A Separation Buffers Platform Set to Facilitate CZE Charge Heterogeneity Method Development for Monoclonal Antibodies

Monoclonal antibodies (mAbs) are complex therapeutic proteins exhibiting heterogeneity due to post-translational modifications, making charge variant analysis essential for defining critical quality attributes. Although ion-exchange chromatography and capillary isoelectric focusing are established techniques, they require extensive optimisation, whereas the widely adopted ε-aminocaproic acid-based capillary zone electrophoresis (CZE) (eACA-CZE) method provides a simpler, robust platform. However, its performance can be limited for mAbs whose charge profiles or isoelectric point (pI) values fall outside the method's optimal range. To expand CZE capabilities while maintaining a platform approach, we developed a series of background electrolytes (BGEs) spanning a range of pH values, polyamine concentrations and buffering capacities. These BGEs were formulated for chemical compatibility, long-term stability and fixed-component composition to ensure consistent pH and reproducible currents. Using a pH 5.7 BGE as the starting point, key components were optimised across the full buffer set. A multivariate approach showed that combining these buffers enhanced resolution, resolving additional impurity peaks in 9 of 10 mAbs. This work establishes a flexible toolkit for screening and optimising charge-variant resolution across diverse mAbs.

ICH Q14; analytical procedure control strategy; capillary zone electrophoresis; charge heterogeneity; critical materials; monoclonal antibody (mAb); ε‐aminocaproic acid (eACA)‐capillary zone electrophoresis (CZE).