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, galactose, or melezitose. 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. The addition of 25 mM melezitose slightly decreased G0F and Man5. qPCR analysis revealed few differences in glycosylation enzyme concentrations between different productivity clones, or when either 50 µM manganese or 25 mM melezitose 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, leading to limitations in complex glycan synthesis.

Controlling process conditions and cell culture media components is essential for maintaining consistent glycosylation. Here, we evaluated the glycan distribution of CHO-K1 clones producing adalimumab, a biosimilar version of the anti-TNF antibody Humira. In an effort to modulate the glycan profile, the cell culture medium was supplemented with manganese, galactose, copper, or melezitose. 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. Copper addition (1.5 mM) increased Man5 levels but reduced G0F, while 25 mM melezitose slightly decreased G0F and Man5. qPCR analysis revealed few differences in glycosylation enzyme concentrations between higher and lower productivity clones, or when either 50 µM manganese or 25 mM melezitose was added. We modeled the glycosylation pathways using a dynamic mathematical model to elucidate the mechanism by which high mannose glycans increased and to develop a more predictive approach to culture modulation. Results suggest that the concentration of activated sugar carriers (i.e., uridine diphosphate, UDP) is limiting and that increased UDP-galactose results in decreased UDP-N-acetylglucosamine, leading to limitations in complex glycan synthesis. These results suggest potential strategies for host cell engineering to obtain more targeted glycan distributions.