Perfusion cell culture serves as a strategic process intensification tool, allowing for continuous monoclonal antibody (mAb) production. Additionally, perfusion processes can offer several advantages over batch or fed-batch processes, including improved space-time yields and quality, enhanced facility flexibility and reduced capital costs. We will demonstrate the feasibility of using the ReadyToProcess WAVE™ 25 rocking bioreactor for steady-state perfusion and can also be used as a scale‑down tool for steady-state perfusion processes.
Introduction
Perfusion processes can be investigated using various methods including spin tubes, the ReadyToProcess WAVE™ 25 rocker bioreactor, and single-use bioreactors like the Xcellerex™ platforms paired with Xcellerex™ automated perfusion system (APS) (Fig 1). The ReadyToProcess WAVE™ 25 rocker bioreactor has already been shown to be highly effective at intensifying seed train applications but with this study we aim to demonstrate its effectiveness in the more challenging steady state perfusion process. The simplicity of the ReadyToProcess WAVE™ 25 system with rapid set up time, multiple bag sizes, and configurations, as well as, having an integrated filter within the single-use bag make both process development and clinical manufacturing straightforward. In addition, the UNICORN™ software and media control feature provide an easy-to-use platform for screening of perfusion medium, CSPR screening, and overall process development.
Fig 1. Example perfusion processes in the (A) spin tubes, (B) ReadyToProcess WAVE™ 25 rocker bioreactor, and (C) single-use bioreactors like the Xcellerex™ platforms paired with Xcellerex™ APS.
To develop a steady-state perfusion process using the ReadyToProcess WAVE™ 25 rocking bioreactor system required modification of a standard perfusion Cellbag™ bioreactor container to incorporate a dip-tube for cell bleeding. In addition to demonstrating feasibility, we aim to show comparability to larger bioreactor systems like the Xcellerex™ 50 XDR single-use bioreactor paired with an Xcellerex™ APS to show potential seamless scalability from small-scale to manufacturing-scale. The data for the perfusion process using an Xcellerex™ bioreactor paired with Xcellerex™ APS was obtained in a previous study. We will show that the ReadyToProcess WAVE™ 25 rocking bioreactor at 0.5 L and 5 L working volumes produce comparable results to larger systems with respect to viable cell density, culture duration, protein titer, and protein quality.
Materials and methods
2 and 20 L Cellbag™ bioreactor containers were used to cultivate a mAb producing CHO cell line using the ReadyToProcess WAVE™ 25 rocker bioreactor system. The CHO cells were cultured using the parameters listed in Table 1.
Table 1. CHO culture parameters for steady-state perfusion process in ReadyToProcess WAVE™ 25 rocker bioreactor system
|
Parameter |
|
|
Working volume |
0.5 L and 5 L |
|
Target inoculation cell concentration |
1 MVC/mL |
|
Perfusion strategy |
Perfusion began on 1 d at a perfusion rate of 40 pL/cells/d for the first 2 d of culture and then lowered to 20 pL/cells/d for the remainder of the culture period |
|
Temperature setpoint |
37°C |
|
DO setpoint |
40% |
|
pH setpoint |
6.8 |
|
Steady-state VCD |
70 MVC/mL (steady-state) |
|
Target viability |
> 95% |
|
Culture medium |
Expansion medium: ActiPro™ Perfusion media: ActiPro™ 10.8% Cell Boost™ 1 (10% w/v stock solution), 12.1% Cell Boost™ 3 (5% w/v stock solution) |
Custom dip-tube
A custom dip-tube was created, autoclaved, and incorporated into a standard Cellbag™ bioreactor to allow cell bleeding (Fig 2).
Fig 2. (A) The dip-tube made from silicone and C-flex tubing. (B) A diagram of the face ports on a 2 L perfusion Cellbag™ bioreactor indicating that P1 was modified to include the sterilized dip-tube. (C) A photo of the dib-tube incorporated into a standard perfusion Cellbag™ bioreactor positioned under the internal perfusion filter.
Results
The 0.5 L and 5 L perfusion cultures were maintained for 10 d in steady-state perfusion mode at the cell density target of 70 MVC/mL. Viability was consistently above 95% throughout the culture period. CHO cell cultured in the ReadyToProcess WAVE™ 25 rocker bioreactor systems performed comparably to those cultured in the Xcellerex™ XDR-50 bioreactor (Fig 3). The cells cultured in the ReadyToProcess WAVE™ 25 rocker bioreactor systems and Xcellerex™ XDR-50 APS system also performed similarly in terms of IgG titer, N-linked glycan profile, and charge variant profile, as seen in Figures 4 through 6.
Fig 3. Cell density and viability for perfusion cell culture in ReadyToProcess WAVE™ 25 rocker bioreactor system and Xcellerex™ XDR-50 APS system.
Fig 4. IgG titer produced by CHO cells during steady-state perfusion in ReadyToProcess WAVE™ 25 rocker bioreactor system and Xcellerex™ XDR-50 APS system.
Fig 5. N-linked glycan profile for perfusion culture in ReadyToProcess WAVE™ 25 rocker bioreactor system and Xcellerex™ XDR-50 APS system.
Fig 6. Charge variant profile for perfusion culture in ReadyToProcess WAVE™ 25 rocker bioreactor system and Xcellerex™ XDR-50 APS system.
Conclusions
We have shown that:
- The ReadyToProcess WAVE™ 25 rocker bioreactor system can be an effective scale down tool for steady-state perfusion processes.
- The internal perfusion filter of the Cellbag™ bioreactor is sufficient for high density cell culture.
- The cell culture media combination used creates an effective perfusion medium.
- The custom dip-tube can be aseptically incorporated into a standard perfusion cell bag.
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REFERENCE
- Poster: Tronnersjö S, Falkman T, Maude H, Anfelt J, and Castan A. Intensification of the fed-batch process using N-1 perfusion. https://cdn.cytivalifesciences.com/api/public/content/u9Gd_F8Uk0SMeqDQf7lhig-pdf?v=a4c0317a. Cytiva, CY21503-02Jul21-PT.