Rapid and Simultaneous Characterization of Drug Conjugation in Heavy and Light Chains of a Monoclonal Antibody Revealed by High-Resolution Ion Mobility Separations in SLIM

Antibody-drug conjugates (ADCs) have recently gained traction in the biomedical community due to their promise for human therapeutics and an alternative to chemotherapy for cancer. Crucial metrics for ADC efficacy, safety, and selectivity are their drug-antibody ratios (DARs). However, DAR characterization (i.e., determining the average number of conjugated drugs on the antibody) through analytical methods remains challenging due to the heterogeneity of drug conjugation as well as the numerous post-translational modifications possible in the monoclonal antibody. Herein, we report on the use of high-resolution ion mobility spectrometry separations in structures for lossless ion manipulations coupled to mass spectrometry (SLIM IMS-MS) for the rapid and simultaneous characterization of the drug load profile (i.e., stoichiometric distribution of the number of conjugated drugs present on the mAb), determination of the weighted average DAR in both the heavy and light chains of a model antibody-drug conjugate, and calculation of the overall DAR of the ADC. After chemical reduction of the ADC and a subsequent 31.5 m SLIM IMS separation, the various drug-bound antibody species could be well resolved for both chains. We also show significantly higher resolution separations were possible for these large ions with SLIM IMS as compared to ones performed on a commercially available (1 m) drift tube IMS-MS platform. We expect high-resolution SLIM IMS separations will augment the existing toolbox for ADC characterization, particularly to enable the rapid optimization of DAR for a given ADC and thus better understand its potential toxicity and potency.