Chemometrics and integrative LC-MS identify compositional variability in cottonseed hydrolysates and its effects on CHO cell growth and antibody productivity

Protein hydrolysates have attracted increasing interest as cost-effective media supplements for mammalian cell culture, including Chinese hamster ovary (CHO) cells used widely in biopharmaceutical production. However, the biological basis of their beneficial effects remains poorly understood because of their compositional complexity and batch-to-batch variability. In this study, time-resolved compositional profiles of culture media supplemented with different batches of cottonseed hydrolysates were analysed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) and related to CHO DG44 cell growth, antibody productivity, cellular metabolism, and antibody glycosylation. During 10-day batch cultures, hydrolysate supplementation prolonged high cell viability and significantly enhanced antibody productivity, despite lower peak viable cell densities than the control. Hydrolysate-supplemented cultures also showed reduced lactate and ammonia accumulation, consistent with altered nutrient utilization and metabolic activity. In addition, cottonseed hydrolysates significantly increased antibody galactosylation to varying degrees. Chemometric analysis further linked hydrolysate compositional variability to culture performance and identified 25 signature features associated with cell growth and antibody production. These findings provide complementary insight into how hydrolysate composition relates to functional performance in CHO cell culture and support the development of a method to identify hydrolysates that support high performance biopharmaceutical manufacturing.