B i o P r o c e s s TECHNICAL

Best Practices for Critical

Sterile Filter Operation
A Case Study

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Yanglin Mok, Lise Besnard, Terri Love, Guillaume Lesage, and Priyabrata Pattnaik

N
A
number of regulatory

O
Figure 1:  Design and layout of Millidisk and Millipak barrier filters
guidelines recommend preuse
integrity testing of critical
sterilizing liquid filters for

N
aseptic processing (1–3). Before Support
Membrane
sterilization, a preuse test will confirm structure

IO
that a filter is installed properly and
was not damaged during shipment or
handling. Performing a preuse test after

S
Three One
sterilization detects damage that may hydrophilic hydrophobic
membranes

IS
have occurred during the sterilization membrane
cycle. Testing after sterilization limits One disk pair
risk, so it is a practice applied based on
M
risk assessment. Because it is perceived
to reduce business loss risk, preuse post-
sterilization integrity testing (PUPSIT)
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is a current industry practice especially Gas

Liquid Air from integrity
in manufacturing products that will be
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Wetting and test and blow-

marketed in the European Union (EU). flushing fluids down can vent
P

Unfortunately, it can be difficult to evacuate to drain from same filter

perform a PUPSIT without breaching
system sterility. A number of methods
H

have been developed for running Outlet

PUPSIT and performing line
IT

conditioning without compromising

sterility. Such methods use a flush use the downstream hold tank as a sterilizing-grade Durapore membranes,
W

bag, catch-can/flush bottle, or filter
arrangement to create a sterile
boundary on the downstream side of
the product filter. Some applications
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sterile boundary. both on the top and bottom of each
Here we describe a robust and disc (Figure 1). Because of that unique
versatile approach to PUPSIT using a combination of different membranes in
self-venting, all-in-one sterile barrier parallel configuration, the devices can

membrane filter from EMD filter condensate, steam, wetting liquid,

IN

Millipore, the life science business of and gases without compromising the
Product Focus:  All biologics Merck KGaA, Darmstadt, Germany, sterility of a steam-sterilized,
Process Focus:  Downstream which operates as MilliporeSigma in autoclaved, or gamma-irradiated
R

processing, fill and finish

the United States and Canada. We system.
also include filtration line design Barrier filters can be used
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Who Should Read:  Process considerations for implementing downstream of sterilizing-grade filters
development and manufacturing barrier filters. to maintain system sterility. Water can
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pass through the hydrophilic discs

Keywords:  Filter integrity testing, Barrier Filters during a flushing sequence; air can
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aseptic processing, multiuse and

Millipak and Millidisk barrier filters pass through the hydrophobic discs
single-use technologies, barrier filters
are stacked-disc devices that combine during integrity-test and drying
Level:  Intermediate hydrophilic and hydrophobic sequences. A barrier filter acts as an

