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

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).