Millistak+HC AppGd_textC.qxd - Millipore

A P P L I C A T I O N G U I D E
Â
Using Millistak+ ®
HC Filters for
Mammalian Cell Culture Clarification
Compress processing steps— saving time, resources and operating costs
Table of Contents
Understanding Millistak+ HC
High Performance Stacked Filters ........................1
How Are Millistak+ HC Filters Used? ....................3
Why Are Millistak+ HC Filters Better? ..................5
Proven Performance for Mammalian
Cell Culture Clarification
Application Test Results ..................................6
Post-Centrifuge ..............................................7
Post-TFF Microfiltration ..................................10
Perfusion Bioreactor......................................11
Whole Cell Culture ......................................13
Summary of Application Test Results ..............14
Evaluation and Optimization
Separation Mechanisms ..............................15
Flux ............................................................17
Scale-up ....................................................19
Guidelines for Process-Scale Operation
Typical Operating Parameters ......................20
Installation ..................................................20
Flushing ......................................................21
Leak-Testing Installed Cartridge Assemblies ....21
Steam-in-Place Sterilization ..........................23
Hot Water Sanitization ................................26
Chemical Compatibility ....................................26
Technical Assistance ........................................26
1

Understanding
Millistak+ HC High
Performance Stacked Filters
Millistak+ HC filters are a unique
clarification/prefiltration tool
developed to meet the critical needs
of the biopharmaceutical industry.
Designed expressly for large-scale
mammalian cell fermentation
processes, Millistak+ HC filters
combine multiple clarification steps
into a single unit operation.
Innovative Dual-Action Filter
The technology is built upon the
superior performance qualities of
the cellulosic filter media available
in the Millistak+ filter product line.
The DE Series filter media consists of
refined cellulose fibers and high-purity
diatomaceous earth. The composite
is chemically treated to impart a
cationic (positive) surface charge,
to enable the capture of negativelycharged
biological particulates and
colloids (cell fragments, protein
agglomerates, etc.).
Millistak+ HC filters represent the
next generation of the stacked-disk,
cellulosic depth filter. The principal
design feature of Millistak+ HC filters
is a graded pore structure, tailored to
maximize the holding capacity of the
filter for the broad range of particle
sizes typical of fermentation fluids.
Two full-thickness cellulosic pads
are employed—a fine grade media
overlaid by a coarser grade—which
together span a far wider range of
pore size than achievable in any
single-grade filter. For particular
configurations of Millistak+ HC filters,
the construction is further enhanced by
the addition of a microporous cellulose
membrane underlying the dual-layer
depth filter media, to further extend
small particle retention.
Millistak+ HC Filter
Open DE Grade
Tighter DE Grade
Cellulosic Membrane
For process-scale operations,
Millistak+ HC filters are available
as a lenticular, stacked-disk cartridge
containing eight double-sided, 16-inch
diameter disks for 1.8 m 2 frontal
Millistak+ HC Filters Application Guide 1
Flow
Figure 1. Media Layers of Millistak+ HC
Filters

Figure 2. Millistak+ HC Stacked-Cell
Cartridges Used in a Large-Scale Process
2
TFF
NFF
Centrifuge
surface area. Up to eight stacked-disk
cartridges can be assembled in a
single housing. For smaller-scale
testing, Millistak+ HC filters are also
available in disposable 650 cm 2
and 23 cm 2 cartridges.
For complete details on the product
line, refer to the Millistak+ HC Filters
Data Sheet (Lit. No. DS1000EN00).
Primary Secondary Sterile
10 – 40 NTU
50 – 200 NTU
200 – 600 NTU
Figure 3. Typical Downstream Recovery Process
0.5–5 NTU
NFF NFF
0.1– 1 NTU
NTU = Normal Turbidity Unit
www.millipore.com

