Sialyllactose supplementation enhances sialylation of Fc-fusion glycoprotein in recombinant Chinese hamster ovary cell culture.

Sialylation during N-glycosylation plays an important role in the half-life of therapeutic glycoproteins in vivo and has sparked interest in the production of therapeutic proteins using recombinant Chinese hamster ovary (rCHO) cells. To improve the sialylation of therapeutic proteins, we examined the effect of sialyllactose supplementation on sialylation of Fc-fusion glycoproteins produced in rCHO cells. Two enzymatically-synthesized sialyllactoses, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), were administered separately to two rCHO cell lines producing the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44, respectively. Two sialyllactoses successfully increased sialylation of Fc-fusion glycoprotein in both cell lines, as evidenced by isoform distribution, sialylated N-glycan formation, and sialic acid content. Increased sialylation by adding sialyllactose was likely the result of increased amount of intracellular CMP-sialic acid (CMP-SA), the direct nucleotide sugar for sialylation. Furthermore, the degree of sialylation enhanced by sialyllactoses was slightly effective or nearly similar compared with the addition of N-acetylmannosamine (ManNAc), a representative nucleotide sugar precursor, to increase sialylation of glycoproteins. The effectiveness of sialyllactose was also confirmed using three commercially available CHO cell culture media. Taken together, these results suggest that enzymatically-synthesized sialyllactose represents a promising candidate for culture media supplementation to increase sialylation of glycoproteins in rCHO cell culture.

Streamlined in vitro screening system of synthetic signal peptides in Chinese hamster ovary cells for therapeutic protein production.

Increasing the screening efficiency and maintaining the N-terminal cleavage pattern are key factors in the development of an in vitro synthetic signal peptide screening system for high therapeutic protein production in Chinese hamster ovary (CHO) cells. This study improved the in vitro screening system of synthetic signal peptides in CHO cells for therapeutic protein production by modifying the expression vector. Incorporating a leaky stop codon with IgG transmembrane and cytoplasmic domains into the expression vector improved the proportion of high producers in establishing stable CHO cell pools. The selected signal peptides from stable CHO cell pools that were generated using degenerate codon-based oligonucleotides with a conserved polar carboxy-terminal domain in the native signal peptide showed similar N-terminal cleavage patterns to the native one. In addition, replacing native signal peptide with selected synthetic signal peptides did not influence the sialylated N-linked glycan formation and biological activity of therapeutic Fc-fusion glycoprotein in CHO cells. Thus, an in vitro synthetic signal peptide screening system can be used for therapeutic Fc-fusion glycoprotein production in CHO cells with an enhanced specific protein productivity while maintaining the N-terminal cleavage pattern similar to the native one.