Abstract
High-resolution native mass spectrometry (MS) provides accurate mass measurements (within 30 ppm) of intact ADCs and can also yield drug load distribution (DLD) and average drug to antibody ratio (DAR) in parallel with hydrophobic interaction chromatography (HIC). Native MS is furthermore unique in its ability to simultaneously detect covalent and noncovalent species in a mixture and for HIC peak identity assessment offline or online.
As an orthogonal method described in this chapter, LC-MS following ADC reduction or IdeS (Fabricator) digestion and reduction can also be used to measure the DLD of light chain and Fd fragments for hinge native cysteine residues such as brentuximab vedotin. Both methods allow also the measurement of average DAR for both monomeric and multimeric species. In addition, the Fc fragments can be analyzed in the same run, providing a complete glycoprofile and the demonstration or absence of additional conjugation of this subdomain involved in FcRn and Fc-gammaR binding.
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References
Beck A, D’Atri V, Ehkirch A et al (2019) Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future. Expert Rev Proteomics 16:337–362. https://doi.org/10.1080/14789450.2019.1578215
Beck A, Terral G, Debaene F et al (2016) Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates. Expert Rev Proteomics 13:157–183. https://doi.org/10.1586/14789450.2016.1132167
Wagner-Rousset E, Janin-Bussat M-C, Colas O et al (2014) Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion. MAbs 6:273–285. https://doi.org/10.4161/mabs.26773
Beck A, Bussat M-C, Zorn N et al (2005) Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells. J Chromatogr B Analyt Technol Biomed Life Sci 819:203–218. https://doi.org/10.1016/j.jchromb.2004.06.052
Xu W, Peng Y, Wang F et al (2013) Method to convert N-terminal glutamine to pyroglutamate for characterization of recombinant monoclonal antibodies. Anal Biochem 436:10–12. https://doi.org/10.1016/j.ab.2013.01.017
Srzentić K, Nagornov KO, Fornelli L et al (2018) Multiplexed middle-down mass spectrometry as a method for revealing light and heavy chain connectivity in a monoclonal antibody. Anal Chem 90:12527–12535. https://doi.org/10.1021/acs.analchem.8b02398
Basa L (2013) Drug-to-antibody ratio (DAR) and drug load distribution by LC-ESI-MS. In: Ducry L (ed) Antibody-drug conjugates. Humana Press, Totowa, NJ, pp 285–293
Chevreux G, Tilly N, Bihoreau N (2011) Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry. Anal Biochem 415:212–214. https://doi.org/10.1016/j.ab.2011.04.030
Sjögren J, Olsson F, Beck A (2016) Rapid and improved characterization of therapeutic antibodies and antibody related products using IdeS digestion and subunit analysis. Analyst 141:3114–3125. https://doi.org/10.1039/C6AN00071A
Firth D, Bell L, Squires M et al (2015) A rapid approach for characterization of thiol-conjugated antibody–drug conjugates and calculation of drug–antibody ratio by liquid chromatography mass spectrometry. Anal Biochem 485:34–42. https://doi.org/10.1016/j.ab.2015.06.001
Friese OV, Smith JN, Brown PW, Rouse JC (2018) Practical approaches for overcoming challenges in heightened characterization of antibody-drug conjugates with new methodologies and ultrahigh-resolution mass spectrometry. MAbs 10:335–345. https://doi.org/10.1080/19420862.2018.1433973
Lin TJ, Beal KM, Brown PW et al (2019) Evolution of a comprehensive, orthogonal approach to sequence variant analysis for biotherapeutics. mAbs 11:1–12. https://doi.org/10.1080/19420862.2018.1531965
Janin-Bussat M-C, Dillenbourg M, Corvaia N et al (2015) Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC–MS analysis. J Chromatogr B 981–982:9–13. https://doi.org/10.1016/j.jchromb.2014.12.017
D’Atri V, Fekete S, Stoll D et al (2018) Characterization of an antibody-drug conjugate by hydrophilic interaction chromatography coupled to mass spectrometry. J Chromatogr B 1080:37–41. https://doi.org/10.1016/j.jchromb.2018.02.026
Said N, Gahoual R, Kuhn L et al (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 918:50–59. https://doi.org/10.1016/j.aca.2016.03.006
Beck A, Goetsch L, Dumontet C, Corvaïa N (2017) Strategies and challenges for the next generation of antibody–drug conjugates. Nat Rev Drug Discov 16:315–337. https://doi.org/10.1038/nrd.2016.268
Botzanowski T, Erb S, Hernandez-Alba O et al (2017) Insights from native mass spectrometry approaches for top- and middle-level characterization of site-specific antibody-drug conjugates. mAbs 9:801–811. https://doi.org/10.1080/19420862.2017.1316914
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Wagner-Rousset, E. et al. (2020). Drug Loading and Distribution of ADCs After Reduction or IdeS Digestion and Reduction. In: Tumey, L. (eds) Antibody-Drug Conjugates. Methods in Molecular Biology, vol 2078. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9929-3_12
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