forming visually observable streams of bubbles. The
pressure at which bubbling commences is referred to as the bubble point
of the membrane, a value which has been successfully used to
characterize the integrity of small area membrane samples, such as the
47 or 90 mm discs often used for retention validation tests.

When passage is observed in a given set of retention validation runs it
must be attributed to the larger pores, no matter what the retention
mechanism is, since the actual retention mechanism is not a variable in
the retention validation studies. This can readily corroborated by
subjecting membrane samples of successively higher bubble-point values
to challenge studies using a defined contaminant such as B. diminuta.
Typically, a clear retention pattern will be observed. At higher
bubble-point values, the retention becomes more "reliable," and a
pressure, or a narrow range, above which "total" (better than the
challenge level or 10\^7 cfu / sq cm) retention is observed, can readily
be identified. This pressure is often referred to as the "critical"
bubble-point value.

Since integrity is the only true variable that will affect the outcome
of a given set of retention validation runs, the FDA Aseptic Guideline
rightfully expects the retention capability of the filter to be
correlated to a meaningful integrity test. Even prior to this official
request, technical support literature and the so-called Validation
Guides published by the filter manufacturers have shown B. diminuta
retention data as a function of the bubble-point pressure of their
sterilizing grade membrane products. Johnston (10) reviewed the
retention data presented in various studies and concluded that for most
types of membrane, a 10% reduction in the bubble-point will reduce the
retention capability by 2 orders of magnitude. This means that if a
given type of membrane has a critical bubble-point of 50 psig, passage
of up to the order of 100 cfu per square centimeter in a 10\^7 cfu / sq
cm challenge test of a 45 psig bubble-point sample could be observed. If
such a disc were to be built into an otherwise "good" cartridge,
challenge of the cartridge to the same bacterial load would again result
in passage of a similar number of organisms, since a chain can not be
stronger than its weakest link.

The FDA Aseptic Guideline also requires that the integrity test be
appropriately scaled up for the filter to be used in the production
setting. The integrity of sterilizing filter cartridges typically used
in the production setting is often established through quantitative gas
flow measurements, often referred to as diffusive flow tests, rather
than the go/no-go visual bubble point approach. If the flow of the test
gas at a pre-established pressure exceeds a value deemed acceptable, the
filter is considered defective and will be excluded from use. While the
approach as such is reasonably sound, it has been a long-standing
tradition that filter cartridges are to be integrity tested at 80% of
the critical bubble-point value. Such a lower test pressure can
obviously assure only that the cartridge is no less than 80% as tight as
it really needs to be, allowing non-retentive areas, such as described
above, to go undetected.

Poor wetting of a membrane filter element may result in failing to meet
the integrity test acceptance criterion. These so-called "wetting
problems" can be distinguished from actual defects by re-testing the
filter after extended flushing or re-wetting it with another liquid of
lower surface tension, but it is not very practical to perform such
additional tests in the production setting. Thus, to circumvent the
issue, some filter users opt not to perform a pre-filtration integrity
test. This practice is safe. After all, the retention observed in
validation runs is correlated to the integrity test results of clean
filters, thus, to expect any retention assurance, the filter must be
tested for integrity prior to becoming plugged with the contaminants it
retains during its use. This is particularly important since it has been
reported that marginal and substandard membrane filters can plug up
during use to the point that after the filtration an acceptable
integrity test result is observed (11). Since untested filters may not
even have met an already compromised 80%-specification, a fair number of
viable cells may well make it through the membrane during the early part
of the filtration process.

**Adequacy of Integrity Control**

Traditions such as lowering the test pressure to improve cartridge
yields or omitting pre-filtration integrity tests to circumvent possible
wetting problems are not safe, because the filter may indeed not be any
tighter than what corresponds to the pressure to which it was tested. As
discussed above, this action will unquestionably reduce the sterility
assurance of the lot of product filtered. Further, deliberately settling
for an avoidable compromise in the sterility assurance level is in
strong contradiction with the FDA "Validation" Guideline (12), which
specifically states: "Each step of the manufacturing process must be
controlled to maximize the probability that the finished product meets
all quality design specifications." Nevertheless, the FDA condones such
practices, making them acceptable in terms of regulatory compliance.

