Conjugation site characterization of antibody-drug conjugates using electron-transfer/higher-energy collision dissociation (EThcD)

Antibody-drug conjugates (ADCs) are formed by binding of cytotoxic drugs to monoclonal antibodies (mAbs) through chemical linkers. A comprehensive evaluation of the critical quality attributes (CQAs) of ADCs is vital for drug development but remains challenging owing to ADC structural heterogeneity than mAbs. Drug conjugation sites can considerably affect ADC properties, such as stability and pharmacokinetics, however, few studies have focused on method development in this area owing to technical challenges. Hybrid electron-transfer/higher-energy collision dissociation (EThcD) produces more fragment ions than conventional higher-energy collision dissociation (HCD) fragmentation, which aids in identifying and localizing post-translational modifications. Herein, we systematically employ EThcD to assess the fragmentation mode impact on conjugation site charac-terization for randomly conjugated and site-specific ADCs. EThcD generates more fragment ions in tandem mass spectrometry (MS/MS) spectra compared with HCD. Additional ions aid in pinpointing the correct conjugation sites that bear complex linker payload structures. Our study may contribute to the quality control of various preclinical and clinical ADCs.

Automated summary

Antibody-drug conjugates (ADCs) are crucial for drug development but assessing their quality attributes remains challenging due to structural heterogeneity. This study focuses on the impact of fragmentation mode on conjugation site characterization using hybrid EThcD. Results show that EThcD generates more fragment ions compared to conventional HCD, aiding in pinpointing correct conjugation sites with complex linker payloads. This study provides valuable insights for quality control of ADCs in preclinical and clinical settings.