Chemometric analysis shows compositional variability in cotton hydrolysates that impacts antibody productivity and glycosylation of CHO cells

Abstract Protein hydrolysates have attracted increasing interest as cost-effective media supplements for mammalian cell culture, including Chinese hamster ovary (CHO) cells used widely for biopharmaceutical production. However, the biological basis of their beneficial effects remains poorly understood due to their structural complexity and batch-to-batch compositional variability. In the current 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 viability, culture longevity, cellular metabolism, antibody productivity, and glycosylation profiles. During 10-day batch cultures, cottonseed hydrolysate supplementation allowed prolonged cell cultivation with consistently high cell viability that enhanced the antibody productivity significantly, despite lower viable cell densities (VCDs) compared to controls. Hydrolysate supplementation altered nutrient utilization and showed metabolic activity with reduced lactate and ammonia accumulation. In addition, the hydrolysate-based cultures produced antibodies with substantially increased galactosylation, although to varying degrees. Chemometric analysis identified molecular features of hydrolysates labelled as mass-to-charge ratio @ retention time (m/z @ RT) that correlated with VCDs and antibody production. The current research provides fundamental insight into how compositional variations of protein hydrolysates relate to CHO cell culture longevity, antibody productivity, and glycosylation. Key points ∙ Protein hydrolysates exhibit structural complexity and compositional variability ∙ Batch-to-batch compositional variations prolong CHO cell viability and productivity ∙ Chemometric analysis identifies candidate features that correlate with CHO bioactivities