How Are Millistak+ HC
Filters Used?
The typical downstream recovery
process for mammalian cell derived
biologics consists of multiple clarification
steps to prepare the liquid-borne
product for either sterile filtration or
direct chromatographic purification.
In the first stage or primary separation
step, the bulk of the cell mass is
removed from the broth—depending
on the volume and type of fermentation
process, different clarification
methods are used, as shown in
Figure 3.
Batch Processing
In large-scale (> 1,000 liter) batch
and fed-batch fermentations, the bulk
of the cell mass is ordinarily separated
by centrifugation or tangential flow
microfiltration. For small batch fermentations
(< 1,000 liter), cartridge depth
filters (NFF) are often used in the pore
size range of 3–5 µm.
Perfused Bioreactor
In contrast to batch processes, perfused
bioreactors are harvested continuously
over extended operating periods
lasting several weeks or months.
For primary clarification, the flowcontrolled
product stream from the
perfusion bioreactor will typically pass
through either a membrane separator
(hollow fiber TFF) or a gravity settler,
with the cell mass recycled to the
fermenter. The crude product is pooled
and then batch-processed for secondary
clarification and final sterile filtration.
A Single Step for Clarification
and Prefiltration
The effluent of these various primary
clarification steps is generally unsuitable
for either sterile filtration or
chromatographic processing. The level
of suspended matter in all cases
(which varies by method) will cause
rapid downstream fouling, with significant
cost impact. The turbidity ranges
noted in the process schematic in
Figure 3 are indicative of the particle
loads present. Even in the case of
TFF microfiltration—typically executed
with a 0.45 – 0.65 µm membrane—
particle loading in the permeate does
not allow for direct sterile filtration at
any reasonable capacity or cost.
Further clarification is required, for
which disposable NFF filters are
normally used (depth or surface types).
The challenge confronting the process
engineer is to arrive at an integrated
clarification and sterile filtration train that
will minimize overall production costs.
In many cases, secondary clarification
of tissue culture fluid involves several
(two or three) serial filtration steps. The
need for multiple separations arises from
the broad range of particle sizes and
Millistak+ HC Filters Application Guide 3

types present in the process stream.
Millistak+ HC filters consolidate these
multiple serial filtration steps into a
single operation.
Media Configurations Specific
to Your Application
Extensive field testing on a variety of
bioprocess streams has led to the
development of three Millistak+ HC
filter configurations, each containing
dual-layer cellulosic depth filter media
in grade combinations best suited
to a particular clarification role
(see table below).
4
More than Cell Culture Clarification
Millistak+ HC filters are an excellent
clarification tool for a number of
associated biopharmaceutical
process applications:
• cell culture growth media
(MEM, peptones)
• plasma fraction products
• vaccines
As in biologics manufacturing,
Millistak+ HC filters offer an economical
and effective means of protecting
downstream sterilizing grade filters,
to achieve optimum overall process
efficiencies.
Media Characteristics Media Construction
A1HC*
For post-TFF Tightest media 60DE + 75DE + RW01
clarification fluids combination
B1HC*
For post-centrifuge A more open 50DE + 75DE + RW01
or settled permeate first layer
containing cellular
particulate
C0HC
For perfusion Two layers of 30DE + 60DE
bioreactor fluid an open Millistak+
DE media
*A1HC and B1HC include the same secondary layer and a membrane layer
proven to protect downstream 0.22 µm membranes.
www.millipore.com

Why Are Millistak+ HC
Filters Better?
Biological solids exhibit poor filter
cake properties—very thin deposits
exhibit very high flow resistance.
The capacity of a clarifying depth
filter is, therefore, highly dependent
on the accessible internal surface
area available for particle deposition.
For a given media type and grade,
capacity increases linearly with
thickness or bed depth. Moreover,
as commonly occurs in biological
suspensions, the particles span a
broad range of sizes—to maximize
capacity, the filter media must be
structured to accommodate these
particle sizes in proportion to their
number. For this reason, a gradient
pore structure or media density
is optimal.
Outperforms the Competition
Based on these design criteria,
Millistak+ HC filters have been
developed to outperform all competitive
depth filters. Each cellulosic
pad within the multilayer composite
possesses a graded pore structure
from front to back. Coupling two
such pads in series expands the
effective pore size range by as
much as several orders of magnitude.
In addition, Millistak+ HC filters
employ two full-thickness cellulosic
pads for an overall bed depth of more
than 0.260 in.—thicker than any
other cellulosic depth filter on the
market today.
Unique Membrane Layer
Improves Performance
A major advance in the design of
stacked-disk prefilters, as offered in
Millistak+ HC filters, is the incorporation
of a microporous membrane layer,
downstream of the cellulosic pads.
The membrane layer delivers two
important advantages. First, it serves
as a final barrier to trap small particles
and colloids that would otherwise
foul a trailing sterile filter. Secondly,
the membrane contributes a modest
hydraulic resistance to the filter,
which significantly improves feed
flow distribution and, consequently,
media utilization. All vertical stackeddisk
designs inherently force a greater
percentage of feed flow to the lower
sections of the filter column, which
are under higher hydrostatic pressure.
The addition of the microporous
membrane to Millistak+ HC filters
helps to minimize this vertical pressure
gradient, to utilize all available filter
area more evenly and efficiently.
Millistak+ HC Filters Application Guide 5

