Modulation of Antibody Glycosylation in Chinese Hamster Ovary Cells: Experimental and Computational Analyses Provide Mechanistic Insights

Process conditions and cell culture media components significantly impact glycosylation in ways that are still poorly understood. Here, we evaluated the glycan distribution of CHO-K1 clones producing adalimumab, a biosimilar version of the anti-TNF antibody Humira. To modulate the glycan profile, the cell culture medium was supplemented with manganese and/or galactose. Both manganese and galactose increased galactosylation, and the effects were synergistic. Notably, the levels of Man5 were higher than in Humira, even when galactosylation was similar. qPCR analysis revealed few differences in glycosylation enzyme concentrations between different productivity clones or when 50 µM manganese was added. We modeled the glycosylation pathways using a dynamic mathematical model to elucidate the mechanisms by which high mannose glycans increased and to develop a more predictive approach to culture modulation. Modeling results suggest that the concentration of uridine triphosphate (UTP), a component of activated sugars, is limiting and that increased uridine diphosphate (UDP)-galactose results in decreased UDP-N-acetylglucosamine, limiting complex glycan synthesis.