Differential effects of N-acetylmannosamine on O-glycan sialylation of therapeutic Fc-fusion glycoproteins in CHO and HEK293 cells.

Sialylation is a critical quality attribute of therapeutic glycoproteins, yet strategies to specifically modulate O-glycan sialylation remain underdeveloped compared to N-glycosylation. While N-acetylmannosamine (ManNAc) is widely utilized as an established enhancer of N-glycan sialylation, its efficacy in modulating O-glycan structures has remained largely unexplored across different mammalian hosts. In this study, we investigated ManNAc supplementation as a practical approach for modulating O-glycan sialylation in recombinant Chinese hamster ovary (CHO) cells. Using etanercept and erythropoietin-Fc-fusion protein as models, we found that ManNAc supplementation resulted in a statistically significant, although modest, increase in the proportion of di-sialylated O-glycoforms and the overall content of O-linked sialic acid without compromising cell growth or protein productivity. This enhancement was accompanied by a substantial expansion of the intracellular CMP-sialic acid (CMP-SA) pool. Interestingly, despite increased CMP-SA levels following ManNAc supplementation in human embryonic kidney 293 (HEK293) cells, O-glycan sialylation remained largely unchanged, indicating that responsiveness to ManNAc supplementation differs among cell lines. Our results suggest that ManNAc supplementation can serve as a useful tool for adjusting O-glycan sialylation in CHO cell-based production systems, providing process-relevant insights for biosimilar quality control. Overall, this study provides fundamental mechanistic insights into the use of metabolic precursors to modify the sialylation profiles of O-glycosylated therapeutic glycoproteins.