N-Glycosylation of Recombinant Human Interferon-γ Produced in Different Animal Expression Systems

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Abstract
Recombinant human interferon-γ (IFN-γ) was expressed in Chinese hamster ovary cells, baculovirus-infected Sf9 insect cells and the mammary gland of transgenic mice. The N-linked carbohydrate populations associated with both Asn25 and Asn97 glycosylation sites were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. A site-specific analysis of dual (2N) and single (IN) site-occupancy variants of IFN-γ derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type. Although Asn25-linked glycans were substituted with a core fucose residue, Asn97 in N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccha-ride chains were also evident. Transgenic mouse derived IFN-γ exhibited considerable site-specific variation in N-glycan structures. Asn97-linked carbohydrates were of the complex, core fucosylated type, Asn25-linked carbohydrates were mainly of the oligomannose type, with smaller proportions of hybrid and complex N-glycans. Carbohydrates associated with both glycosylation sites of IFN-γ from Sf9 insect cells were mainly tri-mannosyl core structures, with fucosylation confined to the ASn25 site. These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.