Isolation and Characterization of an Insect Cell Line Able to Perform Complex N-Linked Glycosylation on Recombinant Proteins

Site specific characterization of the N-glycan structures in human interferon (IFN-) derived from baculovirus-infected insect cells was performed using a combination of reverse-phase, high-performance liquid chromatography (rHPLC) and matrix assisted laser desorption time of flight (MALDI-TOF) mass spectrometry. IFN- was produced in two cell lines, an Estigmena acmz-derived subclone (Ea4), and Spodopterafrugiperda cells (Sf9). Both IFN- N-glycosylation sites (Asn₂₅and Asn₉₇) were characterized. Site-specific differences were observed in both the percentage of sites occupied by N-linked glycans and the types of structure associated with each site. The glycosylation capabilities and glycan processing of Sf9 were limited to the generation of chitobiose [GlcNAc₂], truncated tri-mannose core [Man₃GlcNAc₂], or oligomannose structures. The glycosylation abilities of Ea4 cells were more extensive, producing IFN- molecules incorporating oligosaccharides with GlcNAc and Gal residues on the outer arms (hybrid or complex type N-glycans), as well as oligomannose N-glycans. Incorporation of an l−6 linked fucose residue (<70% in Sf9 and <88% in Ea4) was confined to the Asn₂₅ glycosylation site. These findings demonstrate the more extensive N-glycosylation capabilities of the E₁ acrea−derived Ea4, compared to current insect cell lines used for the expression of recombinant proteins.

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