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 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.
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Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Wang
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D000818:Animals; D001216:Asparagine; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D002846:Chromatography, Affinity; D006224:Cricetinae; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D006031:Glycosylation; D006595:Hexosamines / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011865:Radioisotope Dilution Technique; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins; D012794:Sialic Acids / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection / Q000379:methods; D014316:Tritium
D006595:Hexosamines; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012794:Sialic Acids; D014316:Tritium; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D001216:Asparagine; D007371:Interferon-gamma; D013762:Tetrahydrofolate Dehydrogenase; C002022:N-acetylmannosamine