High-Performance Antigen Affinity Chromatography of Monoclonal Antibodies in One- and Multidimensional Configurations

Abstract The development of monoclonal antibody (mAb) therapeutics requires a thorough understanding of their structure and function. Current target binding assays do not enable direct analysis of the structural composition of the analytes, therefore requiring additional analytics to study the root-cause of impaired drug potency. Here, high-performance analytical affinity chromatography (HPAC) is established as an effective functional separation tool for resolving mAb proteoforms based on interaction strength with their antigen. When coupled to mass spectrometry (MS), HPAC enables simultaneous measurement of target binding affinity and structural attributes, facilitating identification of critical quality attributes (CQAs). Integration of HPAC into multidimensional liquid chromatography (mD-LC) workflows further expands its utility, providing automated multi-attribute analysis within a unified workflow. Trastuzumab was selected as a representative system due to its well-characterized, high-affinity binding to the human epidermal growth factor receptor 2 (HER2). Narrow-bore HPAC columns (2.1 × 20 mm; 3.5 μm) with immobilized HER2 were developed and evaluated under various chromatographic conditions. The analytical performance of the method was assessed using temperature-stressed trastuzumab, exhibiting reduced potency due to aspartate isomerization in heavy chain complementarity determining region 3 (CDR3). The feasibility of one-dimensional (1D) HER2-HPAC-MS for intact- and Fab-level functional profiling of mAb proteoforms is first demonstrated. Building on this capability, a two-dimensional (2D) workflow coupling cation exchange chromatography (CEX) to HPAC enables automated functional characterization of charge variants. Finally, incorporation of HPAC into an online mD-LC−MS workflow supports fully automated middle-up and bottom-up analyses, linking affinity differences directly to unambiguous identification of post-translational modifications (PTMs).