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J Am Soc Mass Spectrom. 2019 Aug 19. doi: 10.1007/s13361-019-02296-2. [Epub ahead of print]

A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate Using Middle-Down Mass Spectrometry.

Author information

1
Laboratoire de Spectrométrie de Masse BioOrganique, CNRS IPHC UMR 7178, Université de Strasbourg, ECPM R5-0 - 25 Rue Becquerel, Cedex 2, 67087, Strasbourg, France.
2
Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA, 95134, USA.
3
Catalent Biologics West, 5703 Hollis Street, Emeryville, CA, 94530, USA.
4
IRPF, Centre d'Immunologie Pierre-Fabre (CIPF), Saint-Julien-en-Genevois, France.
5
Laboratoire de Spectrométrie de Masse BioOrganique, CNRS IPHC UMR 7178, Université de Strasbourg, ECPM R5-0 - 25 Rue Becquerel, Cedex 2, 67087, Strasbourg, France. sarah.cianferani@unistra.fr.

Abstract

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.

KEYWORDS:

Antibody-drug conjugate (ADC); ETD; HCD; Middle-down mass spectrometry (MD MS); Site-specific bioconjugation; UVPD fragmentation

PMID:
31429052
DOI:
10.1007/s13361-019-02296-2

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