Filtration of cell culture media using high performance polyethersulfone (PES) filters

Introduction

Historically, the filters used most often for the sterile filtration of cell culture media (basal and feeds) are hydrophilic polyvinylidene difluoride (PVDF) membranes, rated at 0.2 µm. While PVDF filters are perceived to have lower extractables and leachables profiles, the modern polyethersulfone (PES) membranes offer both a significant increase in capacity and flow, as well as relatively low extractables.

This application note examines the capacity of Cytiva’s range of PES filters – Supor™ EKV, Supor EX ECV, and Supor Prime sterilizing-grade filters. Supor EKV and Supor EX ECV are widely used membranes in a variety of biotechnology applications, while Supor Prime is the latest product in the Supor product family and is designed to deliver even higher performance and efficiency, especially in complex feeds.

Two studies were performed:

• A filterability study compared the throughput performance of Cytiva PES filters with a Fluorodyne™ II DFL (PVDF) sterilizing-grade filter.
• A separate fed-batch cell culture study evaluated the impact of PES-filtered media and feeds on subsequent cell culture performance relative to liquid media and feed controls that were filtered using a sterilizing-grade PVDF filter during manufacturing.

Objective

There were two primary objectives. First, to determine the most effective Cytiva PES filter for the sterile filtration of Cytiva cell culture media, in terms of capacity. Second, it is recognized that changing filters used for cell culture media may introduce process uncertainty, including concerns regarding potential changes to media composition and subsequent cell culture performance. To address this uncertainty, a fed-batch cell culture study was performed to assess potential effects on cell growth, viability, and productivity. The study used HyClone ActiPro™ cell culture media, a chemically defined production medium optimized for CHO cell cultures, as well as HyClone Cell Boost™ 7a and Cell Boost 7b, feed supplements to maximize growth and protein yield. Powdered media and feeds were hydrated and filtered using the PES filters, Supor EKV, Supor EX ECV and Supor Prime, and were then compared with liquid media and feeds that were filtered using a sterilizing-grade PVDF filter during manufacture.

Materials and methods

Two separate studies were performed: a filterability study to evaluate filtration performance and a fed-batch cell culture study to assess the impact of the filtered media and feeds on subsequent cell culture performance.

Filterability testing- scaling study

Filterability testing was performed at bench scale using small-format filter discs and small volumes of freshly hydrated powdered media. The approach was intended to predict full-scale filter performance using the V_MAX test, a well-established, constant-pressure method for estimating filter capacity. The testing was performed using a PendoTECH Normal Flow Filtration (NFF) system to enable automated measurement of filter performance under controlled pressure conditions.

The following 0.2 µm sterilizing grade filters were evaluated:

Table 1. Filters selected for filterability of ActiPro, Cell Boost 7a and Cell Boost 7b.

Cell growth studies

Overview

For this study, Supor EKV, Supor EX ECV, and Supor Prime were evaluated for their ability to filter ActiPro, Cell Boost 7a (CB7a), and Cell Boost 7b (CB7b) while maintaining performance in subsequent cell culture applications. A control was also used, which was liquid media consisting of preformulated liquid media and feeds representing standard off-the-shelf solutions, sourced from and filtered at the Cytiva/HyClone facility using a PVDF filter. A high-level overview of the study design is presented in Figure 1.

Fig 1. Overview of the study design.

Media filtration

Filters were flushed with WFI at ca. 110 mL/min for 2 min to wet the membranes prior to autoclaving, as recommended for PES filters. After gravity draining, all filters were assembled with tubing on outlets and autoclaved (121°C for 30 min). Extra tubing with ReadyMate™ connectors was also autoclaved to connect to pillow bags. Prior to each filtration test, tubing between filter and 2D bag or bottle was welded aseptically.

Filtration flux was set at 500 L/m²/hr corresponding to 167 mL/min flow rate for Supor EKV (200 cm²), 183 mL/min for Supor EX ECV (220 cm²) and 200 mL/min for Supor Prime (240 cm²). The peristaltic pump was calibrated prior to filtration.

