title: Elucidation of N-linked oligosaccharide structures of recombinant human factor VIII using fluorophore-assisted carbohydrate electrophoresis.
abstract: Characterization of the carbohydrate moiety is a critical measure of manufacturing process consistency of recombinant human Factor VIII (rFVIII) in Chinese-hamster ovary (CHO) cells. FVIII, a large (300 kDa) glycoprotein, is employed therapeutically for the correction of haemophilia A. While N-linked and O-linked oligosaccharides are found in this protein, the current study focuses on the N-linked oligosaccharides. The N-linked oligosaccharides from rFVIII were released using either peptide N-glycosidase F or endoglycosidase H, derivatized with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonate, and analysed by fluorophore-assisted carbohydrate electrophoresis (FACE). The electrophoretically resolved oligosaccharide bands were isolated and individual bands subjected to digestion with defined pools of exoglycosidases and re-electrophoresed on FACE sequencing gels. The resulting gel patterns were interpreted, based on band mobility shifts, to obtain the sequence structure of the oligosaccharides. A total of eight acidic and 12 neutral structures were identified, and the majority of the oligosaccharides (approximately 92%) were found to be sialylated. All of the major oligosaccharide structures found in CHO-cell-derived rFVIII have also been reported to be present in plasma-derived FVIII. Among the most abundant are disialylated, biantennary, core-fucosylated (approximately 40%), followed by trisialylated, triantennary, core-fucosylated and monosialo, biantennary, core-fucosylated structures (each approximately 18%). The Gal alpha 1-3Gal structures reported to be present in baby-hamster-kidney-cell-derived rFVIII were not found in the CHO-cell-derived protein. The glycosylation patterns were consistent in six random lots of rFVIII [coefficient of variation (%) 3-14] based on percentage lane luminance data of bands that represent approximately 98% of all asparagine-linked oligosaccharides.
authors.affiliation: Baxter Biotech, Hyland R&D Division, Duarte, CA 91010, USA.
authors.forename: H P
authors.initials: HP
authors.lastname: Kumar
authors.forename: C
authors.initials: C
authors.lastname: Hague
authors.forename: T
authors.initials: T
authors.lastname: Haley
authors.forename: C M
authors.initials: CM
authors.lastname: Starr
authors.forename: M J
authors.initials: MJ
authors.lastname: Besman
authors.forename: R L
authors.initials: RL
authors.lastname: Lundblad
authors.forename: D
authors.initials: D
authors.lastname: Baker
mesh_terms: D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006224:Cricetinae; D004586:Electrophoresis / Q000379:methods*; D005169:Factor VIII / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes; D006596:Hexosaminidases; D006801:Humans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D009844:Oligosaccharides; D011994:Recombinant Proteins; D005169:Factor VIII; D006596:Hexosaminidases