Electrophoresis, Vol.32, No.21, 2979-2985, 2011
Application of partial-filling capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in a therapeutic antibody
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, Gal beta 1-4GlcNAc-specific Erythrina cristagali agglutinin, alpha 1, 6-linked Fuc-specific Aleuria aurantia lectin and Neu5Ac alpha 2-3Gal-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.
Keywords:Glycosidases;Immunoglobulin;Lectins;Oligosaccharides;Partial-filling affinity electrophoresis