28 BioProcess International 14(5) M ay 2016

Figure 2:  Examples of sterile boundary designs
Barrier Filters
P P
Method
diagram
Sterile Boundary Attribute
(All maintain
sterility of
product filter
during preuse
test.)
Retest
Ability to dry
the product
filter
Simple
design
automatic vent during testing and
drying phases. The volume of particle-
free water and air that can pass
through these filters is unlimited.
Flushing and Testing
Critical Product Filters
Although filter rewetting and retesting
should remain an optional activity
when preparing a product final filter
(sterilizing-grade filter) in line, the
PDA Technical Report 26 suggests up
to three repetitions (3). The number of
retests should be considered when
sizing for a flush bag. Barrier filters
could provide a more versatile solution.
Filters that are not wetted efficiently
the first time could give false failed test
results. If rewetting volume is limited,
end users might discard integral filters
that only marginally failed because of
improper wetting. Doing so could lead
to unnecessary quality investigations as
well as downtime associated with
setting up a system again before use.
Using a barrier filter allows for
rewetting and retesting with ease.
Unlimited volumes of particle-free
water can be filtered through them to
wet these filters efficiently. Using a
flush bag and/or catch-can instead
creates a large footprint and could limit
rewetting. Figure 2 summarizes the
advantages and disadvantages of each
sterile boundary method available.
Barrier filters also allow
extractables flushing through a sterile
boundary to drain. A barrier filter also
can be used as a vent in system cooling
after steam-in-place (SIP) sterilization
30 BioProcess International
Catch Can
or Flush Bottle Flush Bag Downstream Filters
P
Gas
Filter
P P
T T Liquid
Filter
and in filter drying after flushing to
minimize product dilution.
Filtration Line
Configuration and Operation
For critical filter applications such as
final product-filling lines, barrier filters
can be used to provide a sterile boundary
while meeting regulatory requirements
for preuse integrity testing. Typically,
such filters are installed downstream of a
product filter (Figure 3).
Figures 4 and 5 illustrate stepwise
use of barrier filters to provide a sterile
boundary in a stainless steel and a
single-use filtration system with a
single product filter. Regulators
recommend redundant filtration as a
risk-mitigation strategy for critical
filtration applications. Redundant
filtration is a type of serial filtration in
which a second product filter is used as
a back-up to protect against the
possibility of an integrity failure for the
primary product filter (3). Each filter
must be independently integrity-
testable in compliance with the relevant
regulations or guidelines. The step-by-
step approaches in Figures 4 and 5 can
be applied to the second product filter
in a redundant filtration system.
Sterilizing: The process begins with
sterilization of a filtration system by
SIP, autoclaving, or gamma-irradiation.
If the chosen method of sterilization is
SIP, then the barrier filter’s low-point
vent on its upstream side will be kept
open during the SIP cycle. Condensate,
steam, and air will pass through the
filter to drain on its outlet. When the
14(5) M ay 2016
Figure 3:  Barrier filters in a typical single
filtration system (filling-line example)
Class C Class B/A
Integrity
tester
k ion
Filling line
Product
tanulat
filter
rm
Fo
Barrier
filter
cycle ends, the low-point vent will be
closed. The system then cools down
with application of compressed gas (to
maintain positive pressure as well as
sterility in the filtrations system).
Wetting: The second step ensures
that a product filter is totally wetted
with particle-free water for its
integrity test. This step also flushes
away extractable residues from a
sterilized product filter element. To
keep a filter train independent, the
vent on the product filter is left open
initially, and the isolation valve to
downstream equipment is closed. The
product filter vent is closed after air in
its housing has been vented. Water is
directed to the drains through the
barrier filters. Isolating the flow path
to the barrier filters can enhance
wetting for increased applied pressure
drop through a product filter.
Testing: PUPSIT is either a
diffusion or bubble-point test
(depending on the product filter). In all
cases, pressurized gas is applied on the
product filter’s upstream side, which is
isolated from the system elements both
upstream and downstream. Only the
drain line with the barrier filters
remains open to allow the free flow of
test gas through the hydrophobic
portion of their membranes.
Drying: Before product is
introduced into the filtration line, the
product filter typically is blown down
and dried to prevent dilution of the
product stream. The associated gas is
vented through the barrier filter.
Barrier Filter Integrity Testing:
Millipak and Millidisk barrier filters
are integrity tested offline using 70/30
isopropyl alcohol (IPA) as the wetting
fluid.
 Process: Once the integrity of the
product filter is confirmed, the sterile
filtration process can begin.
After Processing: After the sterile
filtration process, product recovery
through a sterilizing-grade filter can
be achieved through air blow-down
with application of a low differential
pressure (air or nitrogen) of 5 psi to
the filter. Users can apply a buffer
chase, but product dilution must be
accounted for. At the end of product
recovery, sterilizing-grade filters are
integrity tested with particle-free,
water-based, alcohol (70/30 IPA/
water), or product-based integrity test
specifications. For particle-free–water-
based or alcohol integrity-test
specifications, a filter must be flushed
adequately with the test liquid to
remove residual product before testing.
Product-based integrity-test
specifications can be developed
through support from filter vendors.
Design Considerations
Use of Millipak and Millidisk barrier
filters in a filtration line is simple and
straightforward (Figures 4 and 5).
Similar to all critical applications,
important process steps and conditions
should be reviewed during system and
process design to ensure successful
implementation of this application.
Verification testing should be performed
before implementation of an assembly
with barrier filters for product filtration.
Sterilization of Filtration System:
Sterilization renders a system or
equipment free of microorganisms and
is a critical step, especially for aseptic
manufacturing processes. Filters can be
sterilized through SIP for Millidisk
format (cartridge filters) and
autoclaving or gamma irradiation for
Millipak (disposable capsule) filters
(Figure 6). Thermocouples, radiation
dosimeters, and biological indicators
serve as the worst-case positions within
a filtration system and assembly for
validation of sterilization.
Flushing and Wetting Product
Filters: A filtration system may be
flushed and wetted to remove
extractables after sterilization as well as
for product filter integrity testing. The
flushing or wetting liquid passes
though the barrier filters to a drain.
Flushing/wetting conditions are derived
from vendor recommendations (5, 6) and
can vary among product filters. Inlet
pressures on barrier filters during
flushing/wetting procedures should not
exceed 0.7 bar. If enhanced wetting of a
product filter is required, higher static-
hold pressure can be implemented
across it. However, the downstream
section of that product filter (including
the barrier filter) should be isolated
during such a high-pressure hold step.
Key Verification Point — Efficacy of
Product-Filter Wetting: The efficiency
of wetting a product filter(s) through
barrier filters can be verified by
performing product filter integrity
testing. Results can be compared with
filter specifications and past trending.
Key Verification Point — Gas Flow
Rate of Barrier Filters After Flushing/
Wetting Procedure: Ensuring that the
hydrophobic membrane in a barrier
filter remains dry is critical. Such
dryness can be verified in the filter
following a flushing/wetting procedure

