Technical Note

Bioburden Reduction and Particulate

Retention Using Milligard® PES Filters

Introduction Materials and Methods

Membrane-based filters are used extensively in the
production of biopharmaceutical products to protect Membranes and Devices
the process fluid from microbiological and particulate The studies described in this document compare
contaminants. Milligard® PES filters contain two layers performance of Milligard® PES filters with commercially
of asymmetric polyethersulfone (PES) membranes: available filters of similar pore sizes and composition.
a 1.2 µm upstream layer and a downstream layer Table 2 summarizes the characteristics of the filters
offered in several pore sizes (Table 1). These filters tested. For throughput studies, membranes of
can be used as prefilters upstream of sterilizing filters competitive filters were removed from pleated cartridge
to increase their capacity or can be used stand-alone filters and assembled into OptiScale® 25 capsules
to reduce bioburden and turbidity of process streams. (3.5 cm2); studies with Milligard® PES filters were
Downstream processing comprises multiple operations performed with OptiScale® 25 capsules. Scalability of
that require different levels of microbial control. For throughput performance was assessed using 10-inch
non-critical process steps, where sterile filtration cartridge and Opticap® XL10 capsule filters containing
may not be necessary, Milligard® PES filters offer an Milligard® PES membranes. Effective filtration areas of
alternative to sterilizing-grade filters for bioburden risk filters and smallest membrane pore size are listed in
reduction. Table 2.

Table 1. Milligard® PES Membrane Pores Sizes. Table 2. Summary of filters tested.
Milligard® PES Filter Typical Bioburden Reduction Filter Membrane Characteristics
1.2/0.2 μm nominal ≥6 logs of Brevundimonas diminuta Milligard® PES 1.2/0.2 µm 2-layer asymmetric PES with
1.2/0.45 μm ≥6 logs of Serratia marcescens nominal1 0.2 µm nominal layer

1.2/0.8 μm Not determined Competitive filter A 3-layer asymmetric PES with

0.2 µm nominal layer

The purpose of this application note is to describe the Milligard® PES 1.2/0.45 µm1 2-layer asymmetric PES with
0.45 µm layer
throughput and retention performance of Milligard® PES
filters in various model streams when used stand-alone Competitive filter B 2-layer asymmetric PES with
0.2 µm nominal layer
without a sterilizing filter downstream. In addition,
we demonstrate the scalability of these filters from Milligard® PES 1.2/0.8 µm2 2-layer asymmetric PES with
0.8 µm layer
OptiScale® 25 screening tools to pilot and production-
scale capsule and cartridge filters. Polysep™ II 2-layer borosilicate glass and
mixed esters of 1.2 µm layer.

1
Membrane areas: 10-inch cartridge filter, 0.60 m2;
Opticap® XL10 capsule filters, 0.60 m2
2
Membrane areas: 10-inch cartridge filter, 0.53 m2;
Opticap® XL10 capsule filters, 0.57 m2

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U.S. and Canada.
Challenge Streams setup shown in Figure 2. Throughput performance
of competitive filters in the different streams was
Four different model streams representing a range of normalized relative to the performance of the Milligard®
particle size distributions were used for these studies, PES filter in that stream.
Figure 1. The CHO stream was prepared by clarifying
a CHO harvest containing 1g/L monoclonal antibody
through a Millistak+® D0HC depth filter, then diluting Bioburden Reduction
1:100 in phosphate buffered saline and filtering Milligard® PES 1.2/0.2 µm nominal filters were
through a 5 µm Durapore® membrane. The composition challenged with soy peptone and whey solutions
of the other streams has previously been described1. containing Brevundimonas diminuta at concentrations
Streams were selected to represent the range of of at least 2.3 x 107 colony forming units (cfu)/mL.
particle sizes that might be present in different process Milligard® PES 1.2/0.45 µm filters were similarly
feeds. To minimize the volume of challenge solutions challenged with these same solutions spiked with
required, streams were formulated to achieve 90% flux Serratia marcescens at concentrations of at least 3.9 x
decay at 500-1000 L/m2. 107 cfu/mL. In all tests, the spiked solutions provided a
challenge greater than 107 cfu/cm2 of filtration area. For
each pore size, two membrane lots were each tested in
0.012
the two streams with at least three replicate OptiScale®
Log Normalized Volume Frequency

