VALIDATION GUIDE

Pellicon 2 Cassette Filters

 ProFlux M12 TTangential
angential FFiltration
iltration System

Table of Contents

1. OVER-PRESSURE TESTING ........................................................................................... 12

1.1 Over-Pressure Testing ............................................................................................ 12
1.2 Cassette Feed Channel hydraulic Characterization ................................................. 14
1.3 0.1, 0.5 & 2.5 m2 DevicePerformance Scalability Testing ................................... 18
1.4 Determination of Internal Volumes ........................................................................ 110

2. BIOLOGICAL COMPATIBILITY ..................................................................................... 22

2.1 USP Class VI, Biological Reactivity Tests, in vivo ...................................................... 22
2.2 Preserving Solution FlushabilityUSP Oxidizables Substances ............................... 210
2.3 USP Physiochemical Analysis of Components ........................................................ 211

3. PERFORMANCE CHARACTERISTICS ............................................................................ 32

3.1 Preserving Solution Flushability from a New Cassette .............................................. 32
3.2 Cleaning and Storage Solution Flushability ............................................................. 34
3.3. 1N NaOH ........................................................................................................... 36
3.3.1 Urethane Compatibility .............................................................................. 36
3.3.2 Cleaning Compatibility .............................................................................. 38
3.4 NaOCL Compatibility.......................................................................................... 39
3.5 BSA Retention vs. Air Integrity for BiomaxTM 10K Membrane .................................. 310
3.6 Ethanol Extractables from a New cassette ............................................................ 313

-Contents-
1. Over-Pressure Testing
 Pellicon 2 Cassette FFilters
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1.1 Over-Pressure Testing

Testing

OBJECTIVE
To demonstrate that the urethane encapsulating jacket of the Pellicon 2 cassette filters will not
delaminate or fracture under extreme over-pressure conditions.

SUMMARY
A total of 28 units of various sizes and membrane types were tested at pressures between 100
250 psi and temperatures between 25 C and 50 C. The 28 units have a cumulative total of
1500 hrs. of operation at over-pressurized conditions. None of the units showed any
indications of fracture, delamination or excessive stresses.

METHOD
1. Pellicon 2 cassette filters were installed and torqued, as required, in stainless steel holders manufac-
tured by Millipore. The stainless steel holders were modified to reduce deflection at elevated pres-
sures.

2. Water was recirculated via high pressure pump through both Feed and Permeate sides of the device.

3. Valving was incorporated, such that equal pressure was applied to the feed, retentate and permeate.
This applies maximum stresses on the urethane jacket. Since the silicone sealing gaskets tended to
extrude and leak at approximately 200 psi this was typically the upper limit of the stressing. Testing
was generally done in 810 hour increments, starting at 25 C and gradually increasing to 50 C.

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RESULTS
Pellicon 2 cassette filters are considered to Pass, if upon close visual examination, there is no
evidence of fracture lines, delamination or deformation of the urethane encapsulation.

Size Membrane Maximum Maximum Duration Visual

(m2) Type Pressure Temperature (hours) Examination
(psi) (C)
0.5 plgc 120 50 96 pass
0.5 plbc 120 50 100 pass
2.5 BiomaxTM 10k 200 50 60 pass
2.5 Biomax 10k 250 50 60 pass
0.5 Biomax 10k 250 50 60 pass
0.5 Biomax 10k 250 50 60 pass
2.5 plgc 200 50 65 pass
0.5 Biomax 10k 100 50 24 pass
0.5 Biomax 10k 100 50 24 pass
0.5 Biomax 10k 100 50 24 pass
0.5 Biomax 10k 100 50 24 pass
0.5 Biomax 10k 100 50 24 pass
0.5 Biomax 30k 200 50 30 pass
0.5 Biomax 50k 200 50 30 pass
0.5 plgc 200 50 35 pass
0.5 plgc 200 50 35 pass
0.5 Biomax 50k 200 50 35 pass
0.5 Biomax 10k 200 50 35 pass
0.5 Biomax 50k 200 50 35 pass
0.5 Biomax 30k 200 50 35 pass
0.5 Biomax 10k 150 50 50 pass
0.5 plgc 150 50 50 pass
0.5 pltk 150 50 50 pass
0.1 Biomax 10k 200 50 100 pass
0.1 Biomax 10k 200 50 100 pass
0.1 Biomax 50k 200 50 100 pass
0.1 pltk 200 50 100 pass
0.1 Biomax 10k 200 50 100 pass

