An online four-dimensional HICxSEC-IMxMS methodology for in-depth characterization of antibody drug conjugates

Antibody Drug Conjugates (ADCs) are tripartite molecules consisting of a monoclonal antibody (mAb) onto which highly cytotoxic small molecules are conjugated by cleavable or non-cleavable link- ers. They show better efficiency than canonical unconjugated mAbs, due to the synergic effect of mAb specificity for its target and the efficacy of the highly cytotoxic drug .There are currently two main techniques allowing the analytical characterization of cysteine linked anti- body drug conjugates under non denaturing conditions, namely hydrophobic interaction chromatography (HIC) and native high resolution mass spectrometry.HIC is a chromatographic technique allowing the evaluation of drug load profile and calculation of av- erage drug to antibody ratio (DAR). High resolution mass spectrometry (MS) offers a wealth of information on the biochemical and biophysical properties of ADCs, thanks to accurate mass measurement. On-line coupling of both techniques can potentially be of great interest, but the presence of large amounts of non-volatile salts in HIC mobile phases make them non compatible with MS.Here, we present an innovative multidimensional analytical approach combining comprehensive on-line two dimensional chromatography (HICxSEC) to ion mobility and mass spectrometry (IM-MS) for per- forming analytical characterization of ADCs under non-denaturing conditions. Online hyphenation of non-denaturing 2D chromatography to 2D IM-MS enabled comprehensive and streamlined characteriza- tion of both native and stressed ADC samples.