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.

Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of β(1–3)-linked galactose in glycans

“…Methods of partial filling, plug-plug, and serial plug analyses have been used with either a lectin[19–21] or with an exoglycosidase. [20–23] Combinations of lectins and enzymes in capillary electrophoresis have not been fully exploited. Stationary zones of enzymes with nanogels have recently been described that offer new benefits to in-line analyses including the ability to increase the duration of the substrate incubation with electrophoretic mixing and to extend the lifetime of highly specific exoglycosidases, some of which are costly.…”

Glycosylation analysis of Chinese hamster ovary produced glycoproteins

“…Automated highthroughput systems for CGE-LIF use a 96sample format in parallel that sig nificantly increases throughput of samples for routine analysis [91,93]. This combined with multiplexing with 96 capillaries has very high throughput [94]. Auto mated software analysis is linked to a database can be used for identification of glycans [91].…”

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

“…A partial injection of a NeuGc specific antibody has been used to detect minor oligosaccharides containing NeuGc in IgG pharmaceuticals. 163 Partial filling with α-galactosidase allows on-capillary hydrolysis, which enables detection of the presence of α-Galterminated glycans. The combination of these methods with large-volume sample stacking with an electroosmotic flow pump (LVSEP) enhances the sensitivity of the methods by a factor of 900 times.…”