1. Middle-Down Multi-Attribute Analysis of Antibody-Drug Conjugates with Electron Transfer Dissociation
- Author
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Ziqing Lin, Cexiong Fu, Wayne A. Pritts, Eli J. Larson, Yutong Jin, Zhaorui Zhang, Ying Ge, Bifan Chen, Yanlong Zhu, Qingge Xu, and Qunying Zhang
- Subjects
Brentuximab Vedotin ,Chromatography, Reverse-Phase ,Immunoconjugates ,Chemistry ,Stereochemistry ,Lysine ,Protein subunit ,010401 analytical chemistry ,Conjugated system ,Ado-Trastuzumab Emtansine ,010402 general chemistry ,Mass spectrometry ,Immunoglobulin light chain ,01 natural sciences ,Article ,0104 chemical sciences ,Analytical Chemistry ,Electron Transport ,Electron-transfer dissociation ,Drug development ,Tandem Mass Spectrometry ,Cysteine ,Conjugate - Abstract
Antibody-drug conjugates (ADCs) are designed to combine the target specificity of monoclonal antibodies and potent cytotoxin drugs to achieve better therapeutic outcome. Comprehensive evaluation of the quality attributes of ADCs is critical for drug development but remains challenging due to heterogeneity of the construct. Currently, peptide mapping with reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the predominant approach to characterize ADCs. However, it is suboptimal for sequence characterization and quantification of ADCs because it lacks a comprehensive view of co-existing variants and suffers from varying ionization effects of drug-conjugated peptides compared to unconjugated counterparts. Here, we present the first middle-down RPLC-MS analysis of both cysteine (Adcetris®; BV) and lysine (Kadcyla®; T-DM1) conjugated ADCs at the subunit level (~25 kDa) with electron transfer dissociation (ETD). We successfully achieved high-resolution separation of subunit isomers arising from different drug conjugation and subsequently localized the conjugation sites. Moreover, we obtained a comprehensive overview of the micro-variants associated with each subunits and characterized them such as oxidized variants with different sites. Furthermore, we observed relatively high levels of conjugation near complementarity-determining regions (CDRs) from the heavy chain but no drug conjugation near CDRs of light chain (Lc) from lysine conjugated T-DM1. Based on the extracted ion chromatograms, we measured accurate average drug to antibody ratio (DAR) values and relative occupancy of drug-conjugated subunits. Overall, the middle-down MS approach enables the evaluation of multiple quality attributes including DAR, positional isomers, conjugation sites, occupancy, and micro-variants, which potentially open up a new avenue to characterize ADCs.
- Published
- 2019