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