N-glycosylation is a key product quality attribute that is known to influence critical properties of therapeutic proteins including potency, safety, immunogenicity and pharmacokinetic clearance. Several factors are known to act in tandem in governing N-glycosylation and this interplay of factors often results in weaker predictability and control. We implement a systems biology approach that includes multi-dimensional omics analyses and mathematical modeling to develop a mechanistic understanding of N-glycosylation of proteins produced during fed-batch cultures of CHO cells. First, we show that the amount of galactosylated species produced at a given time point depends on the relative intracellular levels of UDP-Gal and UDP-HexNAc. Second, there exists a non-linear relationship between galactosylation and fucosylation levels. Galactsoylated species increase as fucosylation increase, suggesting that the existence of core fucose might enhance galactosyltransferase activity of B4galt1. In contrast, fucosylated species decrease as galactosylation levels increase, consistent with existing reports that an uncapped GlcNAc is required on the mannose-alpha1,3 branch for Fut8 activity. Third, we also find that the kinetic properties of B4galt1 determine the relative levels of mono (G1) and bigalactosylated (G2) species. Together, these mechanistic insights provide tools for precision control of N-glycosylation in CHO cell fed-batch cultures.

Novel mechanistic insights towards precision control of n-glycosylation in CHO cell fed-batch cultures

2019 Spring National Meeting

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