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during the qualification phase with a their gas flow-rate level at 100 mbar pressure for five minutes. Considering
low-pressure bubble-stream test. This (1.5 psi) pressure, then comparing that the average hydrophobicity level of
includes disconnecting barrier filters to the nominal gas flow-rate level of a polyvinylidene fluoride (PVDF) discs,
from their assembly and determining new filter unit wetted optimally at low flow rates in the 30–100% range of
nominal rate are characteristic of a
Figure 4:  Product filter preparation steps for steamable line, from sterilizing to integrity testing “breathing” unit.
Key Verification Point — Using
Product filter 1 Sterilizing 2 Wetting 3 Testing Wetting Medium Apart from Water for
preparation Integrity test Injection (WFI): Compatibility and
steps for Close valve of product

steamable line: once wetted. filter intrusion pressure/wettability for the

from sterilizing hydrophobic filters within barrier
to integrity Product
filters should be verified before the
testing Steam
in place
filter
Atmospheric
filters are used. Wetting hydrophobic
Water
filters can reduce their air-flow
Barrier (max. capacity, leading to increased pressure
Valves filter 0.5 bar)
drop across the filter assembly during
Open
integrity testing or product-filter
Closed Vent steam through Drain water through Vent test gas
barrier filter. barrier filter. through barrier filter. blow-down. Millipak and Millidisk
barrier filters validation guides provide
4 Drying 5 Barrier Filter 6 Processing Product filter chemical compatibility information
Compressed air
Testing preparation summaries (7–9).
Close steps for
valve
once steamable line: Integrity Testing
wetted.
from drying
of Product Filter
to processing
Filtration assembly designers should
70%/30%
include strategies to minimize product
IPA/water Product hold by reducing piping or tubing
Barrier filter Valves length and to ensure maximum product
tested off-line
Open recovery. The strategy for integrity
Vent air through Closed testing product filters also should be
barrier filter. well thought-out, especially for
redundant filtration assemblies. In
Figure 5:  Product filter preparation steps for single-use assemblies, from sterilizing to integrity 2012, Felo and coworkers at
testing and from drying to process MilliporeSigma provided an in-depth
Product filter look at how product filters can be
1 Sterilizing 2 Wetting 3 Testing integrity tested in a single-use assembly
preparation Water
steps for (max. 0.5 bar) (10). Their strategy can be applied to
single-use Close valve stainless steel systems as well.
Air filter once wetted.
assemblies: Interference on Product Integrity
from sterilizing Testing from Barrier Filters: Barrier
Product
to integrity Product
filter filter Product
filters are placed downstream of a
filter
testing
product filter. To verify the absence of
Valves Barrier interference, the integrity test result of
filter
Open the product filter both with and
Sterilized by autoclave
or gamma irradiated as Drain water through Vent test gas without the barrier filters can be
Closed part of an assembly barrier filter. through barrier filter.
compared. Those results should fall
within 70 mbar for a bubble-point test
4 Drying 5 Barrier Filter 6 Processing Product filter and 5% for diffusion flow.
Testing
Compressed air

preparation Failure Mode Test: To simulate a

Product
steps for worst-case scenario (failure-mode
Close valve
once wetted. single-use test), users can examine how a fully
assemblies:
70%/30% wet barrier filter gas flow rate
IPA/Water from drying
to processing
compromises a product-filter integrity
test. Millipak and Millidisk barrier
Barrier filter filters can be fully wetted by flushing
tested off-line
Valves with WFI at 3 bar.
Open Adaptation of Troubleshooting
Vent air through
barrier filter.
Closed Decision Tree: If a product filter fails
its integrity test, users can apply a
32 BioProcess International 14(5) M ay 2016

