Dissecting the Dynamics of N-Linked Glycosylation in Fed-Batch Cultures of Chinese Hamster Ovary Cells Through Time Course Omics Analyses

N-linked glycosylation is a post-translational modification that affects potency, safety, immunogenicity and pharmacokinetic clearance of several therapeutic proteins including monoclonal antibodies (mAbs). A robust control strategy is needed to accurately dial in appropriate glycosylation profile during the course of a cell culture process. However, N-glycosylation dynamics remain insufficiently understood due to the lack of integrative and quantitative analyses of factors that influence the dynamics, including nucleotide sugar donors (NSDs), glycosyltransferases and glycosidases. Here, an integrative approach is taken employing multiple omics analyses to dissect the temporal dynamics of glycoforms produced during fed-batch cultures of CHO cells. Several pathways linked with NSD metabolism, such as glycolysis, TCA cycle and purine and pyrimidine biosynthesis exhibit significant temporal dynamics over cell culture period. The steps involving galactose and sialic acid addition were determined as temporal bottlenecks in the N-glycosylation process. Our results show that galactose and not manganese is able to mitigate the temporal bottleneck, despite both being known effectors of galactosylation. Another bottleneck step, sialylation, is primarily governed by the sialyltransferases, but is also affected significantly by galactosylated precursors and the auto-regulation of CMP-sialic acid biosynthesis.