ISOLATION AND CHARACTERIZATION OF AN INSECT-CELL LINE ABLE TO PERFORMCOMPLEX N-LINKED GLYCOSYLATION ON RECOMBINANT PROTEINS

Site specific characterization of the N-glycan structures in human int erferon gamma (IFN-gamma) derived from baculovirus-infected insect cel ls was performed using a combination of reverse-phase, high-performanc e liquid chromatography (rHPLC) and matrix assisted laser desorption t ime of flight (MALDI-TOF) mass spectrometry, IFN-gamma was produced in two cell lines, an Estigmena acrea-derived subclone (Ea4), and Spodop tera frugiperda cells (Sf9), Both IFN-gamma N-glycosylation sites (Asn (25) and Asn(97)) were characterized, Site-specific differences were o bserved in both the percentage of sites occupied by N-linked glycans a nd the types of structure associated with each site, The glycosylation capabilities and glycan processing of Sf9 were limited to the generat ion of chitobiose [GlcNAc(2)], truncated tri-mannose core [Man(3)GlcNA c(2)], or oligomannose structures, The glycosylation abilities of Ea4 cells were more extensive, producing IFN-gamma molecules incorporating oligosaccharides with GlcNAc and Gal residues on the outer arms (hybr id or complex type N-glycans), as well as oligomannose N-glycans, Inco rporation of an alpha 1-6 linked fucose residue (<70% in Sf9 and <88% in Ea4) was confined to the Asn(25) glycosylation site. These findings demonstrate the more extensive N-glycosylation capabilities of the E( 1) acrea-derived Ea4, compared to current insect cell lines used for t he expression of recombinant proteins.