Inhibition of poly-LacNAc biosynthesis with release of CMP-Neu5Ac feedback inhibition increases the sialylation of recombinant EPO produced in CHO cells

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Sialylation of recombinant therapeutic glycoproteins modulates their pharmacokinetic properties by affecting their in vivo half-life. N-glycan branching on glycoproteins increases the number of potential attachment sites for sialic acid. Here, we introduce a new approach for increasing the sialylation of recombinant human erythropoietin (rhEPO) produced in CHO cells by modulating poly-N-acetyllactosamine (poly-LacNAc) biosynthesis. We did not observe an increase in rhEPO sialylation, however, until the feedback inhibition by intracellular cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac), which is a limiting factor for sialylation, was released. Thus, we found that a combined approach inhibiting poly-LacNAc biosynthesis and releasing CMP-Neu5Ac feedback inhibition produces the most significant increase in rhEPO sialylation in metabolically engineered CHO cells. Furthermore, a detailed analysis of the resulting N-glycan structures using LC/MS revealed increased tri- and tetra-sialylated N-glycan structures accompanied by a reduction of di-sialylated N-glycan structures. These results validate our new approach for glycosylation engineering, and we expect this approach will be useful in future efforts to enhance the efficacy of other therapeutic glycoproteins.
Publisher
NATURE PUBLISHING GROUP
Issue Date
2018-05
Language
English
Article Type
Article
Keywords

HAMSTER OVARY CELLS; HUMAN INTERFERON-GAMMA; HUMAN ERYTHROPOIETINS; ENHANCED SIALYLATION; MASS-SPECTROMETRY; GLYCOSYLATION; GLYCOPROTEINS; CULTURE; OLIGOSACCHARIDES; PROTEINS

Citation

SCIENTIFIC REPORTS, v.8

ISSN
2045-2322
DOI
10.1038/s41598-018-25580-9
URI
http://hdl.handle.net/10203/242339
Appears in Collection
BS-Journal Papers(저널논문)
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