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Article

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

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Laboratoire de Spectrométrie de Masse BioOrganique, IPHC UMR 7178, Université de Strasbourg, CNRS, 67087 Strasbourg, France
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Infrastructure Nationale de Protéomique ProFI—FR2048, 67087 Strasbourg, France
3
School of Pharmaceutical Sciences, University of Geneva, CMU—Rue Michel-Servet 1, 1211 Geneva, Switzerland
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Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU—Rue Michel-Servet 1, 1211 Geneva, Switzerland
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Genovis AB, SE-220 07 Lund, Sweden
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IRPF—Centre d’Immunologie Pierre-Fabre (CIPF), 74160 Saint-Julien-en-Genevois, France
*
Author to whom correspondence should be addressed.
Academic Editor: Alfredo Berzal-Herranz
Pharmaceuticals 2021, 14(6), 498; https://doi.org/10.3390/ph14060498
Received: 23 March 2021 / Revised: 17 May 2021 / Accepted: 19 May 2021 / Published: 24 May 2021
(This article belongs to the Special Issue Antibody-Drug Conjugates (ADC): 2021)
Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines. View Full-Text
Keywords: native mass spectrometry; size-exclusion chromatography (SEC); ion mobility-mass spectrometry (IM-MS); collision-induced unfolding (CIU); antibody-drug conjugate (ADC); site-specific conjugation native mass spectrometry; size-exclusion chromatography (SEC); ion mobility-mass spectrometry (IM-MS); collision-induced unfolding (CIU); antibody-drug conjugate (ADC); site-specific conjugation
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MDPI and ACS Style

Deslignière, E.; Ehkirch, A.; Duivelshof, B.L.; Toftevall, H.; Sjögren, J.; Guillarme, D.; D’Atri, V.; Beck, A.; Hernandez-Alba, O.; Cianférani, S. State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis. Pharmaceuticals 2021, 14, 498. https://doi.org/10.3390/ph14060498

AMA Style

Deslignière E, Ehkirch A, Duivelshof BL, Toftevall H, Sjögren J, Guillarme D, D’Atri V, Beck A, Hernandez-Alba O, Cianférani S. State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis. Pharmaceuticals. 2021; 14(6):498. https://doi.org/10.3390/ph14060498

Chicago/Turabian Style

Deslignière, Evolène, Anthony Ehkirch, Bastiaan L. Duivelshof, Hanna Toftevall, Jonathan Sjögren, Davy Guillarme, Valentina D’Atri, Alain Beck, Oscar Hernandez-Alba, and Sarah Cianférani. 2021. "State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis" Pharmaceuticals 14, no. 6: 498. https://doi.org/10.3390/ph14060498

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