Weiss_2026_N.Biotechnol__

Hydrolytic host cell proteins (HCPs) from Chinese hamster ovary (CHO) cells, particularly lipoprotein lipase (LPL), pose a persistent challenge in biopharmaceutical manufacturing due to their degrading activity against polysorbate (PS), a commonly used surfactant in therapeutic protein formulations. While downstream purification effectively removes many HCPs, LPL remains difficult to eliminate and upstream cultivation conditions influencing its abundance are poorly understood. Here, we demonstrate that CHO cultivation medium composition is a key-contributor of extracellular LPL levels and PS degradation activity. Using a fluorescence micelle assay, recombinant LPL expression systems and live-cell LPL staining, we show that medium-dependent differences in PS degradation are not attributable to transcription, translation, secretion or proteolytic processing. Instead, LPL-cell surface interactions, probably mediated primarily by heparan sulfate proteoglycans, appear to contribute to LPL retention and release. Experimental medium A promoted strong surface binding and low levels of LPL in the supernatant, whereas experimental medium B enhanced LPL release, correlating with increased PS degradation. Heparin displacement and rapid medium-switch experiments confirmed that these interactions are dynamic, reversible and sensitive to the physicochemical environment. Our findings suggest that the charged components of the culture medium modulate LPL electrostatic binding to the cell surface, thereby controlling its extracellular abundance. From a bioprocessing perspective, these results suggest a novel factor that affects hydrolytic HCP burden by cell culture medium composition and cell surface retention mechanisms. Collectively, this study provides mechanistic insights into LPL regulation in CHO cultures and supports a framework for controlling problematic HCPs to improve the NBE production process.