Characterization of intact antibody–drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography–mass spectrometry

2011 ◽  
Vol 412 (1) ◽  
pp. 56-66 ◽  
Author(s):  
Keyang Xu ◽  
Luna Liu ◽  
Ola M. Saad ◽  
Jakub Baudys ◽  
Lara Williams ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Capillary Liquid Chromatography ◽  
Liquid Chromatography Mass Spectrometry ◽  
Affinity Capture ◽  
Drug Conjugates ◽  
Intact Antibody ◽  
Capillary Liquid ◽  
Antibody Drug

Automated On-tip Affinity Capture Coupled with Mass Spectrometry to Characterize Intact Antibody-Drug Conjugates from Blood

2018 ◽  
Vol 29 (7) ◽  
pp. 1532-1537 ◽  
Author(s):  
Ke Sherry Li ◽  
Phillip Y. Chu ◽  
Aimee Fourie-O’Donohue ◽  
Neha Srikumar ◽  
Katherine R. Kozak ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Affinity Capture ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Intact Antibody ◽  
Antibody Drug

scholarly journals Application of triple quadrupole mass spectrometry for the characterization of antibody–drug conjugates

2019 ◽  
Vol 411 (12) ◽  
pp. 2569-2576 ◽  
Author(s):  
Malin Källsten ◽  
Matthijs Pijnappel ◽  
Rafael Hartmann ◽  
Fredrik Lehmann ◽  
Lucia Kovac ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Triple Quadrupole ◽  
Quadrupole Mass ◽  
Triple Quadrupole Mass Spectrometry ◽  
Antibody Drug Conjugates ◽  
Quadrupole Mass Spectrometry ◽  
Drug Conjugates ◽  
Antibody Drug

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

2021 ◽  
Vol 14 (6) ◽  
pp. 498
Author(s):  
Evolène Deslignière ◽  
Anthony Ehkirch ◽  
Bastiaan L. Duivelshof ◽  
Hanna Toftevall ◽  
Jonathan Sjögren ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Ion Mobility ◽  
State Of The Art ◽  
Native Mass Spectrometry ◽  
Site Specific ◽  
Drug Conjugates ◽  
Conjugation Process ◽  
Antibody Drug

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.

scholarly journals Multilevel characterization of antibody-ligand conjugates by CESI-MS

2020 ◽  
Vol 20 ◽  
Author(s):  
Bryan Fonslow ◽  
Gabor Jarvas ◽  
Marton Szigeti ◽  
Andras Guttman
Keyword(s):  
Mass Spectrometry ◽  
Capillary Electrophoresis ◽  
Peptide Mapping ◽  
Site Occupancy ◽  
Intact Protein ◽  
Ionization Mass ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

Aims: Demonstrating the capabilities of our new capillary electrophoresis – mass spectrometry method, which facilitates highly accurate relative quantitation of modification site occupancy of antibody-ligand (e.g., antibody-drug) conjugates. Background: Antibody-drug conjugates play important roles in medical discovery for imaging and therapeutic intervention. The localization and stoichiometry of the conjugation can affect the orientation, selectivity, specificity, and strength of molecular interactions, influencing biochemical function. Objective: To demonstrate the option to analyze the localization and stoichiometry of antibody-ligand conjugates by using essentially the same method at all levels including ligand infusion, peptide mapping, as well as reduced and intact protein analysis. Materials and Methods: Capillary electrophoresis coupled to electrospray ionization mass spectrometry was used to analyze the antibodyligand conjugates. Results: We identified three prevalent ligand conjugation sites with estimated stoichiometries of 73, 14, and 6% and an average ligand-antibody ratio of 1.37, illustrating the capabilities of CE-ESI-MS for rapid and efficient characterization of antibody-drug conjugates. Conclusion: The developed multilevel analytical method offers a comprehensive way to determine the localization and stoichiometry of antibody-drug conjugates for molecular medicinal applications. In addition, a significant advantage of the reported approach is that small, hydrophilic, unmodified peptides well separated from the neutrals, which is not common with other liquid phase separation methods such as LC.

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