ArticleAnalytical chemistry2019
Middle-Down Multi-Attribute Analysis of Antibody-Drug Conjugates with Electron Transfer Dissociation.
Article in Analytical chemistry, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 20 papers.
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The trial behind it
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Who cites it
20 citing papers in PubMed, 36 citations in OpenAlex.
- Advancements in Dual-Load Antibody-Drug Conjugates and Challenges with Quality Analysis.Pharmaceuticals (Basel, Switzerland) · 2026Review
- New Frontiers in ADC Analysis: Advances in Chromatography, Electrophoresis, and Mass Spectrometry.The AAPS journal · 2025Review
- Top-Down and Middle-Down Mass Spectrometry of Antibodies.Molecular & cellular proteomics : MCP · 2025Review
- Enhanced Payload Localization in Antibody-Drug Conjugates Using a Middle-Down Mass Spectrometry Approach with Proton Transfer Charge Reduction.Analytical chemistry · 2024Article
- Towards a universal method for middle-down analysis of antibodies via proton transfer charge reduction-Orbitrap mass spectrometry.Analytical and bioanalytical chemistry · 2024Article
- Enhanced Characterization of Lysine-Linked Antibody Drug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy Collisional Dissociation-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation.Antibodies (Basel, Switzerland) · 2024Article
- Internal Fragments Enhance Middle-Down Mass Spectrometry Structural Characterization of Monoclonal Antibodies and Antibody-Drug Conjugates.Analytical chemistry · 2024Article
- Direct Comparison of the Hinge-Cleaving Proteases IgdE and BdpK for LC-MS-Based IgG1 Clonal Profiling.Analytical chemistry · 2024Article
- Improved characterization of trastuzumab deruxtecan with PTCR and internal fragments implemented in middle-down MS workflows.Analytical and bioanalytical chemistry · 2024Article
- Top-down proteomics.Nature reviews. Methods primers · 2024Article
- Mass spectrometry characterization of antibodies at the intact and subunit levels: from targeted to large-scale analysis.International journal of mass spectrometry · 2023Article
- Added Value of Internal Fragments for Top-Down Mass Spectrometry of Intact Monoclonal Antibodies and Antibody-Drug Conjugates.Analytical chemistry · 2023Article
- Analytical comparability to evaluate impact of manufacturing changes of ARX788, an Anti-HER2 ADC in late-stage clinical development.PloS one · 2023Article
- Development of a comprehensive approach for performance evaluation of a quantitative multi-attribute method as a quality control method.Analytical sciences : the international journal of the Japan Society for Analytical Chemistry · 2022Article
- High-Throughput Multi-attribute Analysis of Antibody-Drug Conjugates Enabled by Trapped Ion Mobility Spectrometry and Top-Down Mass Spectrometry.Analytical chemistry · 2021Article
- Rapid Analysis of Reduced Antibody Drug Conjugate by Online LC-MS/MS with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.Analytical chemistry · 2020Article
- MASH Explorer: A Universal Software Environment for Top-Down Proteomics.Journal of proteome research · 2020Article
- Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.Journal of the American Society for Mass Spectrometry · 2020Article
- Ion Activation Methods for Peptides and Proteins.Analytical chemistry · 2020Review
- Recent advances in mass spectrometry based clinical proteomics: applications to cancer research.Clinical proteomics · 2020Review
Corrections and comments
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Authors and funding
11 authors at 2 institutions in 1 country.
Funding
Abstract
Antibody-drug conjugates (ADCs) are designed to combine the target specificity of monoclonal antibodies and potent cytotoxin drugs to achieve better therapeutic outcomes. 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 coexisting 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 microvariants 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 accurately measured 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 microvariants, which potentially opens up a new avenue to characterize ADCs.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.