Proven Performance
for Mammalian
Cell Culture Clarification
The benefits of the unique Millistak+
HC filter construction are exemplified
in Figure 4. The test fluid is a typical
mammalian cell culture, which had
been centrifuged for bulk biomass
removal: the centrifugation had
reduced the turbidity of the suspension
to 96 NTU, at which level the prefilter
exhibits little pressure build-up and
overall performance is judged by the
downstream sterile filter capacity.
6
0.22 µm Durapore Resistance
(psid/LMH)
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
CHO Centrate: 96 NTU
0
Figure 4. Effect of Bed Height and Membrane Layer
Application Test Results
The individual contributions of the
three media types that comprise
A1HC were evaluated. As shown in
Figure 4, the 75DE layer was tested
alone, then the combined 60DE/
75DE pairing and finally the A1HC
combination (which includes the microporous
membrane). The benefit of the
increased thickness and expanded
pore-size range of the two pad layers
is evident in the substantial increase in
sterile filter capacity—which was
more than doubled—around the
0.010 psid/LMH level (representing
75DE
60DE + 75DE
A1HC: 60DE + 75DE + RW01
200 400 600 800 1000 1200 1400
0.22 µm Durapore ® Membrane Throughput (L/m 2 )
www.millipore.com

typical operating conditions). The
addition of the membrane layer further
enhances the retention capability of
the A1HC prefilter, to increase sterile
filter protection dramatically.
In order to achieve this level of
sterile filter capacity, the prefilter must
be highly retentive to small particles
and colloidal materials. The correlation
of sterile filter capacity with the turbidity
of prefilter effluent is shown in
Figure 5. The data show that, to reach
sterile filter capacities in the range of
500 L/m 2 , the prefilter must reduce
fluid turbidity to well below 10 NTU.
0.22 µm Durapore Capacity
(L/m2 @ 20 psid)
700
600
500
400
300
200
100
0
0
Clarified Mammalian Cell Culture
Figure 5. Sterile Filter Capacity as a Function of Feed Turbidity
Depending on the primary clarification
process employed (as shown
in the process schematic in Figure 3
above), secondary clarification will
further reduce turbidity levels by a
factor of 5:1—100:1. Note that
monitoring effluent turbidity can be
a useful guide in determining prefilter
performance, but it should not be used
for actual sterile filter sizing, due to
the complexities of membrane fouling.
Post-Centrifuge
Centrifugation is by far the most
widely practiced method of bulk
cell mass removal for fermentation
10 20 30 40 50
Turbidity (NTU)
1.54 PCV,
60% Viability
Millistak+ HC Filters Application Guide 7

oths. For this particular case study,
the centrifugation process reduced the
suspended matter to a level corresponding
to a turbidity of 110 –175 NTU.
Side-by-side tests were run on
each of the three Millistak+ HC filter
device sizes to clarify the cell culture
centrate. Each prefilter device (B1HC
media) was operated in tandem with
a trailing sterile filter under constant
flux conditions, all units drawing on the
same lot of feed material. As shown in
Figure 6, the performance profiles for
the three device sizes—plotted
as total system pressure differential
Figure 6. Prefilter and Sterile Filter Scale-up
8
Total System Pressure
Differential (psid)
35
30
25
20
15
10
5
0
0
Cell Culture: Centrate
Lab-Scale (3 Trials)
Pilot-Scale
Process-Scale
(prefilter + sterile filter) relative to
prefilter throughput or capacity—
were reasonably consistent with
one another. The same figure also
shows that there was some variability
among the three trials that were run
with the smallest, lab-scale device.
This illustrates the need to run multiple
lab-scale tests, in order to normalize
for variability in process stream and
filter media, before scaling up to
pilot-scale and process-scale tests.
This is discussed in more detail later,
under Scale-up, in the section on
Evaluation and Optimization.
50 100 150 200 250 300 350 400
Prefilter Capacity (L/m2)
1.54 PCV,
60% Viability
Feed Turbidity:
80 –100 NTU
Prefilter Flux:
100 –170 LMH
Prefilter: B1HC
www.millipore.com

The moderately higher capacities
achieved with the pilot-scale Opticap
prefilter and process-scale (16-inch)
Although the prefilter accounts for the
majority of the pressure build-up in the
system, the comparatively small sterile
filter area used in the lab-scale tests
exaggerates its pressure contribution.
As a result, the lab tests underestimate
the capacities achieved with the relatively
larger sterile filters used in the
pilot- and process-scale tests.
stacked-disk filter are due to differences
in the relative areas of the sterile filters
used, as shown in the following table:
Prefilter Area Sterile Filter Area Area Ratio
Lab-scale 23 cm 2 4 cm 2 5.7
Pilot-scale 650 cm 2 200 cm 2 3.3
Process-scale 1.8 m 2 0.69 m 2 2.5
These and other tests have produced
the following results in process-scale
trials for secondary clarification of a
CHO cell culture prior to sterile filtration
(0.22 µm). The ratio of prefilter
area to sterile filter area is approximately
2.5 :1. Performance is measured
at a constant feed rate (flux) to
the prefilter of 100–150 LMH:
Capacity Pressure
(L/m 2 ) Differential (psid)
Run 1: 60% Cell Viability
Millistak+ HC B1HC Prefilter 476 30
0.22 µm Durapore Filter 1100 5
Run 2: 80% Cell Viability
Millistak+ HC B1HC Prefilter 443 30
0.22 µm Durapore Filter 1088 5.5
Millistak+ HC Filters Application Guide 9

