Application Note
USD3005
Cleanability and Re-usability of Cadence™
Inline Concentrator Modules
Catherine Casey1, M.Sc., Paul Haberman1, Mufadal Ayubali1, M.Sc.
and Engin Ayturk1, Ph.D.
1 BioPharm Applications R&D
1.
Introduction
Pall’s single-pass tangential flow filtration (SPTFF) is a new technology that utilizes a staged flow path
design to achieve significantly higher concentration factors than a conventional tangential flow filtration
(TFF) system in one pump pass[1-4]. The single pump pass minimizes shear exposure and eliminates
foaming/mixing concerns and reduces the minimum working volume associated with conventional TFF
systems operating with a recirculation loop. Additionally, with the utilization of a smaller pump and tubing
sizes, the reduced system hold-up volumes maximize product recovery with minimal dilution.
The Cadence Inline Concentrator (ILC) module is a holder-less SPTFF device with a built-in retentate
restrictor, designed to achieve concentration factors of 2-4X (or higher)[5]. The ILC module requires only
a feed pressure source (i.e., pump or pressure vessel) and feed pressure measurement device, further
reducing system requirements (Figure 1). Its reduced system footprint eases facility fit concerns where
manufacturing floor space is at a premium.
Figure 1
Cadence Inline Concentrator Set-up
Feed tank
PERMEATE
PERME
ATE
F
P
FEED
RETENTATE
RETENTATE
(to break tank
or inline to next
unit operation)
Table 1 shows the eight Cadence ILC modules that are currently available. They are available in four
format sizes (T01, T02, T12 and T06), which correspond to membrane areas ranging from 0.065 to
3.5 m2, and with Delta membrane (regenerated cellulose) in both 10 and 30 kDa MWCOs.
2
Table 1
Available Cadence Inline Concentrator Modules
Membrane & MWCO
Format Size
Membrane Area (m2)
Part Number
Delta 10 kDa
T01
T02
T12
T06
T01
T02
T12
T06
0.065
0.13
0.7
3.5
0.065
0.13
0.7
3.5
ILD010T010407
ILD010T020407
ILD010T120407
ILD010T060407
ILD030T010407
ILD030T020407
ILD030T120407
ILD030T060407
Delta 30 kDa
The holder-less ILC module design greatly simplifies installation and enhances ease-of-use from a
plug-and-play point of view. However, the ability of the holder-less module to maintain seal integrity
and repeatable performance needs to be further evaluated for applications that require limited re-use.
In this context, this study aimed at better understanding the relationship between storage time and
process performance during multiple runs. In addition, the cleanability of the built-in fixed retentate
restrictor during limited re-use applications was verified in this study via the utilization of current SPTFF
cleaning protocols.
Feed and retentate fluxes and volumetric concentration factors (VCF) of the ILC modules are the
primary determinants for demonstrating repeatability, as they define processing times, vessel sizes, final
concentrations, and required membrane areas. Thus, it is important that these variables be repeatable
so that the end-user will be able to confidently re-use the ILC modules with minimal expected change
in performance. Large performance differences between runs could negatively impact the final process
by extending the processing time, overfilling a hold vessel, or sending an under-concentrated product
stream to the next unit operation. Thus, the main focus of this testing was to demonstrate membrane
and internal seal integrity, process repeatability and cleanability of the ILC modules for limited re-use
applications.
2.
Materials and Methods
The smallest (T01, 0.065 m2) and largest (T06, 3.5 m2) Cadence ILC modules were selected for the
repeatability testing. These ILC modules were chosen to be representative of the entire Cadence ILC
product line. The modules were first pre-conditioned by performing storage solution flush, sanitization,
post-sanitization flush, hydraulic characterization, and buffer conditioning steps. They were then challenged with a protein solution at feed pressures ranging from 20 to 60 psig. It should be noted that the
modules were run in total recirculation mode to conserve solution; however, a typical end-user application
would process the target molecule in a single-pass mode. Multiple data points capturing pressures, flow
rates, and VCFs were recorded at each feed pressure to ensure process stability, which were then used
to generate VCF and flux maps for each run. It is important that the module performance be repeatable
from run-to-run at all possible operating feed pressures, as each feed pressure represents a potential
run condition of an application specific process.
A T01 ILC module with Delta 30 kDa T01 membrane (0.065 m2) was challenged with solutions of 2, 5,
and 10 g/L polyclonal bovine Immunoglobulin G (IgG) in phosphate buffered saline (PBS) on three
separate occasions. These feed concentrations were chosen to represent the majority of case studies
where the Cadence ILC module would typically be implemented between primary clarification and
final concentration steps. Each individual test spanned approximately 3 hours, with all three tests being
executed over a period of 36 days. To further challenge this ILC module, it was run with a 5 g/L bovine
serum albumin (BSA) solution in between the second and third IgG runs. The full test matrix is outlined
in Table 2.
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3
Table 2
Test Matrix for the Cadence Inline Concentrator Module with Delta 30 kDa Membrane in T01 Format
Day Number
Test Solution
Processing Time
1
25
29
36
2, 5, 10 g/L IgG in PBS
2, 5, 10 g/L IgG in PBS
5 g/L BSA in PBS
2, 5, 10 g/L IgG in PBS
~3 hours
Meanwhile, a separate T06 ILC module with Delta 30 kDa membrane (3.5 m2) was also initially challenged with solutions of 2, 5, and 10 g/L IgG in PBS over a test period of approximately 3 hours. Repeat
testing was conducted over a year later with the intermediate concentration of 5 g/L IgG for 1 hour.