Soy Peptone 25 capsules. Testing was performed at 2 psi and filtrate

Whey
0.01 grab samples were collected during the runs and
Clarified CHO
Soy T
assayed for titer. When tests reached 90% flow decay,
0.008 tests were stopped, and samples were collected from
filtrate pools and assayed for titer. Log reduction values
(LRV) for the intermediate samples were determined by
0.006
comparing the log titer in the challenge solutions with
the log titer of the intermediate filtrate samples. For
0.004 the filtrate pools, LRVs were determined by comparing
the log microbial load (concentration x volume) in the
0.002
challenge solution with that of the filtrate pool.

0
Scalability
0.01 0.1 1 10 100 1000
Scalability was assessed by comparing throughput
Particle Size (µm)
performance of Milligard® PES membranes in the
Figure 1. Particle size distributions of the challenge streams. Particle
10-inch cartridge or Opticap® XL10 capsule formats
sizing was performed with Malvern MasterSizer and PMS Liquilaz. to that of the OptiScale® 25 capsules with one non-
plugging (water) and two plugging streams at constant
pressure of 5 psi. This pressure was selected to
Test Methods approximate the typical pressure a prefilter might
experience in a plugging application. Scalability tests
Water Permeability and Throughput compared performance using filters containing the
same membrane lot. All tests were stopped when
Water permeability of Milligard® PES filters was permeability reached 90% flux decay relative to the
measured at 10 psi and 21-25 °C. Filters were then initial water permeability of each filter (all OptiScale®
challenged with the different model streams at 10 psi 25 capsules exceeded 90% flux decay). Scaling factors
until permeability was reduced by 90% compared to represent the throughput of a 10-inch cartridge or
the water permeability (90% flow decay). For all tests, capsule filter relative to the average throughput of five
temperature, pressure and filtrate volume data were OptiScale® 25 capsules.
collected as a function of time, using the experimental

PR P P

Plant air
V1

T
FT

LC

Figure 2. Test setup for throughput tests. Symbols: FT, feed tank; O, OptiScale® 25 filter; LC, load cell;
P, pressure measurement; PR, pressure regulator; T, temperature measurement; V, valve. Temperature,
pressure and filtrate volume were recorded by a data acquisition system.

2
Particle Removal Figure 4 shows the results of similar throughput
tests with Milligard® PES 1.2/0.45 µm filters. In the
Particle removal capability of Milligard® PES filters was soy peptone stream, Milligard® PES 1.2/0.45 µm
determined by comparing the particle concentration filters outperformed the competitive benchmark. In
and size distribution of soy peptone before and after streams containing larger particles, whey and soy T,
processing through OptiScale® 25 and Opticap® XL10 Milligard® PES 1.2/0.45 µm filters showed equivalent
capsules containing Milligard® PES membranes at a performance. However, for the CHO stream where most
constant pressure of 10 psi. Samples of challenge and particles are closest to the filter pore rating, Milligard®
filtrate solutions were analyzed using a Liquilaz Model PES 1.2/0.45 µm filters had lower throughput than
SO2 particle analyzer. competitive filter B.

Results and Discussion

1.8
Throughput 1.6

Normalized to throughput of
Throughput performance of Milligard® PES filters 1.4
was benchmarked against comparable commercially

1.2/0.45 µm filters
1.2

Milligard® PES
available filters using four model streams containing
particle sizes representative of bioprocessing fluid 1.0
streams. Performance of competitive filters was 0.8
normalized to Milligard® PES performance for each
stream. Differences in throughput within 20% are not 0.6