CONCLUSION
The design changes of the Pellicon 2 cassette filters have effectively eliminated the jacket
delamination issues when operated at the maximum recommended operating conditions of 100
psi at 30 C or 60 psi at 50 C.

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1.2 Cassette Feed Channel hydraulic Characterization

OBJECTIVE
To determine the relationship between feed channel pressure drop and average feed flow rate.

SUMMARY
Typical cassette filters with Biomax membrane and A and C screens, and PL membrane with
C screen were tested to determine the feed channel resistance as a function of average feed
flow rate. All units tested showed the expected hydraulic resistance and were within our release
specification.

METHOD
1. All Pellicon 2 cassette filters were tested with clean non-fouling water at 25 C in a recirculating
mode.

2. The feed and permeate flow rates were measured with rotometers. The feed, retentate and perme-
ate pressures were measured with a common, manifolded, digital pressure gauge. Feed channel DP
is defined as the difference between the feed pressure. The retentate pressure is measured as close
as possible to the cassette inlet and outlets. The average feed flow is defined as the feed flow rate
minus 1/2 the permeate rate. This helps to normalize the conditions for membranes with differing
permeabilities.

3. All units were run at the highest feed flow rates first in order to remove any internal air, which may
have an affect on the resistance to flow. The data was collected in decreasing order. All testing was
done in a pneumatic press type holder with the load set to simulate our recommended torquing
conditions of 10,000 pounds force.

RESULTS
Refer to graphs on the following pages, which illustrate Feed Channel DP vs Average Feed Flow
Rate.

CONCLUSION
All units tested performed as expected and fall within our Quality Release Specifications.

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NOTES

1. Testing done with water at 25 C

2. 8 units tested: (4) Catalogue No. P2B010A05 and (4) Catalogue No. P2B030A05

3. Quality Release Specifications: 1120 psi at 3 lpm

4. Test data for other cartridge models can be found on the Certificate of Quality supplied with each
unit.

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NOTES

1. Testing done with water at 25 C

2. 4 units tested: Catalogue No. P2B050C05

3. Unit No. P4CM8979-02, P4CM8979-13, P4DM8988-08 and P4DM8988-20

4. Quality Release Specifications: 511 psi at 3 lpm

5. Test data for other cartridge models can be found on the Certificate of Quality supplied with each
unit.

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NOTES

1. Testing done with water at 25 C

2. 7 units tested: Catalogue No. P2PLTKC05

3. Quality Release Specifications: 511 psi at 3 lpm

4. Test data for other cartridge models can be found on the Certificate of Quality supplied with each
unit.

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1.3 0.1, 0.5 & 2.5 m2 Device Performance Scalability T

DeviceP esting
Testing

OBJECTIVE
To demonstrate that the three sizes 0.1 m2, 0.5 m2 and 2.5 m2 of Pellicon 2 cassetter filters
linearly scale with respect to flux and hydraulic characteristics when operated on a model protein
stream.

SUMMARY
Representative samples of each of the three cassette filter sizes were tested with 10% BSA. The
protein flux (in Lmh) was virtually identical at four different cross-flow conditions. Further, the feed
channel hydraulics, when normalized for membrane area, are the same for all sizes.

