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

  • Oscar Hernandez-Alba
  • Stéphane Houel
  • Steve Hessmann
  • Stéphane Erb
  • David Rabuka
  • Romain Huguet
  • Jonathan Josephs
  • Alain Beck
  • Penelope M. Drake
  • Sarah CianféraniEmail author
Research Article


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.


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



Antibody-drug conjugate


Complementarity-determining region


Critical quality attribute




Drug-to-antibody ratio


Average drug-to-antibody ratio


Drug load distribution




Electron transfer dissociation


Fragment crystallizable




Higher-energy collisional dissociation


Immunoglobulin-degrading enzyme from Streptococcus pyogenes


Immunoglobulin G


Light chain


Liquid chromatography-tandem mass spectrometry


Monoclonal antibody




Mass spectrometry


Post-translational modification


Small molecule drug




Ultraviolet photodissociation



The authors would like to thank Région Alsace for financial support in purchasing an Orbitrap Exactive Plus EMR instrument, the CNRS, the University of Strasbourg, the “Agence Nationale de la Recherche” (ANR) and the French Proteomic Infrastructure (ProFI; ANR-10-INBS-08-03). O.A-H acknowledges the IdeX program of the University of Strasbourg for funding his postdoctoral fellowship.

Supplementary material

13361_2019_2296_MOESM1_ESM.docx (1.9 mb)
ESM 1 (DOCX 1954 kb)


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Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  • Oscar Hernandez-Alba
    • 1
  • Stéphane Houel
    • 2
  • Steve Hessmann
    • 1
  • Stéphane Erb
    • 1
  • David Rabuka
    • 3
  • Romain Huguet
    • 2
  • Jonathan Josephs
    • 2
  • Alain Beck
    • 4
  • Penelope M. Drake
    • 3
  • Sarah Cianférani
    • 1
    Email author
  1. 1.Laboratoire de Spectrométrie de Masse BioOrganique, CNRS IPHC UMR 7178Université de StrasbourgStrasbourgFrance
  2. 2.Thermo Fisher ScientificSan JoseUSA
  3. 3.Catalent Biologics WestCAUSA
  4. 4.IRPFCentre d’Immunologie Pierre-Fabre (CIPF)Saint-Julien-en-GenevoisFrance

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