IMR Press / FBL / Volume 27 / Issue 10 / DOI: 10.31083/j.fbl2710290
Open Access Original Research
A Combination of Native LC-MS Approaches for the Comprehensive Characterization of the Antibody-Drug Conjugate Trastuzumab Deruxtecan
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1 Laboratoire de Spectrométrie de Masse BioOrganique, IPHC UMR 7178, Université de Strasbourg, CNRS, 67087 Strasbourg, France
2 Infrastructure Nationale de Protéomique ProFI – FR2048, 67087 Strasbourg, France
3 Strasbourg Drug Discovery and Development Institute (IMS), University of Strasbourg, 67000 Strasbourg, France
4 Institut de Cancérologie Strasbourg Europe, 67000 Strasbourg, France
*Correspondence: (Sarah Cianférani)
Academic Editor: Sukmook Lee
Front. Biosci. (Landmark Ed) 2022, 27(10), 290;
Submitted: 27 July 2022 | Revised: 26 September 2022 | Accepted: 29 September 2022 | Published: 26 October 2022
(This article belongs to the Special Issue Antibody Drug Conjugates)
Copyright: © 2022 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.

Background: Native mass spectrometry (nMS) approaches appear attractive to complement bottom-up strategies traditionally used in biopharmaceutical industries thanks to their quite straightforward and rapid workflows, especially through online hyphenation of non-denaturing liquid chromatography (LC) to nMS. The present work provides an overview of the state-of-the-art chromatographic tools available for the detailed characterization of monoclonal antibody (mAb) formats, exemplified on the antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd). Methods: T-DXd was first characterized by conventional reversed phase LC (rpLC) and peptide mapping. Couplings of size exclusion chromatography (SEC), cation exchange chromatography (CEX), and hydrophobic interaction chromatography (HIC) to nMS were used to gain further insights into size, hydrophobic, and charge variants of T-DXd and its parental mAb trastuzumab, at intact and middle-up levels. Results: SEC-nMS first offered a direct snapshot of the homogeneous conjugation of T-DXd, with an average drug-to-antibody ratio (DAR) of 8 in agreement with a conjugation on cysteines after reduction of all interchain disulfide bonds. Moreover, SEC-nMS afforded precise identification and quantification of aggregates and fragments. Middle-up level experiments performed after IdeS digestion confirmed that drug conjugation occurs in the Fab region of the mAb, as seen with rpLC. HIC separated two DAR8 species that could not be differentiated by nMS. Although middle-up HIC-nMS proved to be more informative for oxidized forms, the identification of minor variants was still difficult because of poor MS signal quality, showing how the coupling of HIC to nMS remains challenging. Lastly, middle-up CEX-nMS provided accurate determination and localization of post-translational modifications, with several acidic/basic variants within Fab and Fc regions of T-DXd that were also identified by peptide mapping. Conclusions: This study illustrates the strengths and drawbacks of each LC-nMS coupling. By combining SEC-, HIC-, and CEX-nMS, we were able to achieve a comprehensive characterization of T-DXd without extensive sample preparation prior to MS analysis.

native mass spectrometry (MS)
liquid chromatography (LC)
size exclusion chromatography (SEC)
cation exchange chromatography (CEX)
hydrophobic interaction chromatography (HIC)
higher order structures
antibody-drug conjugate (ADC)
ProFI; ANR-10-INBS-08-03/CNRS, the University of Strasbourg, the Agence Nationale de la Recherche, and the French Proteomic Infrastructure
ANR-10-IDEX0002/IdEx Unistra
ANR-20-SFRI0012/SFRI-STRAT’US project
Fig. 1.
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