After processing with protein, the ILC modules were cleaned with 0.1 N NaOH for 1 hour, flushed
with water, re-characterized for normalized water permeability (NWP), and stored in 0.1 N NaOH under
ambient conditions until its next use. ILC modules were pre-conditioned as described earlier before each
subsequent protein challenge.
3.
Results and Discussion
The NWP recoveries for the T01 ILC module with Delta 30 kDa membrane (0.065 m2) are displayed in
Figure 2. This ILC module was tested a total of four times (three times with IgG and once with BSA) over
a period of 36 days. The module recovered 98-99% of its initial NWP after each of the first three tests
and 93.5% of its initial NWP after completion of the fourth and final test. Typically, TFF cassettes and ILC
modules can be re-used until the pre-use NWP decreases to 70-80% of its original value. Therefore, this
ILC module could be even further processed with minimal expected change in performance. Since ILC
module cleanability will be application dependent, it is recommended that specific end-user studies
be executed to determine the limited re-use range of the ILC modules for the intended application.
Figure 2
NWP Recoveries for a T01 ILC Module with Delta 30 kDa Membrane Processed with Solutions
of 2-10 g/L Polyclonal Bovine IgG in PBS and 5 g/L BSA in PBS
120
NWP Recovery (%)
100
98.2%
98.6%
98.3%
Run #1 (IgG)
Run #2 (IgG)
Run #3 (BSA)
92.6%
80
60
40
20
0
4
Run #4 (IgG)
More importantly, process performance was not impacted by the multiple uses. Figure 3 shows
feed fluxes and VCFs at feed pressures between 20 and 60 psig when tested with the intermediate
concentration of 5 g/L IgG in PBS. The average coefficient of variance (CV) for the feed fluxes at each of
the feed pressures was 4.1%. Meanwhile, the average CV for the VCFs was as low as 3.2%, suggesting
minimal variation between all three IgG runs.
As previously noted, this module was also challenged with a 5 g/L BSA solution in between the second
and third IgG tests. Processing with BSA had minimal impact on subsequent IgG performance. Thus, the
ILC modules could possibly be used to process different molecules for rapid performance screening at
process development scale if cross-contamination and product quality was not a concern.
Figure 3
Feed Flux and VCF of T01 ILC Module with Delta 30 kDa Membrane Tested Multiple Times with 5 g/L
Polyclonal Bovine IgG in PBS over a 36-day Period
1,000
Feed Flux (LMH) or VCF (X)
Feed Flux (Run #1)
VCF (Run #1)
Feed Flux (Run #2)
VCF (Run #2)
Feed Flux (Run #4)
VCF (Run #4)
100
10
1
20
30
40
50
60
Feed Pressure (psig)
Furthermore, a T06 ILC module with Delta 30 kDa membrane (3.5 m2) was also re-tested after a
long-term storage period. It was suspected that the largest available ILC module would be worst case
for repeatability because it has the most potential for load relaxation due to the holder-less design.
Module load relaxation could possibly impact re-use performance, including fluxes, VCFs and most
importantly, product retention. Therefore, this T06 ILC module was initially tested and then re-tested
over a year later to represent worst case test conditions. Figure 4 shows that feed fluxes and VCFs were
similar between the two runs. The feed fluxes and the estimated VCFs of the re-test were 3% and 13%
higher than the original values respectively. Thus, indicating the inherent variability in the test method, but
not necessarily a real change in performance. Additionally, the membrane was fully retentive at all feed
pressures, demonstrating that the membrane and the internal seals remained integral after long term
storage. Even though it is expected that the internal sealing force of ILC modules will relax slightly over
time, the module integrity, process performance and product retention remained unaffected within our
test resolution.
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5
Feed Flux (LMH), VCF (X), or Retention (%)
Figure 4
Feed Flux, VCF, and Retention of T06 ILC Module with 30 kDa Membrane Tested with 5 g/L Polyclonal
Bovine IgG in PBS after 1 Year of Storage
4.
1,000
Feed Flux (Initial)
VCF (Initial)
Retention (1 Year Later)
Feed Flux (1 Year Later)
VCF (1 Year Later)
100
10
1
20
30
40
Feed Pressure (psig)
50
60
Conclusions
Based on the findings of this study, the Cadence Inline Concentrator modules could be re-used several
times with minimal change in performance. Module integrity, process performance, and product retention
were unaffected. It is recommended that targeted end-user studies be conducted to identify the limited
re-use range and validate specific re-use requirements per application.
5.
References
[1] Leon Mir and Gaston de los Reyes. Method and apparatus for the filtration of biological solutions.
US Patents 7,384,549 B2 (June 10, 2008), 7,682,511 B2 (March 23, 2010), 7,967,987 B2 (June 28,
2011) and 8,157,999 B2 (April 17, 2012).
[2] Casey, C., Gallos, T., Alekseev, Y., Ayturk, E., and Pearl, S. Protein concentration with Single-Pass
Tangential Flow Filtration, Journal Membrane Science, 384(1-2) (2011) 82-88.
[3] Application Note (USD2789): Cadence Systems Employ New Single-Pass TFF Technology to
Simplify Processes and Lower Costs. Pall Life Sciences.
[4] Dizon-Maspat, J., Bourret, J., D’Agostini, A. and Li, F. Single Pass Tangential Flow Filtration to
Debottleneck Downstream Processing for Therapeutic Antibody Production. Biotechnology and
Bioengineering, 109(4) (2012) 962-970.
[5] Casey, C. and Ayturk, E. Application Note (USTR 2913): Volume Reduction and Process Optimization
with Cadence Inline Concentrator.
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