Conjugation Location-Dependent Stability Shifts in Antibody Drug Conjugate Mimics Revealed by Collision Induced Unfolding

<jats:p>Antibody-drug conjugates (ADCs) represent an emerging class of biotherapeutics that allow the site-specific delivery of bioactive agents. However, ADC production often results in several proteoforms, producing variations in drug to antibody ratio (DAR) and site of conjugation. Current analytical tools for stability and higher-order structures (HOS) analysis of mAbs often evaluate all the variants of ADC present in the sample. This provides limited information on how subtle variations in either the level or site of conjugation can impact mAb HOS and structure. Native ion mobility-mass spectrometry (IMMS) and collision induced unfolding (CIU) have been established as a valuable structural biology tool to examine changes in mAb stability and HOS under various conditions. In this report, we developed a cIM-MS method that allowed for DAR resolved structural analysis of two model ADC systems. We evaluated two ADC mimics that differ in the location of conjugation: CH2 and Fab conjugation. Native collision cross section (TWΩN2) analysis of DAR 2 revealed ~4% increase for Fab conjugated ions, but no significant changes were observed after conjugation at the CH2 domain. Quantitative CIU analysis revealed conjugation in the CH2 domain resulted in stability shifts in higher-energy CIU transitions, while conjugation in the Fab region resulted in stability shifts in transitions observed at lower energies. Middle-level CIU analysis of the Fab fragment revealed conjugation at the N-terminus (VL and VH) impacted to a greater extent (~20% shift) lower energy transitions. CIU analysis after conjugation at the CH2 resulted in altered unfolding pathways, displaying different CIU features. These results demonstrate how CIU can be used to monitor stability changes in location-dependent manner within ADCs. We conclude by discussing the future potential benefits of region-resolved stability assessments on ADC discovery and development alongside future plans for improving CIU methods.</jats:p>