Investigation of Payloads and Related Impurities in the cysteine-linked Antibody-Drug Conjugate Using Mass Spectrometry Techniques.

Loncastuximab tesirine is a cysteine-linked antibody-drug conjugate composed of an anti-CD19 monoclonal antibody linked to the PBD dimer payload SG3249 via a protease-cleavable linker, enabling targeted release of a highly cytotoxic DNA cross-linking agent. NAC-SG3249, a thiol adduct formed with N-acetylcysteine, serves as an important indicator of payload release and ADC stability. In this study, mass spectrometry techniques were employed to systematically investigate the stability and related small-molecule impurities of this ADC. Quantitative analysis of the free payload showed good linearity over 0.05-100 ng/mL (R² ≥ 0.991), with an LLOQ of 0.05 ng/mL, accuracy of 80%-120%, and precision below 5%, and the results were consistent between HRMS and TQMS, both methods can achieve effective quantification. Potential payload-related impurities were further characterized based on predicted hydrolysis pathways using targeted MRM analysis. Forced degradation studies under acidic, alkaline, photolytic, and thermal conditions revealed multiple degradation products using a highly sensitive nanoLC-HRMS method, with alkaline conditions producing the greatest number of impurities. Clinical trial number: not applicable.