Monitoring of recombinant interferon-gamma glycosylation during batch fermentation of CHO cells

A Chinese hamster ovary cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown in a 15-l stirred tank fermenter. N-linked carbohydrate populations associated with both Asn(25) and Asn(97) were isolated by reverse-phase HPLC separation of trypsin-digested IFN-gamma and their structure determined by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MD) in combination with exoglycosidase array sequencing. The predominant oligosaccharide at both glycosylation sites throughout the culture was a complex biantennary structure, Gal(2)GlcNAc(2)Man(3)GlcNAc(2), which was fucosylated when attached to Asn(25) but not to Asn(97). A gradual decrease in this biantennary structure was observed, with a concomitant increase in the proportion of truncated and high-mannose glycans. These experiments demonstrate the relative stability of glycosylation during batch culture and the definitive site-specific glycosylation data that ran be obtained using MALDI-MS as a monitoring technique.