considered to be statistically significant. 0.4

Figure 3 shows that in most model streams, throughput 0.2

of Milligard® PES 1.2/0.2 µm nominal filters is at least 0.0
20% higher than that of competitive filter A. The Soy Peptone CHO Whey Soy T
largest throughput advantage was shown with the soy Milligard® PES 1.2/0.45 µm nominal filter Competitive Filter B
peptone stream, which has the highest concentration of
small particles in the streams tested and is most likely Figure 4. Throughput performance of Milligard® PES 1.2/0.45 µm
filters compared to competitive filter B. Each bar represents the
to challenge the internal structure of the Milligard® average results of two OptiScale® 25 filters.
PES 1.2/0.2 µm nominal membrane. Milligard® PES
filters exhibited lower throughput compared to the
No comparable commercially available filter was
competitive filter in the soy T stream, the model stream
identified for benchmarking Milligard® PES 1.2/0.8 µm
containing the largest-sized particles. The competitive
filter, therefore throughput performance was compared
filter has a different membrane symmetry and pore
to Polysep™ II, our high capacity prefilter (Figure 5).
structure to Milligard® PES filters and is more suited
Similar performance between the filters was shown in
for processing feed streams containing relatively
streams containing the highest concentrations of large
large particles. For these types of solutions, a coarser
and small particles, but in mid-particle size streams
prefilter might be more appropriate.
such as CHO and whey, Polysep™ II filters showed
higher throughput than Milligard® PES 1.2/0.8 µm
1.6
filters. This is likely due to higher particle adsorption
on the borosilicate glass and mixed esters of cellulose
Normalized to throughput of

membrane in Polysep™ II filters. However, in processes

1.2/0.2 µm nominal filters

1.4

1.2 requiring filter compatibility with gamma irradiation or

steaming in place (SIP) sterilization methods, Milligard®
Milligard® PES

1.0
PES filters are an attractive option.
0.8

0.6

0.4

0.2

0.0
Soy Peptone CHO Whey Soy T

Milligard® PES 1.2/0.2 µm nominal filter Competitive Filter A

Figure 3. Throughput performance of Milligard® PES 1.2/0.2 µm

nominal filters compared to competitive filter A. Each bar represents
the average results of two OptiScale® 25 filters.

3
 4.5 10

4.0

log reduction value (LRV)

Normalized to throughput of

8
3.5
1.2/0.8 µm filters

3.0
Milligard® PES

6
2.5

2.0
4
1.5

1.0 2

0.5

0.0 0
Soy Peptone CHO Whey Soy T 0 10 20 30 40 50 60 70 80 90 100

Milligard PES 1.2/0.8µm filter

®
Polysep™ II % Flow Decay

Figure 5. Throughput performance of Milligard PES 1.2/0.8 µm filters®

Figure 6. Retention of triplicate Milligard® PES 1.2/0.2 µm nominal
as compared to Polysep™ II filters. Each bar represents the average filters challenged with soy peptone containing B. diminuta. Arrows
results of two OptiScale® 25 filters. indicate no bacteria was detected in the filtrate.

In summary, these results highlight the throughput The test results of Milligard® PES 1.2/0.45 µm filters
capacity performance of Milligard® PES filters as challenged with the soy peptone stream containing
compared to commercially available filters in four S. marcescens are shown in Figure 7. Bacteria were
model streams of different particle size compositions. not detected in any filtrate grab samples, resulting in
Each application and process fluid will have a different calculated bacterial retention of at least 8 LRV, even at
particle composition, which will affect the capacity of 90% flow decay. Similar results were obtained when
any given filter. However, throughput performance of these filters were challenged with S. marcescens in the
Milligard® PES filters was favorable as compared to whey model stream (data not shown).
alternative commercially available filters in multiple
challenge streams. In practice, we recommend
evaluating process streams using Milligard® PES filters 10
of different pore sizes to identify the preferred filter for
log reduction value (LRV)

maximizing throughput.
8

Bioburden Retention
6
Milligard® PES 1.2/0.2 µm nominal and 1.2/0.45 µm
filters are designed to be used as stand-alone filters
for bioburden control in non-critical process steps. 4

