Antigen affinity chromatography for functional separation and efficient characterization of critical monoclonal...

Monoclonal antibodies (mAbs) are prone to post-translational modifications during manufacturing and storage, which can affect product quality. Modifications within the complementarity-determining regions (CDRs) impairing antigen interaction are considered as critical quality attributes. A modular workflow was developed using analytical antigen affinity chromatography to enable functional separation of mAb variants based on their antigen affinity. The workflow was optimized using trastuzumab and its antigen, human epidermal growth factor receptor-2 (Her2). A linear pH gradient (pH 7.4 - 2.5) was employed to separate binding-deficient variants, which were fractionated and characterized using surface plasmon resonance (SPR), size exclusion chromatography (SEC), ion exchange chromatography (IEC), reducing capillary gel electrophoresis (CGE), and peptide mapping. Critical modifications, such as HC-D102 isomerization and LCN30 succinimide (Asu) formation, were identified as contributors to binding loss, with HC-D102 isomerization showing the most significant impact. Binding was reduced by up to 6 % in deficient fractions. The workflow was applied to two additional mAbs, demonstrating its adaptability. For mAb1 variants, binding activity decreased 10 % with HCCDR isomerization and Asu formation being critical, while for mAb2, distinct variants with binding activity ranging from 2 % to 66 % were resolved. In mAb2, Asu formation in HCCDR and LCCDR was identified as the primary modification contributing to binding loss and was found to be irreversible under physiological-like conditions (37 °C, pH 7.4). This approach enables the targeted identification of critical modifications, supports early risk mitigation as well as control strategies during development, and ensures comprehensive characterization of mAb variants.