(B-75) High-efficiency large-scale dialysis culture of recombinant CHO cells with a rolled scaffold bioreactor with minimum media consumption

Introduction:: With the rapid expansion of the biopharmaceutical market, there is an increasing need for more efficient platforms to lower the production cost of protein therapeutics. To this end, continuous culture for biopharmaceutic production has been actively investigated, where managing concentration of nutrients and cellular bio-wastes in the media is essential for enhanced yield and minimized production cost. Media perfusion has been demonstrated for the continuous culture of recombinant cells, but large consumption of media has been a known issue. In perfusion culture, the culture media containing cellular biowaste as well as target recombinant proteins is continuously exchanged with fresh media. This approach increases the process volume of the downstream process and throws away culture media which can still support cell growth and protein production.

In this work, we present a dialysis rolled scaffold (RS) bioreactor [1,2]. RS is a non-woven matrix based fixed-bed, that can provide large surface area for cell attachment and media transport with low shear stress. The developed bioreactor can effectively remove cellular-biowastes from the media without losing produced proteins, based on the difference of their molecular weights (MW). Low MW bio-wastes in the media are transported through dialysis membrane and removed by low-cost dialysate, whereas large MW proteins remain in the media. Furthermore, the 4-vessel configuration, which separates the dialyzed media and used dialysate from the media before the dialysis and fresh dialysate, significantly improves the dialysis efficiency.


Materials and Methods:: Fig. 1a shows the schematic diagram of the RS dialysis bioreactor with the 4-vessel configuration. Cells are attached to and grow in the RS and the media in the media reservoir 1 flows into the RS to provide nutrients and oxygen. When lactate concentration in the media reached over 25 mM, the media was dialyzed with a hemodialyzer (Revaclear Dialyzer 300, Baxter). The media in the media reservoir 1 is dialyzed and the dialyzed media is collected in the media reservoir 2, while the used dialysate is discarded. When the media reservoir 1 is almost empty, the dialyzed media in the reservoir 2 is pumped into the RS. This process can be repeated while switching back and forth between the media reservoir 1 and the media reservoir 2. We characterized the dialysis efficiency of the 4-vessel configuration as well as the conventional 2-vessel configuration, in which the dialyzed media and the used dialysate are not kept separately.

Recombinant CHO cells producing monoclonal antibody (mAb) were maintained in F-12K with 10% fetal bovine serum (FBS). The RS has an effective surface area of 3150 cm² and 1L of the media was used. The flow rate was controlled to keep the dissolved oxygen (DO) of the media exiting the RS at 3 mg/L. Based on the media flow rate and the DO at upstream and downstream of the RS, the oxygen consumption rate was measured to estimate the total cell population.


Results, Conclusions, and Discussions:: Results

Fig. 1b shows the dialysis efficiency of the two configurations. The 4-vessel configuration shows much higher efficiency than the 2-vessel configuration. Lactate in the media is decreasing rapidly with the 4-vessel configuration, whereas the 2-vessel configuration shows exponential decay saturating at 33% of initial concentration.

Fig. 1c shows the expansion of CHO cells with the dialysis RS bioreactor. 14 million CHO cells were seeded on day 1. Active growth in the initial phase was slowed down on day 6. We believe that this is due to the depletion of signaling molecules and proteins in FBS. After exchanging the media on day 7, the active growth continued until day 10, when the cells were expected to reach full confluency. At the end of day 12, the culture was terminated due to the rupture of the tubing in the peristaltic pump. The maximum oxygen consumption rate was 123 µg/min, indicating 1.3 billion cells in the RS. The average doubling time during the active expansions during day 1~4 and day 8~10 was 23.85 hours and 21.29 hours, respectively.

Throughout the cell culture, we dialyzed the media when lactate accumulated more than 25 mM. The media was dialyzed three times on day 6, 10 and 12. F-12K media with 5% nutrient feed was used as the dialysate. The flow rate ratio of the media and the dialysate was 1:2, and the dialysis took about 30 minutes. The dialysis removed over 85% of the lactate in the media and replenished nutrients of small MW (Fig. 1d).

Conclusions

We successfully demonstrated the efficiency of the new dialysis configuration and the expansion of 1.3 billion CHO cells with the dialysis RS bioreactor.

Discussions

We believe that optimized feeding strategy would accelerate the cell expansion, as well as allow the large-scale cell culture without media exchange. For instance, addition of FBS on day 6 after the dialysis would avoid media exchange and temporal suspension of the cell growth on day 6~8. Currently, we are analyzing the accumulation and production rate of mAb with HPLC.




Acknowledgements (Optional): :

References (Optional): : [1] YekrangSafakar, A., Mehrnezhad, A., Wu, T., & Park, K. (2022). High‐density adherent culture of CHO cells using rolled scaffold bioreactor. Biotechnology and Bioengineering, 119(6), 1498-1508.

[2] YekrangSafakar, A., Hamel, K. M., Mehrnezhad, A., Jung, J. P., & Park, K. (2020). Development of rolled scaffold for high-density adherent cell culture. Biomedical microdevices, 22, 1-10.