Although these filters are not designed to deliver the

same level of microbial protection as filters containing 2
sterilizing-grade membrane, reliable microbial
retention is a performance expectation, even when the
membrane is highly fouled. Assessments of bioburden 0
0 10 20 30 40 50 60 70 80 90 100
reduction performance of Milligard® PES 1.2/0.2 µm
% Flow Decay
nominal and 1.2/0.45 µm filters was limited to the soy
peptone and whey model streams which contained the Figure 7. Retention of triplicate Milligard® PES 1.2/0.45 µm filters
highest concentrations of smaller particles. Although challenged with soy peptone containing S. marcescens at 107 cfu/cm2.
two membrane lots of each Milligard® PES membrane Arrows indicate no bacteria were detected in the filtrate.
pore size were tested in each stream, all test results
were similar, therefore a limited subset of results are Overall, robust bioburden retention was demonstrated
shown. with Milligard® PES filters in two model streams
containing high concentrations of small to medium-
Figure 6 shows the results of retention tests with
sized particles. Importantly, retention performance of
Milligard® PES 1.2/0.2 µm nominal filters challenged
Milligard® PES filters is maintained under conditions
with soy peptone stream containing B. diminuta.
that are typically challenging for membrane filters:
Bacteria were not detected in any filtrate grab samples,
where the membrane pores are highly fouled and
resulting in LRVs of at least 8, even out to 90% flow
permeability is 90% lower than the clean water
decay. A low level of bacteria was detected in one of
permeability2. Table 3 summarizes LRVs in filtrate pools
the filtrate pools, but in all cases, LRVs exceeded 9.4,
after processing model steams across Milligard® PES
Table 3. Similar results were obtained when Milligard®
1.2/0.2 µm nominal and Milligard® PES 1.2/0.45 µm
PES 1.2/0.2 µm nominal filters were challenged with B.
filters.
diminuta in the whey model stream (data not shown).

4
Table 3. Milligard® PES Filter Final pool LRVs.

Throughput Scaling Factor

1.4
Values represent the lowest pool LRV.
1.2
Challenge Fluid
1.0
Microorganism Membrane Soy peptone Whey
0.8
B. diminuta Milligard® PES 9.3 ≥ 9.4
0.6
1.2/0.2 µm
nominal 0.4

S. marcescens Milligard® PES ≥ 9.5 ≥ 9.4 0.2

1.2/0.45 µm
0.0
0 10 20 30 40 50 60
Time (minutes)
Milligard® PES filters are not a substitute for sterilizing Figure 8. Throughput scaling factor as a function of filtration time for
filters in final filtration, but offer an attractive option for Milligard® PES 1.2/0.2 µm nominal cartridge filters in soy peptone.
bioburden risk reduction in non-critical process steps. Error bars show the standard deviation of the mean.

Scalability Scalability of throughput in Milligard® PES 10-inch

cartridge and capsule filters relative to OptiScale® 25
Accurate estimation of filtration area requirements capsules was determined in water and two plugging
for large-scale processes relies on scaling factors that model streams, Table 4. All throughput scaling factors
connect performance of small-scale sizing tools to were established at the time where the cartridge or
larger filter formats. Scaling factors represent the ratio capsule filters were 90% plugged. In a non-plugging
of performance of larger filters to small-scale sizing stream such as water, scaling factors were in the
tools, normalized to membrane area, and ideally should range of 0.4-0.7. Similar low scaling factors have been
be close to 1.0. These factors are influenced by filter reported for other filters and are a consequence of high
design features such as pleat structure as well as the membrane permeability and dense filter pleat structure
plugging characteristics of the process fluid. In practice, which results in added flow resistance3. In these
for filters containing sterilizing-grade membrane, situations, safety factors typically included in filtration
scaling factors are commonly within about 20% of area sizing models could be increased to accommodate
unity, whereas the range for filters used in non-critical non-linear scaling from small to large-scale devices.
process steps might be broadened. Figure 8 shows
how the scaling factor can change with increasing filter In plugging streams, scalability between the
fouling: as the filter fouls, the scaling factor tends to small‑scale devices and larger filters was more linear.
converge towards unity. This is because as the membrane fouls it becomes the
dominant resistance relative to resistances in other
parts of the pleat structure.

Table 4. Milligard® PES filters scaling factors

Non-plugging
Stream Plugging Stream
Product Water Soy peptone Whey
Milligard® PES 1.2/0.2 µm nominal cartridge 0.6 0.9 1.0
Milligard PES 1.2/0.2 µm nominal capsule
®
0.7 0.9 0.9
Milligard® PES 1.2/0.45 µm cartridge 0.5 0.9 1.1
Milligard® PES 1.2/0.45 µm capsule 0.6 0.8 0.9
Milligard PES 1.2/0.8 µm cartridge
®
0.6 1.0 1.1
Milligard® PES 1.2/0.8 µm capsule 0.4 0.7 1.0

5
Particle Reduction A
Milligard® PES 1.2/0.2 µm nominal membrane
Particle retention by Milligard® PES filters was
100%
quantified by measuring particle concentrations and
size distributions in the soy peptone model stream 80%
before and after filtration through both OptiScale® 25

