Antibody Drug Conjugate characterization by HRMS at intact, subunit & peptide levels

Antibody Drug Conjugates (ADCs) represent a rapidly emerging class of biotherapeutic molecules, providing new treatments for a variety of disease classes, including heart disease, arthritis and several types of cancer. ADCs employ the specificity of monoclonal antibodies (mAbs) to selectively deliver highly potent (cytotoxic) drugs. ADCs have been approved for the treatment of various types of cancer, and they continue to be an area of active research and development in the field of oncology.

Similar to all biotherapeutics ADCs require comprehensive analytical characterization since they can be highly complex chemical structures. Liquid chromatography coupled with high resolution mass spectrometry (LC–HRMS) analysis has been widely utilized for protein therapeutic development and characterization. Complementary HRMS-based approaches of peptide mapping and intact mass analysis are needed to facilitate complete characterization of ADCs. Besides, characterizing the Drug-to-Antibody-Ratio (DAR) is a critical quality attribute (CQA) for ADCs since it is pivotal to understand ADC efficacy and its process control across the ADC development and manufacturing process.

In this case study, a commercially available ADC mimic, existing of adalimumab conjugated with 0-4 pairs of dansylcadaverine-SMCC mimic linkers, is characterized at intact, subunit and peptide levels. The ADCs that are characterized here are conjugated at Cys residues of inter-chain disulfide bonds and impart a +667.3481 Da mass shift. A DAR of 4.1 could be experimentally defined at intact protein level. By providing site-specific identification and quantitation of drug conjugates in biotherapeutics, LC-HRMS is one of the most sensitive and powerful technologies to monitor and define the required quality attributes.