Effects of sialylated oligosaccharides on the N- and O-glycosylation of etanercept in recombinant CHO cells

Abstract: In mammalian cell-based bioprocesses, glycosylation of recombinant therapeutic proteins requires stringent control and monitoring as it may affect therapeutic efficacy and safety. The sialylation of glycosylated moieties plays an important role in determining the in vivo clearance of therapeutic proteins by extending their serum half-life. In this study, six enzymatically synthesized oligosaccharides (3′-sialyllactose, 6′-sialyllactose, sialyllacto-N-tetraose a, disialyllacto-N-neotetraose, lacto-N-tetraose, and lacto-N-neotetraose) and lactose were evaluated for their effectiveness on the N- and O-glycan profiles of etanercept, a therapeutic Fc-fusion glycoprotein consisting of 3 N- and 13 O-glycosylation sites, produced in recombinant Chinese hamster ovary (rCHO) cells. Of these seven compounds, only sialylated oligosaccharide supplementation increased the proportion of the acidic isoforms, di-sialylated N-glycan and di-sialylated O-glycan, as well as the total sialic acid content of etanercept, although the degree of the effect varied. Increased sialylation resulted from increased concentrations of intracellular CMP-sialic acid, with the highest increase observed for disialyllacto-N-neotetraose, which contains the most di-sialic acid residues. In contrast, four sialylated oligosaccharides did not enhance the sialylation of etanercept produced by human embryonic kidney 293 (HEK293) cells. Taken together, enzymatically synthesized sialylated oligosaccharides represent novel supplements to enhance the sialylation of therapeutic Fc-fusion glycoproteins in rCHO cell culture. Key points: • Sialylated oligosaccharides supplementation in rCHO cell culture increased the proportion of di-sialylated N- and O-glycans of Fc-fusion glycoprotein. • Among the sialylated oligosaccharides, disialyllacto-N-neotetraose (DSLNnT) increased intracellular CMP-SA the most in two rCHO cells. • The effect of sialylated oligosaccharides on sialylation was also evaluated in HEK293 cells; however, no significant enhancement was observed.