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Biotechnology and Applied Biochemistry (2002) 36, (141–147) (Printed in Great Britain)

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Glycosylation by Chinese hamster ovary cells in dolichol phosphate-supplemented cultures

Inn H. Y. Yuk and Daniel I. C. Wang¹

Key words: batch culture, interferon-g, lipid-linked oligosaccharide.

Abbreviations used: AO/EB, Acridine Orange/ethidium bromide; CHO, Chinese hamster ovary; DPM, disintegrations/min; Dol-P, dolichol monophosphate; ER, endoplasmic reticulum; IFN-g, interferon-g; LLO, lipid-linked oligosaccharide.

¹To whom correspondence should be addressed (e-mail dicwang@mit.edu).

N-linked glycosylation often imparts important properties to protein therapeutics. An essential step in this intracellular process is the transfer of oligosaccharide from dolichol monophosphate (Dol-P) to a potential glycosylation site. Variability in the success rate of this reaction affects the extent of protein glycosylation. The critical role of Dol-P suggests that its availability may influence the extent of glycosylation by limiting the pool of lipid-linked oligosaccharides (LLOs), the glycosyl donor. To test this hypothesis, the impact of Dol-P supplementation on protein glycosylation in Chinese hamster ovary (CHO) cells was investigated. Although exogenous Dol-P was incorporated by CHO cells and processed into LLOs in a dose-dependent manner, Dol-P supplementation had no marked effects on LLO or overall cellular glycosylation levels. While concentrations of exogenous Dol-P exceeding 100 µg/ml were detrimental to CHO cell viability, maximum non-toxic supplemental doses of Dol-P had no significant impact on the glycosylation of recombinant interferon-g produced by batch cultures of CHO cells. These results show that glycosylation in CHO cells cannot be readily enhanced by Dol-P feeding under normal culture conditions.

Received 15 November 2001/1 May 2002; accepted 12 July 2002

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