Hooker, A.D. and Goldman, M.H. and Green, N.H. and James, D.C. and Bull, A.T. and Strange, P.G. and Baines, A.J. and Jenkin, N. (1997) Resolution glycoform analysis of recombinant human interferon-gamma during batch cultures of Chinese hamster ovary cells. In: Funatsu, K. and Shirai, Y. and Matsushita, T., eds. Animal Cell Technology: Basic & Applied Aspects, Vol 8. Kluwer Academic Publ pp. 315-321. ISBN 0-7923-4486-3.
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Abstract
A Chinese hamster ovary cell line expressing recombinant interferon-gamma (IFN-gamma) was grown in 12L batch fermentations under conditions of constant dissolved oxygen tension, pH and temperature. Samples were taken throughout the cultures and IFN-gamma purified from the culture supernatant by immunoaffinity chromatography. Changes in the proportions of site-occupancy variants during cell culture (i.e. glycosylation macroheterogeneity) were determined by densitometry scanning of silver stained SDS-PAGE gels and micellar electrokinetic capillary electrophoresis. Glycosylation deteriorated with time in batch cultures, with a decrease in the doubly-glycosylated (2N) glycoform and a concomitant increase in the singly-glycosylated (1N) glycoform. Matrix-assisted laser-desorption/ionisation mass spectrometry in combination with exoglycosidase arrays allows the changes of individual N-glycan structures at each glycosylation site (i.e. glycosylation microheterogeneity) to be analysed. Both Asn(25) and Asn(97) glycosylation sites are occupied predominantly by complex N-glycan structures, Gal(2)GlcNAc(4)Man(3) being particularly prevalent. These glycans were core alpha 1,6 fucosylated when attached to the Asn(25) glycosylation site, but not at Asn(97). The majority of these complex N-glycan structures were terminally sialylated with N-acetylneuraminic acid. The proportion of the dominant complex biantennary (Gal(2)GlcNAc(4)Man(3) +/- Fuel) decreased by 16-28% during batch culture with a corresponding increase in the proportion of oligomannose and truncated glycans, and in particular Man(5)GlcNAc(2).
| Item Type: | Conference or workshop item (UNSPECIFIED) |
|---|---|
| Subjects: | Q Science Q Science > QH Natural history > QH301 Biology |
| Divisions: | Faculties > Science Technology and Medical Studies > School of Biosciences |
| Depositing User: | M.A. Ziai |
| Date Deposited: | 08 Jun 1914 10:19 |
| Last Modified: | 08 Jun 1914 10:19 |
| Resource URI: | http://kar.kent.ac.uk/id/eprint/18108 (The current URI for this page, for reference purposes) |
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