Engineering Chinese Hamster Ovary Cells to Mitigate Polysorbate Degradation in Biotherapeutics

ABSTRACT A recurring root cause for polysorbate degradation and associated particle observations in liquid biotherapeutics is the presence of residual host cell proteins (HCPs). Some of these HCPs can hydrolyze the polysorbate surfactant to release poorly‐soluble fatty acids that accumulate over time and form particles. To tackle this industry‐wide issue, we disrupted the genes for a plurality of potential polysorbate‐degrading HCPs in CHO cells via two CHO cell engineering approaches. First, we sequentially knocked out six hydrolase genes in a stable recombinant mAb‐expressing cell line and substantially decreased polysorbate degradation (up to ~90% in purified samples) without negatively impacting cell culture performance and mAb product quality. Second, we applied a more efficient multiplex gene disruption method to knockout (KO) 3, 7, and 12 hydrolase‐associated genes from a CHO blank (null) host. The resulting KO hosts were stably transfected to express six different mAbs, and the purified mAb samples exhibited decreased polysorbate degradation (up to ~60%) while maintaining consistent mAb product quality. These representative CHO cell engineering studies demonstrate the feasibility of modulating CHO cells to mitigate product quality risks in biotherapeutics without deleterious effects on the production cells, even with up to 12 hydrolase genes knocked out.