Glycosylation is a critical post-translational modification of proteins in mammaliancells. Glycosylation of monoclonal antibodies (mAbs) is of particular interest due to the effect ithas on the effector function, structure and stability of the mAb, thus making it an importantproduct quality attribute. To achieve a desired therapeutic effect, it is necessary to produce anantibody with a specific glycosylation profile. To produce this antibody, a better understandingof how changes to culturing conditions impact the glycoforms of mAbs is required. Ammoniaconcentrations typically increase during mammalian cell cultures, mainly due to glutamine andother amino acid consumption. 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 varying concentrations of ammonia stress onCHO cells of two cell line ages. To determine the glycan profiles, the culture supernatant wascollected over the course of the run and VRC01 was purified using gravity flow chromatographywith protein A resin. Glycans were fluorescently labeled and released from the mAb and elutedfrom a BEH Amide column using a Waters AQUITY H-class UPLC. We found that increasedammonia concentrations led to a decrease in total and terminal galactosylation, sialylation, andfucosylation of the associated glycans. While age did not have an effect on antibodygalactosylation and sialylation under no ammonia stress, reduced galactosylation andsialylation was more severe for the more aged cell line when ammonia stress was present.This observed reduction in sialylation of glycoproteins following ammonia stress is consistentwith previously published results [1-2]. Antibody fucosylation was found to be reduced in themore aged cell line in all cases, but ammonia stress did not exacerbate this effect.

Combined effect of ammonia stress and cell line age on CHO cell derived VRC01 monoclonal antibody glycosylation

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.