N-GLYCOSYLATION OF RECOMBINANT HUMAN INTERFERON-GAMMA PRODUCED IN DIFFERENT ANIMAL EXPRESSION SYSTEMS

Recombinant human interferon-gamma (IFN-gamma) was expressed in Chines e hamster ovary cells, baculovirus-infected Sf9 insect cells and the m ammary gland of transgenic mice, The N-linked carbohydrate populations associated with both Asn(25), and Asn(97) glycosylation sites were ch aracterized by matrix-assisted laser desorption/ionization-mass spectr ometry (MALDI-MS) in combination with exoglycosidase array sequencing, a site-specific analysis of dual (2N) and single (1N) site-occupancy variants of IFN-gamma derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type, Although Asn(25)-linked glycans were substituted with a core fuc ose residue, Asn(97) N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccharide chains were also e vident, Transgenic mouse derived IFN-gamma exhibited considerable site -specific variation in N-glycan structures, Asn(25)-linked carbohydrat es were of the complex, core fucosylated type, Asn(97)-linked carbohyd rates were mainly of the oligomannose type, with smaller proportions o f hybrid and complex N-glycans, Carbohydrates associated with both gly cosylation sites of IFN-gamma from Sf9 insect cells were mainly tri-ma nnosyl core structures, with fucosylation confined to the Asn(25) site , These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.