A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate Using Middle-Down Mass Spectrometry
| Autor: | Jonathan L. Josephs, Steve Hessmann, Penelope M. Drake, Stéphane Erb, Alain Beck, Stephane Houel, Sarah Cianférani, Romain Huguet, Oscar Hernandez-Alba, David Rabuka |
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| Přispěvatelé: | Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), ThermoFisher Scientific, Thermofisher Scientific, Catalent Biologics West, Centre d'Immunologie Pierre Fabre |
| Rok vydání: | 2019 |
| Předmět: |
Drug
Glycosylation Immunoconjugates medicine.drug_class media_common.quotation_subject Computational biology Mass spectrometry Proteomics Monoclonal antibody 01 natural sciences Peptide Mapping Mass Spectrometry 03 medical and health sciences chemistry.chemical_compound Structural Biology medicine [CHIM]Chemical Sciences Humans Peptide sequence Spectroscopy 030304 developmental biology media_common 0303 health sciences Binding Sites 010401 analytical chemistry 0104 chemical sciences A-site chemistry Conjugate |
| Zdroj: | Journal of The American Society for Mass Spectrometry Journal of The American Society for Mass Spectrometry, Springer Verlag (Germany), 2019, 30 (11), ⟨10.1007/s13361-019-02296-2⟩ |
| ISSN: | 1879-1123 1044-0305 |
| Popis: | International audience; Middle-down mass spectrometry (MD MS) has emerged as a promising alternative to classical bottom-up approaches for protein characterization. Middle-level experiments after enzymatic digestion are routinely used for subunit analysis of monoclonal antibody (mAb)-related compounds, providing information on drug load distribution and average drug-to-antibody ratio (DAR). However, peptide mapping is still the gold standard for primary amino acid sequence assessment, post-translational modifications (PTM), and drug conjugation identification and localization. However, peptide mapping strategies can be challenging when dealing with more complex and heterogeneous mAb formats, like antibody-drug conjugates (ADCs). We report here, for the first time, MD MS analysis of a third-generation site-specific DAR4 ADC using different fragmentation techniques, including higher-energy collisional- (HCD), electron-transfer (ETD) dissociation and 213 nm ultraviolet photodissociation (UVPD). UVPD used as a standalone technique for ADC subunit analysis afforded, within the same liquid chromatography-MS/MS run, enhanced performance in terms of primary sequence coverage compared to HCD- or ETD-based MD approaches, and generated substantially more MS/MS fragments containing either drug conjugation or glycosylation site information, leading to confident drug/glycosylation site identification. In addition, our results highlight the complementarity of ETD and UVPD for both primary sequence validation and drug conjugation/glycosylation site assessment. Altogether, our results highlight the potential of UVPD for ADC MD MS analysis for drug conjugation/glycosylation site assessment, and indicate that MD MS strategies can improve structural characterization of empowered next-generation mAb-based formats, especially for PTMs and drug conjugation sites validation. |
| Databáze: | OpenAIRE |
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