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Elucidation of N‐linked oligosaccharide structures of recombinant human factor VIII using fluorophore‐assisted carbohydrate electrophoresis
Author(s) -
HP Kumar,
Hague C.,
Haley T.,
CM Starr,
MJ Besman,
RL Lundblad,
Baker D.
Publication year - 1996
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1996.tb00399.x
Subject(s) - oligosaccharide , chinese hamster ovary cell , chemistry , endoglycosidase , glycoprotein , biochemistry , glycosylation , exoglycosidase , recombinant dna , carbohydrate conformation , glycan , polysaccharide , gene , receptor
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.