% Rejection
capsules and Opticap® XL10 capsules, Figure 9. Before 60%
testing, the Opticap® XL10 capsules were pre-sterilized
40%
by gamma irradiation.
The soy peptone challenge solution contains particles 20%

that are mostly smaller than 1 µm, with the highest

0%
concentration of particles in the 0.2-0.25 µm range. 0.2-0.3 0.3-0.4 0.4-0.5
Milligard® PES 1.2/0.2 µm nominal filters retained over Particle size (µm)
90% of particles in the 0.2-0.25 µm range, more than OptiScale® 25 filters Opticap® XL10 filters
the other membrane pore size offerings. All the filters
removed greater than 80% of all particles larger than
0.2 µm. B
Milligard® PES 1.2/0.45 µm membrane
This analysis highlights differences in performance 100%
related to membrane pore size. However, filter selection
is generally guided by empirical results: throughput 80%

% Rejection
performance in combination with process needs for
bioburden control and particulate removal. 60%

40%

20%

0%
0.2-0.3 0.3-0.4 0.4-0.5
Particle size (µm)
OptiScale® 25 filters Opticap® XL10 filters

C
Milligard® PES 1.2/0.8 µm membrane
100%

80%
% Rejection

60%

40%

20%

0%
0.2-0.3 0.3-0.4 0.4-0.5
Particle size (µm)
OptiScale® 25 filters Opticap® XL10 filters

Figure 9. Particle removal from soy peptone stream following

processing across OptiScale® 25 and Opticap® XL10 capsule filters
containing Milligard® PES 1.2/0.2 µm nominal membrane (A), Milligard®
PES 1.2/0.45 µm membrane (B), Milligard® PES 1.2/0.8 µm membrane
(C).

6
Conclusions References
1. Performance of High-Area Millipore Express® Cartridge Filters
Milligard® PES filters were demonstrated to provide
Scaling Considerations to Maximize the High-Area Advantage.
high throughput capacity for several different streams Applications Note Lit No. TB12450000
representing wide ranges of particle size distributions. 2. Bolton, G.; Cabatingan, M.; Rubino, M.; Lute, S.; Brorson, K.; and
In model streams containing predominantly small to Bailey, M. “Normal-flow virus filtration: detection and assessment
mid-sized particles (0.1-10 µm), Milligard® PES 1.2/0.2 of the endpoint in bioprocessing.” Biotechnology and Applied
µm nominal and 1.2/0.45 µm filters performed well Biochemistry. (2005) 42, 133–142.

against benchmark competitive filters. However, for 3. Giglia, Sal, and David Yavorsky. “Scaling from discs to pleated
devices.” PDA Journal of Pharmaceutical Science and Technology
streams containing predominantly larger particles 61.4 (2007) 314–323.
(> 10 µm), Milligard® PES 1.2/0.2 µm nominal and
1.2/0.45 µm filters may not be the optimal choice.
For this type of stream, coarser prefilters should be
considered. Milligard® PES 1.2/0.8 µm filters show
similar capacity to Polysep™ II filters in most, but not
all, streams. However, a key advantage of Milligard®
PES filters is their compatibility with gamma irradiation
and thermal sanitization methods.
For non-critical process steps requiring bioburden
control, Milligard® PES 1.2/0.2 µm nominal and
1.2/0.45 µm filters provide reliable bioburden removal,
even under conditions where the filters are highly
fouled. Greater than 6 log removal of B. diminuta
and S. marcescens were demonstrated for Milligard®
PES 1.2/0.2 µm nominal and 1.2/0.45 µm filters
respectively.
Scalability testing of Milligard® PES filters indicated
that as the membrane fouls, the scaling factor between
OptiScale® 25 devices and 10-inch cartridges and
capsules approaches unity, allowing for simple and
reliable filter sizing. For low plugging streams, scaling
factors are available that account for the effect of the
high permeability and dense pleat structure of these
filters.
In summary, these filters combine reliable particle
retention, effective bioburden reduction and
compatibility with thermal and gamma sterilization
methods. For non-critical process steps, they are an
attractive alternative to sterilizing filters for reducing
bioburden and improving processing efficiency.

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