METHOD
Pellicon 2 cassette filters were operated on 10% Bovine Serum Albumin at 8 C in a total
retentate and permeate recirculating mode. To eliminate protein solution variability all units were
run in parallel from the same feed tank. Because of measuring equipment limitations five Pellicon
2, 0.1 m2 mini-cassette filters were run in parallel in a single holder. This allowed higher more
accurately measurable flow rates for the small unit tests. Prior to testing all units were
conditioned at feed pressure of 55 psi and retentate pressure of 35 psi for 60 minutes. Testing
was performed by running TMP excursions at constant feed flow rates of 2.4, 4.8, 6.6, 8.3 and
10 Lpm per square meter of membrane area. Each condition was allowed to stabilize at least
two minutes before readings were taken. Turbine flow meters were used to measure all feed flow
rates (insensitive to viscosity) and rotometers were used for permeate flow rates (viscosity 1 cps).

RESULTS
As shown on the graphs on pg 1-9 the hydraulic resistance for all units is the same. It is
summarized by DP = 4.46 (Qfave.)1.04, where DP is measured in psi and Qfave is measured in
Lpm/m 2.

Maximum Polarized Protein fluxes (Lmh) are:

Feed Flow 0.1 m2 0.5 m2 2.5 m2

(Lpm/m2)
2.4 39.6 42.2 40.5
4.8 56.0 60.5 61.1
6.6 68.9 72.8 70.0
8.3 79.6 83.2 82.9

CONCLUSION
The three sizes of the Pellicon 2 cassette filters linearly scale very well (i.e. normalize by adjusting
for membrane area only) on a typical protein.

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NOTE All units with Biomax 10 K membrane and type A feed screen
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1.4 Determination of Internal Volumes

Volumes

OBJECTIVE

To determine the internal volumes of the Pellicon 2, 0.5 m2 and 0.1 m2 Biomax membranes, A
feed screen, Pellicon 2 cassette filters; to estimate the feed vs. permeate distribution; and to verify
the scalability between sizes.

SUMMARY
Internal volumes of the Pellicon 2 cassette filters were measured by weight of water and the
average results are summarized as follows:

Catalogue Size Total Volume Feed Volume Permeate Volume

Number (m2) (mL) (mL) (mL)
P2B030A01 0.1 31 17 14
P2B010A05 0.5 155 87 68

METHOD
1. The internal volumes were determined by measuring changes in weights before and after filling with
water, assuming 1 gram of water equals 1 mL.

2. Five typical Pellicon 2, 0.5 m2 cassette filters and four typical Pellicon 2, 0.1 m2 mini-cassette filters
were obtained from manufacturing prior to wetting. Dry weights were recorded.

3. The Pellicon 2 0.5 m2 cassette filters were tested individually, while the Pellicon 2 0.1 m 2 cassette
filters were tested 4 up in a single holder. The volumes of the respective holders were determined
(to be subtracted in order to determine the volumes for the filters only).

4. The units were assembled in holders with valves at all ports and the weight of the cassettes, including
the holder assembly were measured.

5. Air at 45 psi was applied to the permeate side of the devices to minimize water flow across the
membrane. The feed side of the device was then filled with water and weighed again. The differ-
ence between initial and final weights is the approximate weight of water contained in the cassette
feed channels.

6. With the feed side porting valve off, the permeate channels were then filled with water and re-
weighed. Again, weight difference determines fluid volume.

7. The Pellicon 2, 0.5 m2 cassette filters were tested individually, one time each; the Pellicon 2, 0.1 m2
mini-cassettes were tested as a group, repeated three times.

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RESULTS
Test results for the Pellicon 2, 0.5 m2 cassette filters are as follows:

Cassette Feed Side Weight Permeate Side Weight Total

Number (grams) (grams) (grams)
147 89 67 156
150 87 69 156
151 80 76 156
152 92 62 154
153 88 67 155
Average 87 68 155

Test results for Pellicon 2, 0.1 m2 cassette filters are as follows:

Cassette Feed Side Weight Permeate Side Weight Total

Number (grams) (grams) (grams)
Test 1 65 58 123
Test 2 68 59 127
Test 3 69 55 124
Average 67 57 125
Per mini-cassette 17 14 31

CONCLUSION
The internal volumes for the Pellicon 2, 0.5 m2 cassette filters and the Pellicon 2, 0.1 m2 mini-
cassette filters were determined and found to scale in the expected 5 to 1 relationship. Since the
Pellicon 2, 2.5 m2 cassette filters are constructed of five individual cassettes, testing is not
necessary. They are equal to five times the value of the Pellicon 2, 0.5 m2 cassette filters.

