Characterization of A Novel Antibody-Drug Conjugate Mimic by Several Modes of Chromatography

Anders Fridstrom, Advanced Analytical, Merck, Stockholm, Sweden
Kevin Ray, Analytical R&d, Millipore-sigma, St Louis, USA
Dave Bell, Analytical Separations R&d , Millipore-sigma, Bellefonte, USA
Cory E., Analytical Separations R&d , Millipore-sigma, Bellefonte, USA

Characterization of the payload drug distribution of an antibody-drug conjugate (ADC) is paramount to understanding efficacy, pharmacokinetics, process control, and to obtaining regulatory approval. However, a significant roadblock to the development of ADC analysis methods is their hazardous nature.

In this study, a non-toxic ADC-mimic with highly similar physicochemical properties to commercial cysteine-conjugated ADCs is characterized using all major separation modes.  The intact ADC-mimic was used to develop native SEC-MS conditions.  The reduced mimic’s heavy and light chains, as well as its IdeS-proteolysis fragments, were used to develop subunit characterization workflows based on denaturing SEC-MS and reversed-phase LC-MS methods. In addition Drug Antibody Ratios was determined using HIC-UV and charge variants analyzed using Ion Exchange Chromatography.

Collectively, these methods enable orthogonal characterization of antibody drug conjugates.