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

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.

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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.

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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.

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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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.

Results and Discussion
The NWP recoveries for the T01 ILC module with Delta 30 kDa membrane (0.065 m2) are displayed in Figure 2.

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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.
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.

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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.
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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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.

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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