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2. Biological Compatibility
 Pellicon 2 Cassette FFilters
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2.1 USP Class VI, Biological Reactivity T

Reactivity ests, in vivo
Tests,

OBJECTIVE
To demonstrate that the materials of construction used in the Pellicon 2 cassette filters meet the
requirements of the USP Class VI, Biological Test for Plastics.

SUMMARY
The materials of construction for the Pellicon 2 cassette filters were submitted to an independent
testing laboratory for biological reactivity testing. All materials of construction meet the
requirements for USP Class VI Biological Testing for Plastics.

COMPONENTS TESTED
Biomax 5K membrane, PBCC00000, Roll number 070694CPC-1 (Representative of Biomax 5K
and 8K membranes)

Biomax 10K membrane, PBGC00000, Roll number 061593-4

Regenerated cellulosic 10 K membrane, PLGC00000, Roll number 082288PL1E

(Representative of all PL-series membranes

Polypropylene screen, P34892 (Representative of Types A, B, C and V screens)

Urethane, cured (mixed parts A and B) P60006 and P60007

Silicone gasket sheet material, P60011

METHOD
1. Components were extracted in four specified solutions at 50 C for 24 hours.

2. Samples of the extract and components were injected and implanted into mice and/or rabbits. Refer
to the tests results on pages 2-3 thru 2-9, and the U.S. Pharmacopeia for details of the procedures.

RESULTS
Neither the injections nor the implants showed signs of toxicity. See the test results on pages 2-3
thru 2-9 for detals.

CONCLUSION
All test articles meet the requirements of USP Class VI50 C Biological Test for Plastics.

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2.2 Preserving Solution FlushabilityUSP Oxidizable Substances

OBJECTIVE
To demonstrate that the U.S.P. Oxidizable Substances extracted from Pellicon 2 cassette filter,
after a specified flushing procedure, are within acceptable limits.

SUMMARY
Three Pellicon 2 cassette filters, one representing each nominal size0.1 m2, 0.5 m2 and 2.5
m2were flushed and then dynamically extracted with water for 4 hours with 20 liters per square
meter of membrane area

Samples from the extracts of the three devices passed the USP XXII Oxidizable Substances
testing.

METHOD
Representative samples of the Pellicon 2 cassette filter with Biomax membranes were flushed as
described in the recommended flushing procedure:

Representative Nominal Size Lot Unit

Catalogue Number (m2) Number Number
P2B030A25 2.5 P4CM8986 02
P2B010A05 0.5 P4CM9232 19
P2B010A01 0.1 P4BM8974 03

After flushing the prescribed volumes to drain, the devices were then dynamically extracted for 4
hours by recirculating deionized water at 25-30 C under the following conditions:

Size Ext. Volume Inlet Pressure Retentate Rate Permeate Rate

(m2) (L) (psi) (Lpm) (Lpm)
2.5 50 25 7.5 8*
0.5 10 25 1.5 3
0.1 2 25 0.3 0.6

* Limited by system restrictions

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ANALYSIS
100 mL samples of each extraction volume were analyzed for Oxidizable Substances as
described in page 1457 of the U.S.P. XXII, using the method for containers over 50 mL internal
volume.

RESULTS:
All samples passed, indicating that the Oxidizable Substances in the extraction volumes were
within the USP allowable limits.

2.3 USP Physiochemical Analysis of Components

OBJECTIVE
To demonstrate that the materials of construction used in the Pellicon 2 cassette filters meet the
requirements of the USP Physicochemical Test for Plastics

SUMMARY
The materials of construction for the Pellicon 2 cassette filters were submitted to an independent
testing laboratory for analysis. All submitted materials of construction meet the requirements for
USP Physicochemical Test for Plastics.