Post-TFF Microfiltration
Mammalian cell cultures clarified by
means of crossflow (TFF) microfiltration
will typically utilize a membrane in
the 0.45–0.65 µm range. Even at
this high degree of separation, the
treated effluent (permeate) frequently
requires further clarification in order
to achieve acceptable capacities in
subsequent sterile filtration. The preferred
Millistak+ HC prefilter for these
low solids fluids is the A1HC. When
applied to a post-TFF process stream,
this prefilter will ordinarily develop little
pressure build-up, yet will provide a
remarkable improvement in downstream
sterile filter throughput.
Sterile Filter (0.22 µm)
Capacity (L/m2 )
10
700
600
500
400
300
200
100
0
CHO Cell Culture: TFF/MF Permeate
Competitor A
Single Layer
Media
Competitor A
Dual Layer
Media
Figure 7. Competitive Prefilter Analysis: Sterile Filter Protection
In one example, a fed-batch CHO
cell culture was first processed using a
0.65 µm TFF/MF (Prostak ) filter system
with Durapore membrane and the
permeate was batch-filtered through
the A1HC, followed by a 0.22 µm
Durapore sterilizing filter (prefilter flux =
150–160 LMH). The A1HC was able
to achieve a process capacity of over
500 L/m 2 , while never exceeding
a pressure differential of 5 psid. The
throughput on the trailing sterile filter
was greater than 3000 L/m 2 .
Figure 7 illustrates the performance
benefit of Millistak+ HC filters over
competitive products, in side-by-side
trials on a mammalian cell culture
Competitor B
Single Layer
Media
Millistak+
A1HC
Prefilter Flux:
37 LMH
Sterile Filter Flux:
200 LMH
www.millipore.com

after microfiltration (0.65 µm). The
competitive products represent similar
stacked-disk prefilters, based on both
single- and double-layer cellulosic
media. In all tests, the prefilter exhibited
little or no pressure build-up—
performance was measured according
to the volume of filtrate that could be
processed subsequently through a
sterilizing-grade (0.22 µm) filter.
As shown, the A1HC outperformed
all other products tested, demonstrating
that the proper design of depth filter
media and bi-layer configuration will
result in superior sterile filter protection.
Perfusion
Bioreactor
Biomass
300 – 400 NTU
Depth
Filter
Figure 8. Processing of Perfusion Bioreactor Product
Perfusion Bioreactor
A side-by-side test of Millistak+ HC
filters and several competitive single
and multilayer stacked-disk products
was conducted on a mammalian cell
(CHO) perfusion process. As shown in
Figure 8, the perfusion process generates
two types of material, which are
accumulated and batch-processed.
The majority of the process fluid
(~70%) is a crude product, harvested
from the bioreactor through a screening
mechanism, which must be further
clarified before final filtration through
a 0.45 µm membrane. In addition,
the bioreactor is periodically purged
Spin
Filter
Cell Harvest
40 NTU
0.45 µm
Sterile Filter
Millistak+ HC Filters Application Guide 11

to control cell density and contaminant
levels. This raw cell culture is, similarly,
clarified and sterile filtered to recover
contained product. The suspended
solids content of the raw cell culture is
many times that of the prefiltered harvest
fluid, as indicated by their relative
turbidities (300 NTU versus 40 NTU).
All tests were run at constant
flow (150–200 L/m 2 /hr, based
on prefilter area), with the clarifying
prefilter and sterile filter operated
in tandem.
During the early growth phase of
the culture with cell counts low, the
12
Sterile Filter Pressure
Differential (psid)
16
14
12
10
8
6
4
2
prefilters displayed little or no pressure
build-up: the capacity of the filter train
was dictated by the pressure build-up
on the downstream 0.45 µm filter.
Figure 9 shows the pressure-volume
profiles for the Millistak+ C0HC filter
and two trial runs of the competitive
product, using process-scale multilayer
stacked-disk filter cartridges. The
C0HC prefilters clearly demonstrated
a capacity to retain not only the
preclarified cell culture (harvest fluid)
but also a relatively large portion of
the untreated culture, in far greater
volumes than the competitive product.
Mammalian Cell Culture Perfusate Clarification
C0HC: Preclarified Harvest
C0HC: Unclarified Harvest
Competitor: Harvest, Run 1
Competitor: Harvest, Run 2
0
0 50 100 150 200 250 300 350 400
Process Volume (L)
Figure 9. Competitive Prefilter Analysis for Perfusion Bioreactor Product (Early)
www.millipore.com

