In-depth characterization of lysine-conjugated antibody-drug conjugates (ADCs) by a multiplexed MS/MS data acquisition strategy combined with multi-enzyme digestion

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Liuxi Chen, Henry Shion, Ying Qing, Weibin Chen
Waters Corporaton
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The reactivity of primary amines and their compositional availability in monoclonal antibodies (mAbs) make them a popular target for chemical conjugation in antibody-drug conjugates (ADCs). However, conjugation reactions of lysine residues result in highly heterogeneous mixtures with drugs in many combinations at different lysine sites on the mAb. Hence, the structural complexity and intrinsic heterogeneity of lysine-conjugated ADCs impose a prominent analytical challenge to current characterization methods.

In this study, we present an integrated approach that combines multiplexed MS/MS data acquisition strategy with multi enzyme digestion for the in-depth characterization of lysine-conjugated ADCs. Both data-independent acquisition and data-dependent acquisition (DDA) methods were used to identify the lysine-conjugated peptides, confirm the conjugation sites and determine the drug occupancy ratio.

The peptide digest mixtures were analyzed by a Xevo G2-XS QTof Mass Spectrometer coupled with an ACQUITY UPLC H-Class Bio System. The acquired data were processed using UNIFI 1.8 Software, in which the conjugation sites were confirmed and the drug occupancy ratio was calculated automatically. A detailed description of the identification of conjugation sites and comparisons between different samples is presented.

The multiplexed MS/MS data acquisition strategy in combination with multi-enzyme digestion provides an extensive characterization of lysine-conjugated ADCs, which serves as a vital tool for ADC research and development.