A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation.

Ioscani Jimenez Del Val,Karen M Polizzi,Cleo Kontoravdi

doi:10.1038/srep28547

Ioscani Jimenez Del Val, Karen M Polizzi + Show 1 more

Open Access

https://doi.org/10.1038/srep28547

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Abstract

Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation.

Highlights

Glycosylation is the most prevalent and impactful post-translational modification of recombinant therapeutic proteins[1,2]
Our results indicate that nucleotide sugars (NSs) consumption rates towards cellular and recombinant therapeutic proteins (rTPs) glycosylation are within the same order of magnitude, suggesting that considering both is imperative to achieve a comprehensive description of NS metabolism
The estimate combines the relative abundance of individual CHO host proteins with the number of N-linked and O-GalNAc glycosylation sites present on each host cell proteins (HCPs) and the reported CHO HCP glycome

Summary

Introduction

Glycosylation is the most prevalent and impactful post-translational modification of recombinant therapeutic proteins (rTP)[1,2]. The glycosylation of recombinant product (rTP), host cell proteins (HCPs), and lipids occurs simultaneously. This is substantiated by the close correlation observed between cell surface and rTP glycans reported recently[12]. Despite providing useful initial approximations, these estimates have limitations given that they do not account for the relative abundance of each protein within the host organism’s proteome or the number of glycosylation sites on each protein. These estimates have not included the consumption of NSs towards glycolipid synthesis

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