Tests run later in the perfusion cycle
(when cell densities and contaminant
levels are higher), showed significant
pressure build-up on both prefilters and
sterile filters for competitive products
(see Figure 10). The Millistak+ C0HC
filter repeatedly demonstrated far
greater capacities for both preclarified
and untreated biomass over
competitive prefilter products.
Whole Cell Culture
A series of lab-scale tests were
conducted on a CHO cell culture
processed directly from a batch
Capacity (L/m 2 )
500
400
300
200
100
0
20 psid
Competitor A
Multilayer
Biomass
Cell Harvest
20 psid
Competitor A
High Capacity
Single Layer
20 psid
Competitor A
Multilayer
fermenter: the broth contained a cell
density of 6 x 10 6 /mL, corresponding
to 1.5% packed cell volume
(cell viability 90%). The C0HC
prefilter was applied in tandem with
a 0.22 µm sterile filter (area ratio 6:1)
at a constant prefilter flux of 160 LMH.
The C0HC reached a capacity of
215 L/m 2 , on average, at a pressure
differential of 20 psid: the sterile filter
(which showed no significant pressure
build-up) reached a corresponding
capacity of 1100 L/m 2 .
20 psid
B1HC C0HC C0HC C0HC
Filter Type
4.5 psid 4.5 psid
Figure 10. Competitive Prefilter Analysis for Perfusion Bioreactor Product (Late)
6.5 psid
Millistak+ HC Filters Application Guide 13

Summary of Application Test Results
The following table presents a summary
of the various applications tested with
Millistak+ HC filters. Values are given
0.2 µm
Millistak+ HC Millistak+ HC Sterile Filter
Media Capacity Capacity
Application Grades (L/m 2 ) (L/m 2 )
Human Serum A1HC 120 —
E. coli Lysate: Centrate B1HC 35 —
E. coli (20% Viability) C0HC 20 120
Mammalian Cell Culture (10% FBS) C0HC 60 130
Mammalian Cell Culture C0HC 125 > 200
Mammalian Cell Culture:
post-MF (0.65 µm) B1HC 400 1500
Bacterial Fermentation: Centrate B1HC 130 —
14
for the approximate prefilter and
trailing sterile filter capacities
achieved in these tests.
www.millipore.com

Evaluation and
Optimization
Separation Mechanisms
The particle removal achieved by
Millistak+ HC filters is the consequence
of two distinct separation mechanisms.
The first of these mechanisms is
mechanical sieving, in which particles
are captured within the prefilter due to
their physical size and/or orientation.
In the case of a depth filter, the path
through the media matrix is tortuous
and varies greatly in effective pore
dimensions, providing ample opportunity
for particle retention by entrapment.
The outward effect, typical of
most filters, is a build-up in pressure
differential in the direction of flow.
The second particle capture
mechanism for Millistak+ HC filters
is adsorption: the physical/chemical
binding of particles to the filter media.
This process is aided by the net
positive charge on the surface of the
Millistak+ HC filter media. Colloidal
particles in a biological suspension
(cell wall fragments, agglomerated
proteins, polynucleic acids, etc.)
exhibit a net negative electrostatic
charge on their surface at typical
near-neutral pH conditions. The
cationic charge on the Millistak+ HC
filter media allows these particles to
be captured, when otherwise they
might escape retention due to their
small size.
This electrostatic retention mechanism
allows the Millistak+ HC prefilter
to be effective in removing particles
well below the smallest pore size
of the media and thereby provides
additional protection to the sterile filter.
This capture mechanism does not
however reveal itself as a pressure
build-up on the prefilter. Instead, the
user must carefully monitor the quality
of the filtrate, to detect the point at
which all available adsorption sites
within the media have been used.
This can be done either by monitoring
filtrate turbidity in relation to process
throughput or by measuring the
capacity of the downstream sterile
filter in situ or off-line (with a post-run
Vmax SM test).
Depending on the properties of
the feed stream (and the consequent
relative contribution of the two separation
mechanisms described above),
two distinct types of behavior may
be observed for the Millistak+ HC
prefilter. These two distinct profiles
Millistak+ HC Filters Application Guide 15

