Contributions of Chinese hamster ovary cell derived extracellular vesicles and other cellular materials to hollow fiber filter fouling during perfusion manufacturing of monoclonal antibodies.

Hollow fiber filter fouling is a common issue plaguing perfusion production process for biologics therapeutics, but the nature of filter foulant has been elusive. Here we studied cell culture materials especially Chinese hamster ovary (CHO) cell-derived extracellular vesicles in perfusion process to determine their role in filter fouling. We found that the decrease of CHO-derived small extracellular vesicles (sEVs) with 50-200 nm in diameter in perfusion permeates always preceded the increase in transmembrane pressure (TMP) and subsequent decrease in product sieving, suggesting that sEVs might have been retained inside filters and contributed to filter fouling. Using scanning electron microscopy and helium ion microscopy, we found sEV-like structures in pores and on foulant patches of hollow fiber tangential flow filtration filter (HF-TFF) membranes. We also observed that the Day 28 TMP of perfusion culture correlated positively with the percentage of foulant patch areas. In addition, energy dispersive X-ray spectroscopy-based elemental mapping microscopy and spectroscopy analysis suggests that foulant patches had enriched cellular materials but not antifoam. Fluorescent staining results further indicate that these cellular materials could be DNA, proteins, and even adherent CHO cells. Lastly, in a small-scale HF-TFF model, addition of CHO-specific sEVs in CHO culture simulated filter fouling behaviors in a concentration-dependent manner. Based on these results, we proposed a mechanism of HF-TFF fouling, in which filter pore constriction by CHO sEVs is followed by cake formation of cellular materials on filter membrane.

Effective bioreactor pH control using only sparging gases.

pH control is critical in bioreactor operations, typically realized through a two-sided control loop, where CO2 sparging and base addition are used in bicarbonate-buffered media. Though a common approach, base addition could compromise culture performance due to the potential impact from pH excursions and osmolality increase in large-scale bioreactors. In this study, the feasibility of utilizing control of sparge gas composition as part of the pH control loop was assessed in Chinese hamster ovary (CHO) fed-batch cultures. Fine pH control was evaluated in multiple processes at different setpoints in small-scale ambr®250 bioreactors. Desired culture pH setpoints were successfully maintained via air sparge feedback control. As part of the pH control loop, air sparging was increased to improve CO2 removal automatically, hence increase culture pH, and vice versa. The effectiveness of this pH control strategy was seamlessly transferred from ambr®250 to 200 L scale, demonstrating scalability of the proposed methodology. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2743, 2019.