Resolution glycoform analysis of recombinant human interferon-gamma during batch cultures of Chinese hamster ovary cells

Hooker, Andrew D. and Goldman, Merlin H. and Green, Nicola H. and James, David C. and Bull, Alan T. and Strange, Philip G. and Baines, Anthony J. and Jenkins, Nigel (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. (The full text of this publication is not available from this repository)

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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 (Paper)
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: 16 Apr 2014 15:43
Resource URI: http://kar.kent.ac.uk/id/eprint/18108 (The current URI for this page, for reference purposes)
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