Figure 11. Two Types of Behavior Observed for the Millistak+ HC Prefilter
16
Breakthrough Limited
Pressure Differential (psid)
Pressure Limited
Pressure Differential (psid)
25
20
15
10
5
25
20
15
10
5
Turbidity
Pressure Differential
0
0
0 5 10 15 20 25
Volume Filtered per Unit Area
(L/m 2 )
Turbidity
Pressure Differential
10
0
0
0 5 10 15 20 25
Volume Filtered per Unit Area
(L/m 2 )
10
8
6
4
2
8
6
4
2
Turbidity (NTU)
Turbidity (NTU)
www.millipore.com

are shown in Figure 11. When the
dominant capture mechanism is
adsorption, filtrate turbidity will be
seen to rise markedly, as the capacity
of the prefilter is reached, while the
pressure differential across it rises more
slowly; this profile is known as “breakthrough
limited.” When the dominant
capture mechanism is mechanical
sieving, the pressure differential
across the prefilter will be seen
to rise markedly, as its capacity is
reached, while filtrate turbidity rises
Feed
Pump
Figure 12. Test Configuration for
Millistak+ HC Filter
Prefilter
Millistak+ HC Filters Application Guide
P
P
Sterile
Filter
more slowly; this profile is known as
“pressure limited.” Testing is required to
determine which of these applies and,
therefore, whether a prefilter “pressure
limit” or a “breakthrough limit” should
be employed in overall filter train sizing.
It is strongly recommended that,
during testing of a Millistak+ HC filter,
it should be coupled with a sterilizing
grade filter in series (e.g., 0.22 µm
hydrophilic Durapore cartridge), as
shown in Figure 12. While simulating
the typical filtration process, this test
configuration can tell the user whether
the prefilter is performing correctly, or
if a change needs to be made either
to the prefilter type or to the ratio of
prefilter area to sterile filter area.
If creating a serial filtration test,
as illustrated above, is not feasible,
samples of filtrate from a Millistak+ HC
filter can be collected and tested
off-line, as prescribed by Millipore’s
Vmax sizing method.
Flux
Flux is an important process parameter,
especially for depth filtration. Flux is
flow rate normalized for filtration area
and is usually expressed in LMH
(liters per hour, per square meter).
Flux is significant also with regard
to the adsorptive contribution of
the Millistak+ HC filter media. The
faster a particle is flowing through a
17

Figure 13. Effect of Flux on Capacity of Millistak+ HC Filters
Millistak+ HC filter, the less likely it is
to interact electrostatically with the
charged surfaces, and consequently,
the more likely it is to pass directly
through the filter. If, however, the flow
rate (or velocity) is slowed, and the
particle is given additional time to
migrate to the surface of the media,
the chances of it being removed from
the solution are increased. This effect
18
Pressure Differential
(psid)
30 75DE Media
25
Flux (LMH) = 734 491 258 108
0.375 g/L Dairy
Whey Solution
20
Solution Turbidity:
44 NTU
15
10
Duplicate Tests,
Each Flux Level
5
0
0 50
100 150 200 250
Capacity (L/m 2 )
is depicted in Figure 13, which shows
that the capacity of the Millistak+ HC
prefilter is proportionately increased,
as the flux is reduced. Process designers
should search for the optimum flux at
which a given process should be run,
to minimize process time and maximize
filter efficiency. The initial recommended
flux range for Millistak+ HC filters is
150–400 LMH.
www.millipore.com

Scale-up
During the optimization process, tests
should be run at three separate scales,
and Millistak+ HC filters are available
in device sizes to match these steps.
Initial experiments can be run on
the two smaller Millistak+ HC filters.
Care should be taken to operate
in a constant-flux manner, recording
the pressure increase. If desired,
Millipore’s Pmax SM system can be
employed, to increase the efficiency
of interpreting the test data.
First, to determine the correct type
of Millistak+ HC filters to incorporate
into the process, screening with test
volumes of 100–1000 mL can be
performed with the Millistak+ HC Mini
Capsule (23 cm 2 ). To normalize for
variability in filter media and process
stream, multiple tests (at least three)
are recommended, with different
batches of test fluid.
The next step would be to process
5–25 L of solution through an Opticap
capsule filter with Millistak+ HC media
(650 cm 2 ). Again, testing multiple
devices with multiple process samples
will ensure that the sizing is as accurate
as possible.
When the process has been well
characterized, the process-scale
device can be tested, on volumes
greater than 50 L. Each processscale
16-inch 8-cell cartridge
provides 1.8 m 2 of filtration area.
Care should be taken to
simulate as closely as possible the
final process conditions. Since biological
process streams are very complex
mixtures, their properties could change
drastically if any process parameters
have been changed. If the temperature
of the fluid is not kept constant,
if the age of the fermentation is
changed or if it is stored overnight
under refrigeration, the solution may
change—precipitants may form,
proteins may denature, etc.—and
the sample will, consequently, no
longer be representative of the final
process fluid.
Millistak+ HC Filters Application Guide 19

