CE methods for charge variant analysis of mAbs and complex format biotherapeutics

Abstract

AbstractCharge heterogeneity analysis of monoclonal antibodies (mAbs) and complex formats, such as bispecifics, is crucial for therapeutic applications. In this study, we developed two capillary electrophoresis (CE)‐based methods, capillary zone electrophoresis (CZE) and imaged capillary isoelectric focusing (iCIEF), for analyzing a broad spectrum of mAbs and complex mAb formats. For CZE, we introduced a new buffer system and optimized the background electrolyte (BGE) with an alternative dynamic coating agent and a superior polymeric additive. The pH of the BGE was increased, leading to enhanced resolution of high pI and complex format mAbs. In iCIEF, we identified an ampholyte combination offering a highly linear pH gradient and covering a suitable pH range. We also investigated alternatives to denaturing stabilizers and found that non‐detergent sulfobetaine 195 exhibited excellent properties for iCIEF applications. These optimized methods provide a framework for the charge heterogeneity analysis of therapeutic mAbs and complex formats.