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Some of these HCPs can hydrolyze the polysorbate surfactant to release poorly\u2010soluble fatty acids that accumulate over time and form particles. To tackle this industry\u2010wide issue, we disrupted the genes for a plurality of potential polysorbate\u2010degrading HCPs in CHO cells via two CHO cell engineering approaches. First, we sequentially knocked out six hydrolase genes in a stable recombinant mAb\u2010expressing 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\u2010associated 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.<\/jats:p>","DOI":"10.1002\/bit.70037","type":"journal-article","created":{"date-parts":[[2025,8,6]],"date-time":"2025-08-06T07:20:37Z","timestamp":1754464837000},"page":"3139-3159","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Engineering Chinese Hamster Ovary Cells to Mitigate Polysorbate Degradation in Biotherapeutics"],"prefix":"10.1002","volume":"122","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4698-0115","authenticated-orcid":false,"given":"Inn H.","family":"Yuk","sequence":"first","affiliation":[{"name":"Pharma Technical Development South San Francisco California 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