The FDA "Validation Guideline" also specifically states that "In
assessing the suitability of a given piece of equipment, it is usually
insufficient to rely solely upon the representations of the equipment
supplier," presumably in order to increase the validity of validations
and reduce the potential for conflict of interest. Again, sterile
filtration stands out as a regulatory exception, as a high percentage of
filter users rely heavily on data generated on a contract basis by
filter manufacturers, even product-specific validation data. The FDA
"Aseptic Guideline," in recognition that "Some of the more complex
filter validation tests may be beyond the capabilities of some filter
users," accepts this practice at the expense of complacency among filter
users. This exemption has kept the number of adequately trained and
knowledgeable filter users at such a low level that filter manufacturers
are often called upon to answer questions the FDA has about their
client's process. The fact that some users may not be able to answer
such questions on their own is a poor reflection of the level of control
our industry has managed to acquire over such a critical process step.

The technical flaws in the currently accepted integrity test practices
make it mathematically impossible to consistently expect the advertised
retention level of better than 10\^7 cfu / sq cm. In fact, a few filter
users have observed and reported discrepancies when attempting to
reproduce manufacturers' retention data on their own (13,14,15), and
even the FDA has reported observing such discrepancies in validation and
actual production filtration settings (16,17). The possibility that
occasionally a batch of filtered product may be lost for failing the
sterility test on account of poor filter retention performance cannot be
ruled out. None-the-less, it has to be recognized that the process of
sterile filtration serves the pharmaceutical industry incredibly well on
a day-to-day basis. The reasons for this include the fact that on
account of sound hygiene and microbial control practices, including
pre-filtration and redundant filtration, the bioburden level that
reaches the final filter in most production lots is well below the 10\^7
/ sq cm level used in the validation studies, allowing for ample safety
margins. Further, in many cases, the actual filter used might well be
far more integral than the test at 80% of the critical bubble-point
would indicate. And last, the statistical limitation of the sterility
test would most likely cover for imperfections in the sterile filtration
process, much like it did for other sterilization processes prior to the
mandated implementation sound validation programs in the 1970's.

**Discussion**

For all the above reasons, it must be concluded that sterile filtration
is not in a state of real compliance. In fact, the state of the art of
this process is similar to the state of the art that prevailed for other
sterilization processes over 25 years ago, prior to the general
requirement for process validation. Although the validation of sterile
filtration is obviously required by cGMP regulations, the currently
accepted approach differs considerably from the validation of other
processes. For instance, the validated cycle time is a parameter as key
to moist heat sterilization as the critical bubble point pressure for a
given filter-product combination is for sterile filtration. While a
reduction to 80% of the validated cycle time for the corresponding
production sterilization run would be totally out of the question, the
critical bubble-point for most products sterilized by filtration is
often not even taken into consideration.

This lack of understanding is akin to accepting the validity of
statements to the effect that "A 20-minute cycle will sterilize most
loads" found in the instruction manuals of most old autoclaves. When
such statements were found to be misleading, rightfully so, the FDA did
not question the validity of moist heat as a sterilization tool, but
"encouraged" autoclave users to gain better understanding and control of
key processing parameters through targeted validation studies. Some of
the cycles in use at the time were indeed found to be marginal, if not
unsafe, when validated against what are now commonly accepted sterility
assurance level targets. Corrections were made and "real compliance"
control was gained through the appropriately run validation studies. The
net effect was an overall improvement in the sterility assurance of
terminally sterilized products.