Guidelines for
Process-Scale Operation
This section contains typical operating
parameters, general installation considerations
and standard procedures
for flushing, leak-testing, manual SIP
sterilization and hot water sanitization.
Your Applications Specialist or Process
Development Specialist can advise
you in detail concerning process
development. For additional sources
of information, see the Technical
Assistance section at the back of
this Application Guide.
Typical Operating Parameters
Flush Volume (L/m 2 ) 100
Flush Rate (L/min/m 2 ) 10
Typical Process Flux 80 – 300
(L/m 2 /hr)
Hold-Up Volume 7–7.5 fully wet
(L/m 2 ) 4–5 after blow-down
Maximum Pressure 30 @ 25 °C
Differential (psid) 15 @ 80 °C
3 @ 123 °C
Installation
Millistak+ HC filters, due to their
increased packing density, have
different physical dimensions from
the current single-layer Millistak+ CE,
DE and A filters. Millistak+ HC filters
are sold in 8-cell stacks, and are
assembled to a different stack height
from the single-layer filters. Since
two 8-cell stacks are taller than a
16-cell stack of the single-layer type,
adapters will be needed to ensure
a proper fit, in housings built to the
height requirements of single-layer
Millistak+ filters or to the dimensions
of a competitor’s single-layer devices.
Please refer to your Millistak+ housing
manual for installation details.
20 www.millipore.com

Flushing
Millistak+ HC filters require a 100 L/m 2
water flush (RO or WFI) prior to use.
The water used for flushing should be
sent directly to drain, since there may
be a small quantity of particles released
during the flush.
To ensure that the media is fully
wetted, a flux of 1 Lpm/ft 2 (645 LMH)
is recommended. To flush a full 8-cell
stack, use the following procedure:
1. Open the vent, to purge any air
from inside the housing.
2. Fill the housing at a low flow rate,
equivalent to about 0.25 Lpm/ft2 (162 LMH).
3. Once the housing is filled, close
the vent. Flow will now be sent
through the filter.
4. Over about 30 seconds, gradually
increase the flow, to achieve a flux
of 1 Lpm/ft2 (645 LMH).
5. Once the full flux (645 LMH) has
been reached, start the flush.
Millistak+ HC Filters Application Guide
Leak-Testing Installed
Cartridge Assemblies
To check that an assembled stack
has been put together correctly,
use the following procedure:
1. Wet out the filters as described
above.
2. Drain the housing. (This may be
done with a connection to drain
through the inlet to the housing.)
3. Once the housing is empty, apply
a forward pressure of 1 – 2 psid
of air pressure.
4. Wait 3 – 5 minutes for stabilization,
since the air pressure will force
some of the retained water within
the media through the stack.
5. Measure the flow of air into the
housing.
There will be some air flow due to the
low bubble point of the media, and
hence some air permeation through
the media will occur. A typical value
of air flow through a fully-wetted
8-cell stack (1.8 m2 ), with a pressure
differential of 2.0 psid, is 100 to
400 cc/min.
21

A larger air flow may indicate a
leak. In this case, refill the housing,
and repeat the flushing step.
If a second test shows a leak,
remove the housing bell, and
determine the source of the leak by
visual inspection. (There may be an
improperly seated gasket within the
stack or the retaining spring may
have lost compression.)
Bear in mind that this test is not
correlated to retention, but is a useful
Air Flow (cc/min)
7000
6000
5000
4000
3000
2000
1000
0
0
Pressure Differential (psid)
way to verify that the stacks are properly
assembled into the housing. The
sensitivity of the test was demonstrated
during product qualification by introducing
a pin hole into the filter media,
which resulted in elevated air diffusion
values of 2000 cc/min at 0.5 psid
and 6000 cc/min at 3 psid. The
graph shown in Figure 14 represents
detection capability at various test
pressure differentials.
0.5 1 1.5 2 2.5 3
3.5
Figure 14. Millistak+ HC Air Diffusion Leak Test: Pin Hole Detection
22 www.millipore.com

Steam-in-Place Sterilization
While working with steam, all
necessary precautions should be
taken to prevent injury. All hot lines
and housings should be labeled and,
if possible, insulated. Proper signage
should be displayed, warning of any
hot surfaces. All persons involved in
the steaming operation should be
outfitted with appropriate personal
protective equipment.
Before starting the steam-in-place
(SIP) procedure, check for the following:
• The filter housing is installed and
the correct product filter is in place.
• Filter retainer plates are in place
above and below the Millistak+ HC
cartridges.
Millistak+ HC Filters Application Guide
• The product filter is dry.
Note: If the assembled stack has
been tested for leaks, as described
above, a blow-down to remove
residual water from within the
filter media is required. This is
accomplished by applying a 5 psi
forward pressure to the feed side
of the housing to force the water
through for approximately 30
minutes, or until the water flow
is observed to have stopped.
• All valves are closed. Silicone
tubing is attached to the bleed
valves and directed to a
condensate drain.
The manual operations described in
this procedure should be performed
in the given sequence. For automatic
SIP procedures, refer to the Principles
of Steam-In-Place Technical Brief
(Lit. No. ET011EN00).
23

