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Application of partial‐filling capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in a therapeutic antibody
Author(s) -
Yagi Yuki,
Yamamoto Sachio,
Kakehi Kazuaki,
Hayakawa Takao,
Ohyama Yukihito,
Suzuki Shigeo
Publication year - 2011
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100126
Subject(s) - lectin , chemistry , capillary electrophoresis , electropherogram , antibody , glycosylation , agglutinin , chromatography , oligosaccharide , biochemistry , glycan , glycoprotein , biology , immunology
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, Galβ1–4GlcNAc‐specific Erythrina cristagali agglutinin, α1,6‐linked Fuc‐specific Aleuria aurantia lectin and Neu5Acα2–3Gal‐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.