Different volumes of each product were filtered through each test filter to match the volume per filter area. The planned throughput was decided based on sterile filtration parameters currently used in the manufacturing of Cytiva liquid media. Each filtrate was collected in a 2D bag (Cell Boost 7a, Cell Boost 7b) or a sterile glass bottle (ActiPro) of adequate size for the expected volume filtered. During filtration of each product, differential filter pressure and filtrate weight were manually recorded at regular intervals.

Cell culture study

Cell thaw and seed train

For evaluating the filtered media, a cell-culture study using an internal mAb-expressing, CHO-K1 clone and standard fed-batch process was performed.

One vial of CHO cells was thawed from an internal cell bank. The cells were then sub-cultured for 3 passages in ActiPro media with 37.5 µM L-methionine sulfoximine (MSX). Passaging was adjusted to stay within a viable cell density (VCD) range of 0.3 to 5 MVC/mL. Cells were cultured in an incubator using settings listed in Table 2.

Table 2. Incubator settings for cells cultured in seed train and fed-batch.

Fed-batch culture

For the fed-batch study, 5×3 shake flasks were seeded and fed with filtered media according to the overview shown in Figure 2 and cultured using incubator settings listed in Table 2 according to the process described in Table 3.

All cultures were sampled daily to monitor viable cell density, viability, metabolite concentrations, osmolality, and IgG titer.

Fig 2. Overview of the shake-flask study performed on the different filtrates.

Table 3. Process parameters for the shake-flask study in fed-batch.

Results

Filter throughput results

Table 4. Filter throughput results for ActiPro, Cell Boost 7a and Cell Boost 7b.

Fig 3. Filter throughput.

Cell growth study results

After hydration, the media and feeds were sampled and analyzed for osmolality and pH before filtration. The measured values are presented in Table 5. For all preparations, all parameters were within the specification.

Table 5. Media specifications.

The cell growth study was conducted according to the method described in the Materials and methods section above.

Fig 4. Comparison of VCD (a), viability (b), IgG titer (c), glucose (d), lactate (e), and glutamate (f) profiles for the biological replicate groups EKV, ECV, Prime, and PVDF filtered during manufacturing liquid media from day 0 to 12. Each data point represents the group mean from replicated, with error bars indicating the standard deviation.

The data is further summarized by the comparison of the main parameters peak VCD and final titer listed in Table 6.

Table 6. Summary of peak VCD and final IgG titer for cultures grown in media filtered using PES and PVDF filters.

* One EKV replicate was excluded from the analysis as an unintended double-feed event took place. EKV results therefore represent n=2, while the remaining groups represent n=3.

Discussion and conclusions

The data highlights that all tested PES filters provided higher throughput than the PVDF filter under the tested conditions and are suitable for cell culture media filtration showing no negative impact on cell growth, viability and productivity.

Throughput performance varied by media type. For ActiPro, Supor EX ECV showed the highest throughput, followed by Supor Prime. For Cell Boost 7a and Cell Boost 7b, Supor Prime showed the highest throughput. These results demonstrate that filter selection can be optimized for individual media requirements. Where a common filter is preferred across the three evaluated media, Supor Prime can be considered a potential single-platform filtration solution, subject to process-specific filterability and sizing studies.

The cell culture studies support the use of Supor Prime and other Cytiva PES filters for cell culture media filtration. Although Supor Prime showed a lower VCD from day 8 to 12 in this study, the final IgG titer remained comparable across conditions and IgG titer is the key parameter when evaluating cell culture media. Similar variability was observed within all shake-flask groups as cultures progressed, consistent with the inherent variability associated with shake-flask cultivation. While there were observed differences in lactate concentration from day 7 to 10, these differences did not correlate with differences in IgG titer, suggesting that the differences did not affect productivity. Viability, glutamate and glucose concentrations remained consistent across all groups with minimal variation.

Table 7 lists the Supor Prime filters recommended for the filtration of ActiPro, Cell Boost 7a and Cell Boost 7b based upon the throughput studies performed. For other basal and feed media, the use of V_MAX testing is recommended to confirm sizing.

Table 7. Recommended Supor Prime part numbers and recommended flow rates based on throughput studies of ActiPro, Cell Boost 7a, and Cell Boost 7b.

* Indicates optional inlet and outlet, vent and drain options. Please consult Cytiva.com and search “Supor Prime filters” for full details.