Enhanced Sialylation of Recombinant Erythropoietin in CHO Cells by Human Glycosyltransferase Expression

Abstract

Sialylation, the attachment of sialic acid residues to a protein, can affect the biological activity and in vivo circulatory half-life of glycoproteins. Human alpha 2,3-sialyltransferase (alpha 2,3-ST) and beta 1,4-galactosyltransferase (beta 1,4-GT) are responsible for terminal sialylation and gallactosylation, respectively. Enhanced sialylation of human erythropoietin (EPO) by the expression of a2,3-ST and beta 1,4-GT was achieved using recombinant Chinese hamster ovary (CHO) cells (EC1). The sialic acid content and sialylation of N-glycans were evaluated by HPLC. When a2,3-ST was expressed in CHO cells (EC1-ST2), the sialic acid content (moles of sialic acid/mole of EPO) increased from 6.7 to 7.5. In addition, the amount of trisialylated glycans increased from 17.3% to 26.1%. When alpha 2,3-ST and beta 1,4-GT were coexpressed in CHO cells (EC1-GTST15), the degree of sialylation was greater than that in EC1-ST2 cells. In the case of EC1-GTST15 cells, the sialic acid content increased to 8.2 and the proportion of trisialylated glycans was markedly increased from 17.3% to 35.5%. Interestingly, the amount of asialoglycans decreased only in the case of GTST15 cells (21.4% to 14.2%). These results show that coexpression of alpha 2,3-ST and beta 1,4-GT is more effective than the expression of alpha 2,3-ST alone. Coexpression of alpha 2,3-ST and beta 1,4-GT did not affect CHO cell growth and metabolism or EPO production. Thus, coexpression of alpha 2,3-ST and beta 1,4-GT may be beneficial for producing therapeutic glycoproteins with enhanced sialylation in CHO cells.