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

Evolène Deslignière (1) , Anthony Ehkirch (1) , Bastiaan L Duivelshof (2) , Hanna Toftevall (3) , Jonathan Sjögren (3) , Davy Guillarme (2) , Valentina d'Atri (2) , Alain Beck (4) , Oscar Hernandez-Alba (1) , Sarah Cianférani (1)

1 DSA-IPHC - Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires
2 UNIGE - Université de Genève = University of Geneva
3 Genovis AB
4 CIPF - Centre d'Immunologie Pierre Fabre

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 ratio (DAR). Sitespecific 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 sitespecific 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 solution and gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation empowered mAbderived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

Mots clés: native mass spectrometry; size-exclusion chromatography (SEC); ion mobility-mass spectrometry (IM-MS); collision-induced unfolding (CIU); antibody-drug conjugate (ADC); sitespecific conjugation

Domaines: Sciences pharmaceutiques; Chimie analytique

Evolène Deslignière, Anthony Ehkirch, Bastiaan L Duivelshof, Hanna Toftevall, Jonathan Sjögren, et al.. State of the Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis. Pharmaceuticals, 2021, 14 (6), pp.498. ⟨10.3390/ph14060498⟩. ⟨hal-03287311⟩