Improvement of recombinant EPO sialylation in chinese hamster ovary cells by combinatorial genetic engineering

Abstract

The importance of sialic acids, which are occupied the terminal sites of $\It{N}$-linked glycans in glycoproteins, is well known from many researches. The galactose-exposed glycoproteins are cleared rapidly from blood streams by capturing asialoglycoprotein receptors in hepatocytes. Therefore we explored to maximize the sialylation of human erythropoietin by genetic engineering of sialylation pathway in CHO cells. Sialylation is catalyzed by the various sialyltransferases using CMP-sialic acid as an active sugar donor in lumen of the Golgi apparatus. Thus, the activity of sialyltransferase is the key factor to determine the sialylation level of glycoproteins. In this study, we compared the effect of various sialyltransferase overexpression in rhEPO-producing CHO cells. Different from the human cells, Chinese hamster cells lack the genes encoding $\alpha$2,6-sialyltransferase ($\alpha$2,6-ST). Thus, human $\alpha$2,6-ST was overexpressed in CHO cells to produce the much human-like glycoproteins. Chinese hamster $\alpha$2,3-ST was overexpressed to demonstrate the necessity of signal peptide for the localization of sialyltransferase. The sialic acid content of rhEPO which produced from Chinese hamster $\alpha$2,3-ST overexpressed CHO cells showed the similar increment as compared that of human $\alpha$2,3-ST overexpressed CHO cell lines. The sialic acid contents of rhEPO was most significantly increased when the human $\alpha$2,3-ST and human $\alpha$2,6-ST was overexpressed simultaneously up to 20.4%. The sialic acid content of rhEPO produced from EC2-1H9-H26ST 8 cell lines was increased up to 13.0%. The overexpression of human $\alpha$2,6-ST in CHO cells typically showed the much higher increment of sialic acid contents in produced rhEPO. However, there were various reports about the effect of $\alpha$2,6-linked sialic acid in pharmacokinetics of therapeutic glycoproteins. Thus, the accurate effects and functions of $\alpha$2,6-linked sialylation have to be de...