Enhancement of recombinant EPO sialylation produced in CHO cells by combinatorial genetic engineering of glycosyltransferases

Abstract

Most of the biopharmaceuticals are glycoproteins. The glycosylation of recombinant therapeutic proteins is one of the most important factors that determines various pharmacological properties. Especially, sialylation regulates the in vivo half-life of recombinant therapeutic glycoproteins, affecting their therapeutic efficacy. Here, we show that the sialylation level of recombinant human erythropoietin (rhEPO) was increased by enhancing N-glycan antenna branching and sialylation pathway. Initially, we introduced heterogenous Nacetylglucosaminyltransferases (GNT) to maximize the N-glycan branching up to hexa-antennary, the theoretical maximum level. We were able to obtain rhEPO with penta-antenna N-glycan structure only in GNTVI from Hen oviduct introduced CHO cells. However, determining whether a glycan composition is penta-antennary or polyLacNAc was challenging especially in highly branched N-glycans. Hence, we tried to eliminate polyLacNAc portion on rhEPO by inhibiting expression of β-1,3-N-acetylglucosaminyltransferase 2 (β3gnt2) to reduce complexity of N-glycan with high level of branch. Although we successfully reduced polyLacNAc on rhEPO, ratio of the penta-antenna structure was only found up to 4% of total N-glycan which is not significant level. Fortunately, however, we found that repression of the key polyLacNAc biosynthesis enzyme β3gnt2 increases the multi antennary (tri-, tetra-) N-linked glycan structures which have enlarged capacity for sialic acid. We noticed that inhibition of polyLacNAc biosynthesis by depletion of β3gnt2 induced N-glycan branching, however, sialylation of rhEPO was not significantly increased because of negative feedback by intracellular cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac). Because CMP-Neu5Ac is a limiting factor for sialylation, we observed a significant increase in the sialylation of rhEPO produced in metabolically engineered CHO cells when we combined the inhibition of polyLacNAc biosynthesis with a release of CMP-Neu5Ac feedback inhibition. Upon further analysis of the resulting N-glycosylation pattern, we discovered that the enhanced rhEPO sialylation could be attributed to a decrease in di-sialylated N-glycans and an increase in tri- and tetra sialylated N-glycans. Our results suggest the novel way of improving glycoprotein sialylation by shunting GlcNAc and Gal away from polyLacNAc biosynthesis but toward N-glycan branch formation.