COMPONENTS TESTED
Biomax 10K membrane, PBGC00000, Lot number P4CM9232-46 (representative of Biomax
10K, 30K and 50K membranes)

Biomax 5K membrane, PBCC00000, Lot number 070694CPC-21 (representative of Biomax 5K

and 8K membranes)

Polypropylene Screen, P34892 (representative of Types A ,B , C and V screens)

Urethane, cured (mixed parts A and B), P60006 and P60007

Silicone Gasket Sheet Material, P60011

METHOD
Components were extracted in sterile Water for Injection at 70 C for 24 hours. Samples of the
extraction water were then analyzed for Non-Volatile Residue, Residue on Ignition, Heavy Metals
and Buffering Capacity. Refer to the attached test reports and the U.S. Pharmacopeia for details
of the procedures.

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RESULTS
The tests that are of most interest to typical applications of Pellicon 2 cassette filters are the Non-
Volatile Residue and Heavy Metals. The results are summarized below. See test reports on
pages 2-13 thru 2-17 for complete results.

Component NVR Heavy Metals

(mg)
Biomax 10 K membrane 3.6 Passed
Biomax 5 K membrane 0.4 Passed
Urethane 1.6 Passed
Gasket 0.0 Passed
Polypropylene Screen 0.4 Passed

CONCLUSION
All test articles meet the requirements for USP Physiochemical Test for Plastics.

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3. Performance
Characteristics
 Pellicon 2 Cassette FFilters
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3. PERFORMANCE CHARACTERISTICS

3.1 Preserving Solution Flushability from a New Cassette

OBJECTIVE
To verify that the membrane preserving solution is effectively removed from the Pellicon 2 cassette
filters with a recommended flushing procedure.

SUMMARY
Samples from a 4 hour recirculating, 25 C water extraction, after a recommended flushing step
showed:

Total Organic Carbon = 9 ppmC

Non-Volatile Residue = 4 mg/L

FTIR analysis of nonvolatile residue identifies it as glycerine

Passed USP Oxidizables Testing

METHOD
A Pellicon 2, 0.5 m2 cassette filter (catalogue number P2B010A05 and unit number P4CM8984-
08) preserved per SOP number 0060455 SO with 0.1% Lysol, 20% Glycerine and 79% water
was initially water flushed as follows:

1. 5 minutes at feed pressure = 50 psi

Retentate pressure = 25 psi

Approximate volume thru retentate = 6 liters

Approximate volume thru permeate = 24 liters

2. Recirculated for 1 hour at 40 C to simulate the recommended pre-cleaning step

3. Flushed per procedure, which recommends 12 liters at feed pressure of 15 psi (6 L permeate and 6
L retentate) and then flushed approximately 50 liters at Feed pressure = 25 psi and Retentate
pressure = 10 psi

The unit was then dynamically water extracted in a recirculating mode for 4 hours at 25 C with
10 L of water . A Test Sample was collected for TOC, NVR (with FTIR) and USP Oxidizables.

NOTE Prior to extracting the test sample the system was recirculated for 1 hour with a PVDF,
low extractables cassette mock-up. A sample of the recirculating water was
collected and labeled System Blank. This blank will be used to determine the
approximate system background contamination levels.

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RESULTS
Sample Description TOC NVR Oxidizables FTIR
(ppmC) (mg/L)

System Blank 0.81 3.3 Pass

Test Sample 9.7 6.9 Pass glycerine
Net Difference 8.9 3.6
(amt. contributed by cassette)

CONCLUSION
The recommended procedure flushes a new Pellicon 2 cassette filter, such that the effluent is
reduced to < 10 ppm TOC and NVR (residue identified as glycerine), and passes the USP
Oxidizable testing.

