Analysis of Identity, Charge Variants, and Disulfide Isomers of Monoclonal Antibodies with Capillary Zone Electrophoresis in an Uncoated Capillary Column

Abstract

A set of related capillary zone electrophoresis (CZE) methods have been developed for the analysis of identity, charge variants, and disulfide isoforms of IgG monoclonal antibodies (mAbs). These methods utilize an uncoated capillary column. The combined use of concentrated zwitterionic (e-amino-caproic acid) buffer and acid flushing was effective in minimizing the adsorption of protein to the inner wall of a bare capillary. Under these conditions, a selective and reproducible separation of multiple IgG1 and IgG2 monoclonal antibodies (mAbs) was obtained with a long capillary column (40 cm effective length), allowing the reliable identification of different mAbs by migration time. A rapid ( approximately 10 min) and selective separation of charged variants of IgG mAbs was attained using a short capillary column (10 cm effective length). Finally, the addition of urea in the separation buffer resulted in the separation of disulfide isoforms of IgG2 mAbs by CZE. CZE methods using an uncoated capillary column offer a versatile, generic, and economical approach to the evaluation of identity, charge heterogeneity, and disulfide isoforms of IgG antibodies.