troubleshooting decision tree such as
the example given in PDA’s Technical
Report #26 (3).
Drying of Filtration System
To minimize product dilution or
contact of product with the wetting
liquid (either buffer or water) before
filtration, the assembly may be blown
down to remove wetting liquid. The
current industry practice of blowing
down a filtration system ranges from
30 minutes to three hours.
Duration of Drying: Exact drying
times should be verified on site and
determined during qualification by
weight and visual checks. The same
time taken to reach the “dry weight”
of the assembly will be required for
drying the assembly during operation.
Acceptable Applied Pressures:
Typical pressures applied for drying
filtration assemblies are 0.5 bar higher
than the bubble-point pressure of a
product filter. Such pressures should
not exceed the maximum allowable
pressure of the “weakest link” in an
assembly. That might be silicone
tubing, a connector, or a barrier filter
(4.1 bar for Millipak and Millidisk
formats), for example. If the required
pressure is greater than what the
weakest link allows, then blow-down
pressure should be reduced, and an
extended drying time can be applied.
Absence of Air-Flow Interference:
Restriction of air flow through fittings,
connectors, tubing, or piping used in an
assembly should be minimized. As a
product filter dries, the air-flow rate
will increase and pressure drop across
the product filter will decrease.
Integrity Testing
of Barrier Filters
Barrier filters are integrity tested
offline with 70/30 IPA/water as a
wetting medium and bubble-point test
specification of ≥1,280 mbar
(18.5 psi). These filters can be wetted
by dynamic flushing or static-soak
methods. The wetting procedure of
barrier filters can be found in a
technical guide (5). A 15-minute static
soak can be applied to either Millipak
or Millidisk barrier filters.
For critical product applications in
which resources are readily available, a
Figure 6:  Sterilization considerations for Millidisk and Millipak barrier filters
Millidisk Barrier Format
Filter Format
Steam-in-Place
Important Sterilization
Sterilization process validation required
Considerations
Maximum 135 °C for 60 minutes,
up to four times
Steamed in forward direction with 5 psi Has no vent (self-venting) and serves as
(340 mbar) maximum pressure drop
Housing should a include housing drain autoclaved assembly
for condensate removal.
barrier filter should be integrity tested
after the product filter has passed
integrity but before product filtration.
This minimizes the risk of
reprocessing product because of a poor
installation or nonintegral barrier
filter caused by mishandling. For
situations in which resources are
limited and product can be
reprocessed, barrier filters can be
integrity tested after product filtration.
For Best Practices
Barrier filters help enable best
practices of aseptic filtration lines for
flushing/wetting and preuse integrity
testing of product filters. In particular,
implementation of Millipak and
Millidisk barrier filters is easy and
provides flexibility and versatility to
the filtration line.
References
1 Annex 1: Manufacture of Sterile
Medicinal Products. Volume 4, EU Guidelines to
Good Manufacturing Practice Medicinal Products
for Human and Veterinary Use. European
Commission: Brussels, Belgium, November
2008; http://ec.europa.eu/health/files/eudralex/
vol-4/2008_11_25_gmp-an1_en.pdf.
2 CBER/CDER/ORA. Sterile Drug Products
Produced By Aseptic Processing: Current Good
Manufacturing Practice. US Food and Drug
Administration: Rockville, MD, September 2004.
3 Technical Report No. 26: Sterilizing
Filtration of Liquids. Parenteral Drug
Association: Bethesda, MD, 2008.
4 ASTM Standard F838-83: Standard
Test Method for Determining Bacterial Retention
of Membrane Filters Utilized for Liquid
Filtration. American Society for Testing and
Materials: Philadelphia, PA, 1983.
Millipak Barrier Format
Autoclave
Maximum 123 °C for 90 minutes,
up to three times
the sterile vent for air and steam of the
5 P35515 Rev G: Wetting Instructions for
Filter Units with Durapore Membrane. EMD
Millipore: Billerica, MA, April 2012.
6 RF1510EN00: Hydrophilic Durapore
Cartridges and Capsules User Guide. EMD
Millipore: Billerica, MA, January 2002.
7 VG026 rev 2: Millidisk Cartridge Filter
Units with Hydrophilic Durapore Membrane
Validation Guide. EMD Millipore: December
1999.
8 VG033 Rev D: Millipak Disposable Filter
Units Validation Guide. EMD Millipore:
Billerica, MA, May 2012.
9 VG2000EN00: Millidisk Barrier Filter
Validation Guide. EMD Millipore: Billerica,
MA, May 2002.
10 Felo M, Oulundsen G, Patil R. Single-
2012: 38–41. •
Use Redundant Filtration. BioPharm Int. 25(4)
Corresponding author Yanglin Mok, BE, is a
senior process engineer and technical
manager of the Biomanufacturing Sciences
Network, 1 Science Park Road, #02-10/11 The
Capricorn, Singapore 117528; 65-6403-5313,
fax 65-6403-5322; yanglin.mok@
merckgroup.com. Lise Besnard, MSc, is a
process development scientist at Sanofi
Pasteur, 1541 Avenue Marcel Mérieux, 69280
Marcy l’Etoile, France. Terri Love, BSc, is a
biomanufacturing engineer; Guillaume
Lesage, MSc, is a biosafety technical
consultant; and Priyabrata Pattnaik, PhD,
is director of the worldwide vaccine initiative;
all with the life-science business of Merck
KGaA, Darmstadt, Germany, which operates
as MilliporeSigma in the United States and
Canada. Millipak, Millidisk, and Durapore
are registered trademarks of MilliporeSigma.
For reprints, contact Rhonda Brown of Foster
Printing Service, rhondab@fosterprinting.com,
1-866-879-9144 x194.
M ay 2016 14(5) BioProcess International 33