Conjugation Site Analysis of Lysine-Conjugated ADCs

Hua Sang, Ning Wan, Gaoyuan Lu, Yang Tian, Guangji Wang, Hui Ye*

Abstract: Lysine-conjugated antibody-drug conjugates (ADCs) are formed by attaching cytotoxic drugs to reactive lysine residues of monoclonal antibodies (mAbs) through chemical linkers. During production, the payloads are conjugated nonspecifically to lysine residues in mAbs, resulting in a heterogeneous mixture of ADCs with both different number and conjugation sites of drug payloads per mAb. On account of the drug conjugation sites and levels that both have significant influences on physical and pharmaceutical properties of ADCs, a reliable and straightforward approach for conjugation site analysis for ADCs is highly demanded. Herein, we used a lysine-conjugated ADC, Trastuzumab-MCC-DM1 (T-DM1), as a model ADC, and described an integrative strategy that combines the signature ion fingerprinting method for rapid and reliable filtering of DM1-conjugated peptides, and the normalized area quantitation approach for accurately gauging the conjugation levels for each identified site. This approach is believed to be readily applicable to other maytansinoid derivatives-modified ADCs, and more importantly, universally applicable to lysine-conjugated ADCs for both the recognition of conjugation sites and the measurement of conjugation levels.