POSTTRANSLATIONAL PROCESSING OF RECOMBINANT HUMAN INTERFERON-GAMMA INANIMAL EXPRESSION SYSTEMS

We have characterized the heterogeneity of recombinant human interfero n-gamma (IFN-gamma) produced by three expression systems: Chinese hams ter ovary cells, the mammary gland of transgenic mice, and baculovirus -infected Spodoptera frugiperda (Sf9) insect cells. Analyses of whole IFN-gamma proteins by electrospray ionization-mass spectrometry (ESI-M S) from each recombinant source revealed heterogeneous populations of IFN-gamma molecules resulting from variations in N-glycosylation and C -terminal polypeptide cleavages. A series of more specific analyses as sisted interpretation of maximum entropy deconvoluted ESI-mass spectra of whole IFN-gamma proteins; MALDI-MS analyses of released, desialyla ted N-glycans and of deglycosylated IFN-gamma polypeptides were combin ed with analyses of 2-aminobenzamide labeled sialylated N-glycans by c ation-exchange high-performance liquid chromatography. These analyses enabled identification of specific polypeptide cleavage sites and char acterization of associated N-glycans. Production of recombinant IFN-ga mma in the mammalian expression systems yielded polypeptides C-termina lly truncated at dibasic amino acid sites. Mammalian cell derived IFN- gamma molecules displayed oligosaccharides with monosaccharide composi tions equivalent to complex, sialylated, or high-mannose type N-glycan s. In contrast, IFN-gamma derived from baculovirus-infected Sf9 insect cells was truncated further toward the C-terminus and was associated with neutral (nonsialylated) N-glycans. These data demonstrate the pro found influence of host cell type on posttranslational processing of r ecombinant proteins produced in eukaryotic systems.