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3.2 Cleaning and Storage Solution Flushability

OBJECTIVE
To determine the flush volume required to remove cleaning and storage solution from the Pellicon
2 cassette filters

SUMMARY
The testing showed that the effluent pH from a Pellicon 2, 0.1 m2 mini-cassette filter and a Pellicon
2, 0.5 m2 cassette filter filled with NaOH was equal to the incoming flush water pH, after
approximately 20 liters of flushing.

METHOD
1. A Pellicon 2, 0.5 m2 cassette filter, A screen with Biomax 10K membrane, unit number BE-104
not previously preserved, was pumped full of caustic, 0.5N NaOH and total internal volume of 140
mLs. The filled unit was then flushed with UF water pH 7, 35 C at the following conditions:

Feed Pressure = 40 psi

Retentate Pressure = 0 psi (minimal restrictions)

Permeate Pressure = 0 psi (minimal restrictions)

Retentate Flow Rate = 2.3 Lpm

Permeate Flow Rate = 3.0 Lpm

2. Samples of Feed, Retentate and Permeate were collected and the pH was measured.

3. The same testing was also done on a Pellicon 2, 0.1 m2 mini-cassette filter. Flushing was done at
the same pressures, which results in approximately 20% of the above flow rates. The Pellicon 2
mini-cassette filter has one-fifth the membrane area and internal volumes.

RESULTS
The effluent pH, retentate and permeate initially at pH 13 from both the Cassette and Mini-
cassette, flushed down to equal the incoming UF water pH 7. See curves on page 3-5

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3.3. 1N NaOH

3.3.1 Urethane Compatibility

OBJECTIVE
To demonstrate the compatibility of the urethane from the Pellicon 2 cassette filter with 1N
NaOH.

SUMMARY
Samples of the urethane from the Pellicon 2 cassette filter were exposed to 1N caustic at 50 C
for approximately 3500 hours. Testing shows no measurable change in hardness or weight gain
after exposure.

METHOD
1. Samples of the urethane from the new Pellicon 2 cassette filter were mixed and cured per our
standard manufacturing procedure.

2. The cured urethane was die cut into samples of approximately 4-inch length x 1-inch wide x 0.1-
inch thick (weights were in the 8-11 gram range).

3. Five test samples were soaked in a polyethylene container with 1 liter of 1N NaOH, held at 50 C.

4. Five control samples were similarly soaked in 20 C water.

5. Both the test specimens and the controls were periodically removed, blotted dry and measured for
weight in grams and hardness in Shore D changes.

6. The measurements from the 5 samples were averaged and compared to the original Time = 0
values prior to soaking. The Time = 0 values are reported as 100% and all subsequent measure-
ments are reported as % of Original.

RESULTS
See Hardness and Weights graphs on page 3-7 for details. The 1N NaOH exposed samples
showed no measurable differences in weight or hardness.

CONCLUSION
Thirty-five hundred hours of exposure to 1N NaOH at 50 C show no measurable changes in
weight or hardness, indicating no obvious degradation by the caustic.

To demonstrate that 380 hours, approximately 1 year of cleaning cycles, 1N caustic exposure do
not degrade performance of a Pellicon 2 cassette filter.

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3.3.2 Cleaning Compatibility

OBJECTIVE
To demonstrate that 380 hours, approximately 1 year of cleaning cycles, 1N caustic exposure
does not degrade performance of a Pellicon 2 cassette filter.

SUMMARY
A Pellicon 2 cassette filter with Biomax 10K membrane was tested for Air Integrity, BSA flux and
BSA retention at Time = 0 , and 160 and 380 hours of NaOH exposure. Test results show
changes well within the reproducibility of the procedures, indicating no measurable changes after
caustic exposure.

METHOD
1. Unit Number P4AM8798-07 was flushed and loaded into a standard Millipore stainless steel holder
per our recommended procedures. At time = 0 with no NaOH exposure, air integrity was per-
formed at 30 psi on the water flushed cassette. Retention and flux testing was performed with 6%
BSA at 8 C at feed pressure = 70 psi and retentate pressure = 30 psi.

