title: Application of partial-filling capillary  electrophoresis using lectins and  glycosidases for the characterization of  oligosaccharides in a therapeutic antibody.
abstract: Oligosaccharides in therapeutic recombinant antibodies play important roles in regulation of various biological functions. To monitor the glycosylation profiles of antibody pharmaceuticals in the manufacturing process, a highly sensitive and specific method is required. We extended partial-filling techniques using lectins and exoglycosidases in capillary electrophoresis for the characterization of 8-aminopylene-1,3,6-trisulfonic acid labeled N-linked oligosaccharides derived from the therapeutic antibody rituximab. In the lectin-filling method, Galb1–4GlcNAc-specific Erythrina cristagali agglutinin, a1, 6-linked Fuc-specific Aleuria aurantia lectin and Neu5Aca2–3Gal-specific Maackia amurensis lectin were used. The oligosaccharides migrated through the lectin plug during separation; the changes in separation profiles were observed according to the interaction with the lectins. The glycosidase-filling method allowed rapid digestion as suggested by the electropherograms. Partial-filling CE methods can avoid tedious hands-on procedures such as overnight incubation and optimization reaction condition with lectins and exoglycosidases. Combination of these partial-filling capillary electrophoresis methods makes the characterization of oligosaccharide profiles of therapeutic antibodies easier and faster.
authors.affiliation: Kyowa Hakko Kirin Co., Ltd., Takasaki, Japan. yuuki.yagi@kyowa-kirin.co.jp
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yagi
authors.forename: Sachio
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Kakehi
authors.forename: Takao
authors.initials: T
authors.lastname: Hayakawa
authors.forename: Yukihito
authors.initials: Y
authors.lastname: Ohyama
authors.forename: Shigeo
authors.initials: S
authors.lastname: Suzuki
mesh_terms: D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis*; D004364:Pharmaceutical Preparations / Q000737:chemistry* / Q000592:standards; D011721:Pyrenes / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D000069283:Rituximab; D013997:Time Factors
chemical_list: C103430:8-aminopyrene-1,3,6-trisulfonic acid; D058846:Antibodies, Monoclonal, Murine-Derived; D006023:Glycoproteins; D037102:Lectins; D009844:Oligosaccharides; D004364:Pharmaceutical Preparations; D011721:Pyrenes; D011994:Recombinant Proteins; D000069283:Rituximab; D006026:Glycoside Hydrolases