IMPROVEMENT OF INTERFERON-GAMMA SIALYLATION IN CHINESE-HAMSTER OVARY CELL-CULTURE BY FEEDING OF N-ACETYLMANNOSAMINE

Because the presence of sialic acid can extend circulatory lifetime, a high degree of sialylation is often a desirable feature of therapeuti c glycoproteins. In this study, the incomplete intracellular sialylati on 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 sial ic acid synthesis. By introducing 20 mM ManNAc into the culture medium , incompletely sialylated biantennary glycan structures were reduced f rom 35% to 20% at the Asn(97) glycosylation site. This effect was achi eved without affecting cell growth or product yield. The intracellular pool of CMP-sialic acid, the nucleotide sugar substrate for sialyltra nsferase, 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 ManN Ac was used to trace the incorporation of the precursor, it was found that supplemental ManNAc was exclusively incorporated into IFN-gamma a s sialic acid and that, at 20 mM ManNAc feeding, nearly 100% of produc t sialylation originated from the supplemental precursor. (C) 1998 Joh n Wiley & Sons, Inc.