2. The cassette was then exposed to 1N NaOH, 55 C, Feed pressure = 55 psi and Retentate pres-
sure = 35 psi for 160 hours

3. The above retention and flux testing was then repeated.

4. The cassette was then exposed to the NaOH for an additional 220 hours, a total of 380 hours and
the reference tests were then repeated for a third time.

RESULTS

NaOH EXPOSURE TIME

Reference Test T = 0 hours T = 160 hours T = 380 hours

(Initial)
Air Integrity (cc/min) at 30psi 9 14
Maximum BSA Flux (Lmh) at 70/30 psi 122 117 112
BSA Retention (%) at 70/30 psi 99.98 99.95 99.95

CONCLUSION
The differences in the reference testing are considered to be within the range of reproducibility
and it is concluded that the 360 hours of 1N NaOH is not a measurable affect on the
performance of the Pellicon 2 cassette filter with Biomax 10K membrane.

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3.4 NaOCL Compatibility

OBJECTIVE
To investigate the affects of 60,000 ppm hour active chlorine exposure on air integrity and BSA
retention of a cassette with Biomax 10K membrane.

SUMMARY
Two Pellicon 2 cassette filters with Biomax 10K membrane were exposed to average
concentration of 600 ppm active chlorine at 45 C for 100 hours. BSA retention and air integrity
were tested before and after chlorine exposure with no measurable degradation.

METHOD
1. Two units (catalogue number P2B010A05) were tested for water wet air integrity at 30 psi and
retention tested with a 6% BSA solution with the feed pressure set at 50 psi and retentate pressure
equal to 10 psi.

2. Permeate samples were collected and analyzed for protein passage with a visible spectrophotometer
at 562 nm.

3. The units were then assembled in a standard acrylic holder with valves attached to all ports, manu-
factured by Millipore .

4. The cassettes were filled with a solution of 900 ppm active chlorine made from NaOCL and valved
off. The entire assembly was then submerged in a 45 C water bath.

5. At the end of 24 hours the assembly was drained and refilled with a new 900 ppm chlorine solution.
The drained solution was measured and found to typically contain 300 ppm active chlorine, for an
average of 600 ppm over the 24 hours or 14400 ppm hours per day of testing.

6. The units were re-submerged for the next 24 hour cycle. This was repeated for a total of 100 hours
for a total of 60,000 ppm hours of exposure.

7. At the end of the chlorine exposure the units were retested for air integrity and BSA retention using
the same procedures as previously.

RESULTS

Unit # 138 Unit #142

Air Integrity (sccm) prior to exposure 7 8
BSA Retention (5%) prior to exposure 99.97 99.97
Air Integrity (sccm) after exposure 3 10
BSA Retention (%) after exposure 99.97 99.97

CONCLUSION
The 60,000 ppm active chlorine exposure did not cause measurable changes in either the air
integrity or protein retention of the Pellicon 2 cassette filter with Biomax membrane.
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3.5 BSA Retention vs. Air Integrity for Biomax 10K Membrane

OBJECTIVE
To determine the relationship between BSA retention and Air Integrity testing for the Pellicon 2
cassette filters with Biomax 10K membrane

SUMMARY
All units with air flow rates below 25 scc/min showed a 99.9% retention of BSA. Units with very
poor integrity, 290 scc/min, had > 99% retention. Our published specification is 18 scc/min at
30 psi.

METHOD
Twenty-five Pellicon 2 cassette filters with Biomax 10 K membrane units with varying air integrity
values were tested on BSA. Testing consisted of:

1. Challenging the devices with 6% BSA at Feed Pressure = 70 psi and Retentate Pressure = 30 psi

2. The units were loaded and torqued per standard recommendations.

3. The BSA was run in a total recycle mode at 8 C.

4. All units were preconditioned by operating at P (feed) = 55 psi and P (retentate) = 35 psi for 1
hour prior to taking samples.

