Rapid Analysis of Reduced Antibody Drug Conjugate by Online LC-MS/MS with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
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Antibody drug conjugates (ADCs), which harness the high targeting specificity of monoclonal antibodies (mAb) with the potency of small molecule therapeutics, are one of the fastest growing pharmaceutical classes. Nevertheless, ADC conjugation techniques and processes may introduce intrinsic heterogeneity including primary sequence variants, varied drug-to-antibody ratio (DAR) species, and drug positional isomers, which must be monitored to ensure the safety and efficacy of ADCs. Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for characterization of ADCs. However, the conventional bottom-up MS analysis workflows require an enzymatic digestion step which can be time consuming and may introduce artifactual modifications. Herein, we develop an online LC-MS/MS method for rapid analysis of reduced ADCs without digestion, enabling determination of DAR, characterization of the primary sequence, and localization of the drug conjugation site of the ADC using high-resolution Fourier transform ion cyclotron resonance (FTICR) MS. Specifically, a model cysteine-linked ADC was reduced to generate six unique subunits: light chain (Lc) without drug (Lc0), Lc with 1 drug (Lc1), heavy chain (Hc) without drug (Hc0), and Hc with 1-3 drugs (Hc1-3, respectively). A concurrent reduction strategy is applied to assess ADC subunits in both the partially reduced (intrachain disulfide bonds remain intact) and fully reduced (all disulfide bonds are cleaved) forms. The entire procedure including the sample preparation and LC-MS/MS takes less than 55 min, enabling rapid multiattribute analysis of ADCs.
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Analysis of antibodies by top-down can quickly determine modification state, drug conjugate ratio, quality control screening, and more.
“For example, analysis of antibodies by top-down can quickly determine the modification state [81], drug conjugate ratio [161], quality control screening [162], and more [163,164].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [161]
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- Authors
- Larson, E. J., Zhu, Y., Wu, Z., Chen, B., Zhang, Z., Zhou, S., Han, L., Zhang, Q., & Ge, Y.
- Affiliations
- University of Wisconsin–Madison (US)
- AbbVie (United States) (US)
- Corresponding author
- Ge, Ying
- ORCID
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2020-10-27
- Volume
- 92
- Issue
- 22
- Pages
- 15096-15103
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC9463631
- OpenAlex
- W3095006435
- Cited by
- 21
- References
- 35
- Citations by year
- 2026: 2 · 2025: 7 · 2024: 5 · 2023: 2 · 2022: 2 · 2021: 2 · 2020: 1
- FWCI
- 0.67
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- AbbVie
- National Institute of General Medical Sciences (GM125085)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Immunology and Microbiology › Immunology › Biosimilars and Bioanalytical Methods
- Keywords
- antibody-drug conjugates, drug-to-antibody ratio, disulfide bond cleavage, monoclonal antibodies, FT-ICR mass spectrometry, LC-MS/MS
- MeSH terms
- Chromatography, Liquid, Fourier Analysis, Isomerism, Time Factors, Cyclotrons, Immunoconjugates, Tandem Mass Spectrometry
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