The validity of sterile filtration as a tool must also not be
questioned, but some of the currently accepted practices need to be
revised in order to achieve a state of real compliance. A key element in
the success of such a revision is a more responsible involvement in
sterile filtration issues by members of our own filter user community.
Validation of autoclaves by the user was also beyond the capability of
some members of our industry 25 years ago, but the first Parenteral Drug
Association Technical Report on Validation of Moist Heat Sterilization
was instrumental in assisting autoclave users in this task. The more
recent Technical Report No. 26 on Sterile Filtration can help the filter
user overcome most of the shortcomings discussed. This industry guidance
document, prepared with cooperation from the FDA, membrane filter
manufacturers and, albeit, relatively few members of the filter user
community, represents a great step towards providing the basic tools
required by filter users to bring sterile filtration into a controlled
state of real compliance. The document encourages filter users to become
more aware and more involved in the filter selection process, which
ultimately will reduce the bias and potential for conflict of interest
that shadows the present approach to filter validation. Most
importantly, the document encourages filter users to take more
responsibility for product-specific retention test and to make sure that
the membranes used for such tests are commensurate with the minimum
integrity specified by the filter manufacturer or their own production
test parameters.

It should be evident from the above discussions that the integrity test
parameter selection must be taken more seriously than it is in current
practice in order to optimize the sterility assurance of sterile
products produced by aseptic processing techniques that involve sterile
filtration. Other than convenience, mainly for the filter manufacturer,
there is no excuse to test the integrity of sterilizing filters in the
production setting at pressures below the critical bubble point value.
As discussed elsewhere in greater detail (18), if the critical
bubble-point for a given filter-product combination is not known, the
lowest bubble-point value at which the desired, typically total,
retention is observed in a series of product-specific challenge tests
must be used as the diffusive flow test pressure for cartridge filters
to be used in production. This is the case even if the manufacturer has
qualified the membrane as sterilizing grade by means of B. diminuta
challenges. After all, the retention may be affected by the nature of
the product and is a function of the true integrity of the membrane, not
an arbitrary rating. Further, the retention capability at lower
bubble-point values was not established, and unknown retention
performance must be avoided to err on the safe side. For the reasons
discussed above, the integrity of filter cartridges used in the
production setting must also be verified before and after the filtration
process to assure retention equivalent to the level observed in the
validation runs.

For decades, filter users have been lead to believe that sterilizing
filters are capable of retaining to the specified level of 10\^7 cfu /
sq cm of filter area, but this goal can not be substantiated in light of
current practices. By bringing the process into a controlled state of
real compliance, this retention level can more likely be achieved and
validated appropriately, making products sterilized by filtration that
much safer. The fact that there have been no major catastrophes that can
be traced to sterile filtration should, by rights, not stand in the way
of a safer real compliance approach. However, progress in this area
might be slow. As Madsen points out, "The primary obstacles to real
compliance are inertia, people and the regulators..." (3).

**References**

1.  Journal of GXP Compliance, Institute of Validation Technology

2.  J. Berger, "Assessing the Quality Assurance of your Aseptic
    Processing Operations", American Pharmaceutical Review, **4**, (4),
    31 (2001).

3.  R. E. Madsen, "Real Compliance and How to Achieve It", PDA J. Pharm.
    Sci. Technol. **55** (2): 59 (2001).

4.  FDA \"Guideline on Sterile Drug Products Produced by Aseptic
    Processing\", Division of Manufacturing and Product Quality,
    Rockville, MD, June 1987.

5.  M. W. Jornitz, J. P. Agalloco, J. E. Akers, R. E. Madsen and T. H.
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6.  M. W. Jornitz, J. P. Agalloco, J. E. Akers, R. E. Madsen and T. H.
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7.  S. Sundaram, J. Eisenhuth, G. Howard Jr., and H. Brandwein,
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9.  S. Sundaram, J. Eisenhuth, G. Howard Jr., and H. Brandwein,
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10. P. R. Johnston, Fluid Sterilization by Filtration, Interpharm Press,
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11. T. H. Meltzer, "The Need for Initial or Prefiltration Integrity
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12. FDA "Guideline on General Principles of Process Validation,"
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13. K. H. Wallhausser, \"Is the Removal of Microorganisms by Filtration
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14. K. H. Wallhausser, \"Bacterial Filtration in Practice\", Drugs Made
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15. C. E. Weatherford, \"A User\'s Perspective on Bacterial Retention
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16. E. M. Fry, \"FDA Update on Aseptic Processing Guideline,\" J.
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17. P. Cooney, Oral Presentation, FDA / PDA Sterile Filtration
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18. H. G. Schroeder, "Rationalization and Valid Scale-Up of Integrity
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