5. Permeate samples were collected and analyzed for protein passage with a visible spectrophotometer
at 562 nm.

6. Sensitivity was increased by using a BCA (Bicinchoninic Acid) detection reagent on all permeate
samples.

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RESULTS
The actual data is shown below and summarized in the following table.

Lot Number Unit Number Air Integrity at 30 psi BSA Retention

(cc/min) (%)
P4EM9248 50 7 99.93
P4EM9248 16 293 99.04
P4EM9248 05 53 99.82
P4EM9248 42 18 99.93
P4EM9248 45 19 99.92
P4EM9248 44 26 99.92
P4DM9249 82 8 99.94
P4DM9249 89 18 99.91
P4DM9249 46 18 99.96
P4DM9249 38 18 99.94
P4DM9249 52 19 99.92
P4DM9249 36 22 99.93
P4DM9249 19 24 99.90
P4DM9249 18 24 99.93
P4DM9249 14 26 99.89
P4DM9249 13 30 99.89
P4DM9249 42 31 99.91
P4DM9249 16 33 99.89
P4DM9249 43 45 99.85
P4CM8984 93 6 99.96
P4AM8798 07 9 99.96
P4AM8798 10 19 99.96
P4AM8797 03 9 99.97
P4AM8799 08 8 99.98
P4CM9232 17 9 99.95

CONCLUSION
The Air Integrity specification for Pellicon 2 cassette filter with Biomax 10K membrane of 18 cc/
min at 30 psi results in BSA retentions of 99.9%. For other membranes, please refer to the
Integrity Release Criteria indicated on the Certificate of Quality included with each unit.

3-10
 Pellicon 2 Cassette FFilters
ilters

3-11
 Millipore Validation Guide
Validation

3.6 Ethanol Extractables from a New Cassette

Objective
To determine the level and source of Non-Volatile Residue that can be extracted from a cassette
filter in 40% ethanol

SUMMARY
After an initial water flush, Pellicon 2, 0.5 m2 cassette filters were statically extracted in 40%
ethanol for 8 hour at room temperature. Analysis showed that the total amount of NVR was 100
mg/cassette and it was identified as components of the preserving solution (glycerin and
Benzalkonium chloride)

METHOD
1. Five Pellicon 2, 0.5 m2 cassettes (catalogue number P2B010A05) were preserved per SOP number
0060455 SO with 0.1% Roccal (Benzalkonium chloride (BAK) active ingredient), 20% Glycerine
and 79% water was initially water flushed as follows:

5 minutes at feed pressure = 50 psi

Retentate pressure = 25 psi

Approximate volume thru retentate = 6 liters

Approximate volume thru permeate = 24 liters

2. Recirculated for 1 hour at 40 C to simulate the recommended pre-cleaning step

3. Flushed per procedure, which recommends 12 liters at feed pressure of 15 psi (6 L permeate and 6
L retentate) and then flushed approximately 50 liters at Feed pressure = 25 psi and Retentate
pressure = 10 psi.

4. The units were assembled into a holder as a group and filled with a 40% ethanol at a total volume
of 750 mLs. The assembly was allowed to extract at room temperature of 20 C for 8 hours. At the
end of the extraction period the ethanol was collected.

5. A 250 mL sample was evaluated for nonvolatile residue and the residue was then analyzed by FTIR.
In addition a portion of the sample was analyzed by HPLC in order to quantify the amount of BAK.

RESULTS

The total nonvolatile residue = 507 mg for (5) cassettes

= 100 mg / cassette
and this is identified as = 30 mg BAK /cassette
= 70 mg of glycerin /cassette

3-12
 Pellicon 2 Cassette FFilters
ilters

CONCLUSION
The only detectable ethanol extractables are due to the preserving solution used in the Pellicon 2
cassette filters.

3-13
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Millipore and Pellicon are registered trademarks of Millipore Corporation or an affiliated company. Biomax
is a trademark of Millipore Corporation or an affiliated company. Lit. No. VG059 Rev 10/98. 1998
Millipore Corporation or an affiliated company. All rights reserved.