Characterization of the Primary Structure of Cysteine-Linked Antibody-Drug Conjugates Using Capillary Electrophoresis with Mass Spectrometry
Capillary electrophoresis-mass spectrometry (CE-MS) enables the characterization of the primary structure of ADCs. An analytical method based on a derived bottom-up proteomic workflow is designed to provide detailed information about the amino acid sequence, the glycosylation profiling, and the location on the peptide backbone of the conjugated drugs. Here we describe the experimental protocol applied on the characterization of cysteine-linked brentuximab vedotin (Adcetris®).
Key wordsAntibody-drug conjugate Brentuximab vedotin Cytotoxic drug Capillary electrophoresis–mass spectrometry Structural characterization Drug-loaded peptide
The authors would like to thank Sciex separations Inc. for lending a CESI8000 system and a 5600 TripleTOF. They would like to thank Bruker Daltonics for lending a Maxis 4G. They would like also to express their gratitude to Dr. E. Wagner-Rousset, M. C. Janin-Bussat, and O. Colas (Centre d’Immunologie Pierre Fabre, St Julien en Genevois, France) for helpful discussions. This work was supported by the CNRS (UMR 7140) and the University of Strasbourg.
- 3.Jen EY, Ko C-W, Lee JE et al (2018) FDA approval: Gemtuzumab ozogamicin for the treatment of adults with newly-diagnosed CD33-positive acute myeloid leukemia. Clin. Cancer Res 24(14):3242–3246Google Scholar
- 12.Gahoual R, Busnel J-M, Beck A, François Y-N, Leize-Wagner E (2014) Full antibody primary structure and microvariant characterization in a single injection using transient Isotachophoresis and Sheathless capillary electrophoresis–tandem mass spectrometry. Anal Chem 86(18):9074–9081CrossRefGoogle Scholar
- 16.Said N, Gahoual R, Kuhn L, Beck A, François Y-N, Leize-Wagner E (2016) Structural characterization of antibody drug conjugate by a combination of intact, middle-up and bottom-up techniques using sheathless capillary electrophoresis—tandem mass spectrometry as nanoESI infusion platform and separation method. Anal Chim Acta 918:50–59CrossRefGoogle Scholar
- 18.Dada OO, Zhao YM, Jaya N, Salas-Solano O (2017) High-resolution capillary zone electrophoresis with mass spectrometry peptide mapping of therapeutic proteins: peptide recovery and post-translational modification analysis in monoclonal antibodies and antibody-drug conjugates. Anal Chem 89(21):11236–11242CrossRefGoogle Scholar