Characterizing Antibody-Drug Conjugates with High Molecular Heterogeneity by Native Direct Mass Technology

Mass spectrometry (MS) has emerged as a powerful tool for characterizing antibody-drug conjugates (ADCs), enabling the determination of the drug-to-antibody ratio (DAR) and the monitoring of ADC degradation. Here we present a native direct mass technology (DMT) approach, an Orbitrap-based charge detection MS integrated with the SampleStream platform and Newomics' MnESI source, providing automated, highly sensitive analysis of DAR and stability for two interchain-modified high DAR ADCs (DAR 14) in native conditions. We applied this platform to samples from formulation buffer, as well as from ex vivo and in vivo conditions, where ADC concentrations were substantially lower. Owing to the pronounced heterogeneity of these DAR-14 ADCs, direct DAR characterization was only achievable using native DMT, underscoring the limitations of conventional native MS in studying heterogeneous ADCs. Furthermore, the interchain stability assessments from biomatrices demonstrate that native DMT delivers a robust, high-resolution method for characterizing interchain-modified high DAR ADCs across diverse biological environments.