Manual Procedure
The configuration for steam sterilization
is shown in Figure 15.
1. Check that the steam supply and
compressed gas pressures are set
at the required values. For SIP at
123 °C, steam pressure should
be set at 1.18 bar (17.1 psi).
2. Open V1 and V2 and purge
the steam line until condensate
is completely absent.
3. Fully open V4 and V5 to allow
for subsequent air and condensate
evacuation.
4. Slowly open V3 to progressively
introduce steam and heat up
the filter.
Sterile
Air/N 2
WFI/
Product
Steam
V8
V9
V1
V2 V4
Figure 15. Configuration for Steam-in-Place Sterilization
V3
P1
5. Partially close bleed valves V2,
V4 and V5, so that a wisp of steam
and a continuous drip of water can
be seen exiting from each valve.
6. Open V6 and then crack open
bleed valve V7, to establish a
steady flow of steam and to allow
for condensate drainage and air
removal from the filter housing.
Note: It is very important to control
the difference between pressure
gauges P1 and P2, to keep the
∆P across the filter below
350 mbar (5 psid). Too high
a pressure differential across
the filter at elevated temperatures
may cause damage to the device.
To tank
24 www.millipore.com
V5
P2
V6 V10
V7
T1

7. To ensure that all air and condensate
are effectively removed, adjust
V2, V4, V5 and V7 as required,
so that a wisp of steam and a
continuous drip of water can be
seen exiting from each valve.
8. When the temperature downstream
of the product filter, as measured
by temperature gauge T1,
exceeds 123 °C, start the timer.
The sterilization time should be
at least 30 minutes (or longer,
as established during validation).
During the sterilization phase,
both pressure and temperature
should be recorded regularly.
9. At completion of the sterilization
cycle, close the steam supply
valve V1 and slowly open V8
to introduce compressed gas
into the system.
Millistak+ HC Filters Application Guide
Caution: Make sure that the system
remains under positive pressure (as
indicated by pressure gauges P1 and
P2) and that the pressure differential
across the filter does not exceed
350 mbar (5 psid).
10. Allow for steam purge from all
bleed valves and close valves
V2 and V4, to increase the
flow of gas through the system.
Maintain the minimum gas flow
required to cool the system,
until the temperature gauge T1
indicates approximately 30 °C.
11. In the following order, close valves
V7, V6 and V5. Keep V8 and V3
open, to maintain positive pressure
into the sterile filter system while it
is not in use.
12. After steam sterilization, repeat the
procedures for flushing and testing
the assembled stack, described
above.
25

Hot Water Sanitization
In some processes, a hot water sanitization
step may be required. The stack
should first be wetted, flushed and
tested for leaks, as outlined above.
A supply of 80 °C clean (RO or WFI)
water is required.
To heat the housing and filters, run
hot water through the housing and
monitor the temperature at the outlet
of the housing. Once the outlet temperature
has reached 80 °C, start
the timer: sanitization time should
be at least 30 minutes (or longer, as
established during validation). During
sanitization, the inlet and outlet temperatures
should be recorded regularly
and the pressure differential should
not exceed 15 psid. To ensure that
the entire housing remains at 80 °C,
the flow of hot water should be
sufficient to achieve a flux of at least
0.25 Lpm/ft 2 (162 LMH). Once the
time required for sanitization has been
reached, the filters and housing can
be dynamically cooled by switching
the flow over to cold, clean (RO or
WFI) water.
Chemical Compatibility
For information on chemical compatibility,
refer to the Millistak+ Filter Chemical
Compatibility Chart (Lit. No. PF088).
Technical Assistance
For complete product specifications,
refer to the Millistak+ HC Filters
Data Sheet (Lit. No. DS1000EN00).
For more information, contact the
Millipore office nearest you. In
the U.S., call 1-800-MILLIPORE
(1-800-645-5476). Outside the
U.S., see your Millipore catalogue
for the phone number of the office
nearest you or go to our web site
at www.millipore.com/offices for
up-to-date worldwide contact
information. You can also visit the
tech service page on our web site
at www.millipore.com/techservice.
Millipore, Millistak+ and Durapore are registered
trademarks of Millipore Corporation.
Opticap and Prostak are trademarks of Millipore
Corporation.
Pmax and Vmax are service marks of Millipore
Corporation.
Lit. No. AN1100EN00
Doc. No. P36464 Rev. C 9/03 03-299
© 2003 Millipore Corporation. All rights reserved.
Printed in U.S.A.
26 www.millipore.com

Â
Lit. No. AN1100EN00 P36464, Rev. C 9/03