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

Chung-Geun Lee,Myung Jin Oh,Seung-Yeol Park,Hyun Joo An,Jung Hoe Kim

doi:10.1038/s41598-018-25580-9

Abstract

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.

Highlights

N-acetylglucosamine (GlcNAc) is the initial glycan residue in N-glycan branches
We introduce a new approach for increasing recombinant human erythropoietin (rhEPO) sialylation by modulating N-glycan branch synthesis
Recombinant interferon (IFN)-γ and rhEPO with more antennary structures were obtained via the overexpression of MGAT-4 and −5, which are responsible for the formation of tri- and tetra-antennary structures[17,18]

Summary

Introduction

N-acetylglucosamine (GlcNAc) is the initial glycan residue in N-glycan branches. Initial N-glycan branches are extended by the sequential attachment of galactosyl- and sialyl- residues. Poly-LacNAc consists of repeated N-acetyllactosamine (Galβ1-4GlcNAc)n residues formed as GlcNAc residues are attached to galactosyl termini via the enzymatic activity of β-1,3 N-acetylglucosaminyltransferase (β3gnt) (Fig. 1). We hypothesized that rhEPO sialylation may increase when poly-LacNAc synthesis is inhibited because this would increase the availability of GlcNAc and galactosyl residues for N-glycan branch synthesis. We found that β3gnt2-depleted CHO cells produce rhEPO without poly-LacNAc, but with increased tri- and tetra-antennary N-glycans. Sialylation of rhEPO from β3gnt2-depleted CHO cells were still not increased due to feedback regulation by intracellular CMP-Neu5Ac20,21. By modifying CHO cells to increase N-glycan branching synthesis and reduce feedback inhibition by CMP-Neu5Ac, we were able to produce rhEPO with significantly more N-glycan branching and sialylation

Methods

Results

Conclusion