Glycosylation is a critical post-translational modification of proteins in mammalian cells. Glycosylation of monoclonal antibodies (mAbs) is of particular interest due to the effect it has on the effector function, structure and stability of the mAb, thus making it an important product quality attribute. To achieve a desired therapeutic effect, it is necessary to produce an antibody with a specific glycosylation profile. To produce this antibody, a better understanding of how changes to culturing conditions impact the glycoforms of mAbs is required. In this work we detect significant changes to glycosylation following ammonia stress of CHO produced VRC01- a broadly neutralizing antibody undergoing clinical trials to treat HIV. Ambr250 bioreactors were run in parallel with increasing concentrations of ammonia stress (0, 10 or 30 mM) on both young and aged CHO cells, with high and low gas flow rates. The determine the glycan profiles, the culture supernatant was collected over the course of the run and VRC01 was purified using gravity flow chromatography with protein A resin. Glycans were fluorescently labeled and released from the mAb and eluted from a BEH Amide column using a Waters AQUITY H-class UPLC. We found that increased ammonia concentrations measured in the culture for all conditions tested led to a decrease in total and terminal galactosylation, as well as a decrease in sialylation of the associated glycans. This observed reduction in sialylation of glycoproteins following ammonia stress is consistent with previously published results [1-3]. Future work will look into how the conserved Asn297 Fc glycan is impacted compared to Fab glycans under various culturing conditions

Impact of ammonia stress on glycosylation of the VRC01 monoclonal antibody produced in CHO cells

ACS Fall 2020 Virtual Meeting

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