Ion Mobility-Mass Spectrometry and Collision Induced Unfolding Reveal Linker-Payload Effects on Antibody-Drug...

Antibody-drug conjugates are a promising class of therapeutics that offer targeted cancer treatments with fewer side effects. Optimizing the mAb, linker, payload, conjugation site, and drug-to-antibody ratio (DAR) are crucial for developing ADC candidates. However, assessing the influence of each factor on the higher-order structure (HOS) of the ADC remains challenging due to their complexity and heterogeneity, and the development of high-throughput workflows capable of detecting subtle changes in structure and stability would be advantageous. Here, we used ion mobility-mass spectrometry (IM-MS) and collision induced unfolding (CIU) to investigate the impact of linker-payload (LP) structure and conjugation strategy on the HOS and stability of ADCs. Site-specific conjugation to engineered cysteines revealed that LPs with different structures differentially affect the gas-phase stability of the Fc domain, and a correlation between gas-phase stability (CIU₅₀) and thermal stability (T_m) of the ADC was observed. Conjugation at interchain cysteine residues disrupted the HOS of the ADC to a greater extent compared to site-specific conjugates, as evidenced by higher root-mean-square deviation values. CIU data revealed differences in conformation and stability in ADCs with different DARs and different positional isomers with the same DAR. These findings demonstrate the utility of IM-MS and CIU as a high-throughput, information-rich assay for probing the HOS and stability of ADCs during their discovery and development.