Assessing the Impact of CDR Deamidation and Isomerization on mAb Target Binding Using Affinity-Resolved 2D-SEC-SCX-MS...

Post-translational modifications (PTMs) within the Complementarity-Determining Regions (CDRs) of monoclonal antibodies (mAbs) can impact their target-binding capabilities, making them potential critical quality attributes (CQAs) during therapeutic mAb development. Conventional methods for assessing PTM criticality often involve variant enrichment followed by target-binding testing, which face limitations in throughput and complexity. To address these challenges, affinity enrichment-based strategies have emerged, offering a valuable alternative for PTM assessment. Notably, the combination of competitive binding, size exclusion chromatography (SEC) separation, and MS detection, has proven highly effective in assessing the criticality of PTMs in a multiplexed fashion. Recently, we introduced a new technique termed affinity-resolved SEC-MS, which employs SEC to separate free and target-bound mAbs, followed by postcolumn denaturation (PCD)-assisted intact mass measurements. While highly effective in interrogating PTMs associated with significant mass shifts, this technique is less suitable for studying PTMs with subtle mass changes, such as asparagine (Asn) deamidation and aspartic acid (Asp) isomerization - two prevalent and often critical PTMs found in therapeutic mAbs. To overcome this limitation, we combined affinity-resolved SEC separation with online strong cation exchange chromatography-MS (SCX-MS) analysis. This 2D-LC-MS approach leverages the excellent selectivity of SCX separation for mAb CDR modifications at the Fab fragment level, enabling effective evaluation of Asn deamidation and Asp isomerization with site-specific resolution. The utility of this new approach was demonstrated through two case studies: examining a single Asn deamidation in an in-house mAb and assessing multiple site-specific Asn deamidation and Asp isomerization in trastuzumab, all occurring within the CDRs. Additionally, we detailed a quantitative approach to estimate the relative fold change in dissociation constant (K_D) for antigen-antibody interactions resulting from each individual CDR modification.