Improvement of interferon‐γ sialylation in Chinese hamster ovary cell culture by feeding of N‐acetylmannosamine

Abstract

Because the presence of sialic acid can extend circulatory lifetime, a high degree of sialylation is often a desirable feature of therapeutic glycoproteins. In this study, the incomplete intracellular sialylation of interferon-gamma (IFN-gamma), produced by Chinese hamster ovary cell culture, was minimized by supplementing the culture medium with N-acetylmannosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis. By introducing 20 mM ManNAc into the culture medium, incompletely sialylated biantennary glycan structures were reduced from 35% to 20% at the Asn97 glycosylation site. This effect was achieved without affecting cell growth or product yield. The intracellular pool of CMP-sialic acid, the nucleotide sugar substrate for sialyltransferase, was also extracted and quantified by HPLC. Feeding of 20 mM ManNAc increased this intracellular pool of CMP-sialic acid by nearly thirtyfold compared with unsupplemented medium. When radiolabeled ManNAc was used to trace the incorporation of the precursor, it was found that supplemental ManNAc was exclusively incorporated into IFN-gamma as sialic acid and that, at 20 mM ManNAc feeding, nearly 100% of product sialylation originated from the supplemental precursor.

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…The results showed a linear correlation between the time period of glucose depletion during 24 h and galactosylation and sialylation indexes, suggesting that the decrease in galactosylation and sialylation could be elicited by depletion of activated monosacharides as dolichol phosphate or nucleotide derivates, mostly glucose-derived substrates. Previous studies revealed that feeding N-glycosylation precursors or substrates could improve sialylation or galactosylation of interferon-γ expressed in CHO (Gu and Wang, 1998;Jones et al, 2010). In addition, a feeding regime of uridine, manganese and galactose as media supplements improves GI of CHO-EG2 cells (Liu et al, 2014).…”

Epigenetic Switching by the Metabolism-sensing Factors in the Generation of Orexin Neurons from Mouse Embryonic Stem Cells

“…ManNAc is incorporated into mammalian cells easily rather than Neu5AC in culture conditions (51). In the cells, at least, two molecules recognize ManNAc: glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase and GlcNAc 2-epimerase.…”

A Novel Type of Macrothrombocytopenia Associated with a Defect in α2,3-Sialylation

“…34,35 We therefore extensively investigated the effect of exogenous ManNAc (in concentrations ranging from 2 to 50 mmol/L) on sLe x expression in cultures of patient granulocytes and monocytes using incubation times of 12 to 72 hours. In neither case did the sialic acid precursor increase sLe x expression (not shown).…”