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Analysis of ADCs by Native Mass Spectrometry

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Antibody-Drug Conjugates

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2078))

Abstract

Mass spectrometry performed in nondenaturing conditions (native MS) has proven its utility for the quantitative and qualitative analysis of antibody-drug conjugates (ADCs), especially when ADCs’ subunits involve noncovalent interactions (i.e., cysteine-conjugated ADCs). Its hyphenation to ion mobility spectrometry (IM-MS) allows differentiation of gas-phase ions based on their rotationally averaged collision cross section providing an additional dimension of conformational characterization of ADCs. More recently, size exclusion chromatography (SEC) appeared as an interesting technique to perform online buffer exchange in an automated way prior to native MS/IM-MS analysis. Online SEC-native MS/IM-MS allows the global structural characterization of ADCs and the assessment of some critical quality attributes (CQAs) required for ADC release on the market, such as drug load distribution (DLD), drug-to-antibody ratio (DAR), the average DAR (DARav), and the relative amount of unconjugated mAb.

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References

  1. Chari RVJ (2008) Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc Chem Res 41(1):98–107

    Article  CAS  Google Scholar 

  2. Carter PJ, Lazar GA (2018) Next generation antibody drugs: pursuit of the ‘high-hanging fruit’. Nat Rev Drug Discov 17(3):197–223

    Article  CAS  Google Scholar 

  3. Valliere-Douglass JF, McFee WA, Salas-Solano O (2012) Native intact mass determination of antibodies conjugated with monomethyl Auristatin E and F at interchain cysteine residues. Anal Chem 84(6):2843–2849

    Article  CAS  Google Scholar 

  4. Chen J, Yin S, Wu YJ, Ouyang J (2013) Development of a native nanoelectrospray mass spectrometry method for determination of the drug-to-antibody ratio of antibody-drug conjugates. Anal Chem 85(3):1699–1704

    Article  CAS  Google Scholar 

  5. Hengel SM, Sanderson R, Valliere-Douglass J, Nicholas N, Leiske C, Alley SC (2014) Measurement of in vivo drug load distribution of cysteine-linked antibody-drug conjugates using microscale liquid chromatography mass spectrometry. Anal Chem 86(7):3420–3425

    Article  CAS  Google Scholar 

  6. Debaene F, Boeuf A, Wagner-Rousset E, Colas O, Ayoub D, Corvaia N, Van Dorsselaer A, Beck A, Cianferani S (2014) Innovative native MS methodologies for antibody drug conjugate characterization: high resolution native MS and IM-MS for average DAR and DAR distribution assessment. Anal Chem 86(21):10674–10683

    Article  CAS  Google Scholar 

  7. Marcoux J, Champion T, Colas O, Wagner-Rousset E, Corvaïa N, Van Dorsselaer A, Beck A, Cianférani S (2015) Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate. Protein Sci 24(8):1210–1223

    Article  CAS  Google Scholar 

  8. Botzanowski T, Erb S, Hernandez-Alba O, Ehkirch A, Colas O, Wagner-Rousset E, Rabuka D, Beck A, Drake PM, Cianférani S (2017) Insights from native mass spectrometry approaches for top- and middle-level characterization of site-specific antibody-drug conjugates. MAbs 9(5):801–811

    Article  CAS  Google Scholar 

  9. Ehkirch A, Hernandez-Alba O, Colas O, Beck A, Guillarme D, Cianférani S (2018) Hyphenation of size exclusion chromatography to native ion mobility mass spectrometry for the analytical characterization of therapeutic antibodies and related products. J Chromatogr B 1086:176–183

    Article  CAS  Google Scholar 

  10. Ehkirch A, Goyon A, Hernandez-Alba O, Rouviere F, D’Atri V, Dreyfus C, Haeuw J-F, Diemer H, Beck A, Heinisch S, Guillarme D, Cianferani S (2018) A novel online four-dimensional SEC×SEC-IM×MS methodology for characterization of monoclonal antibody size variants. Anal Chem 90(23):13929–13937

    Article  CAS  Google Scholar 

  11. Ehkirch A, D’Atri V, Rouviere F, Hernandez-Alba O, Goyon A, Colas O, Sarrut M, Beck A, Guillarme D, Heinisch S, Cianferani S (2018) An online four-dimensional HIC×SEC-IM×MS methodology for proof-of-concept characterization of antibody drug conjugates. Anal Chem 90(3):1578–1586

    Article  CAS  Google Scholar 

  12. D'Atri V, Causon T, Hernandez-Alba O, Mutabazi A, Veuthey JL, Cianferani S, Guillarme D (2018) Adding a new separation dimension to MS and LC–MS: what is the utility of ion mobility spectrometry? J Sep Sci 41(1):20–67

    Article  CAS  Google Scholar 

  13. Beck A, D’Atri V, Ehkirch A, Fekete S, Hernandez-Alba O, Gahoual R, Leize-Wagner E, François Y, Guillarme D, Cianférani S (2019) Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future. Expert Rev Proteomics 16:337–362

    Article  CAS  Google Scholar 

  14. Ramstrom H, Sanglier S, Leize-Wagner E, Philippe C, Van Dorsselaer A, Haiech J (2003) Properties and regulation of the bifunctional enzyme HPr kinase/phosphatase in Bacillus subtilis. J Biol Chem 278(2):1174–1185

    Article  Google Scholar 

  15. Atmanene C, Petiot-Becard S, Zeyer D, Van Dorsselaer A, Hannah VV, Sanglier-Cianferani S (2012) Exploring key parameters to detect subtle ligand-induced protein conformational changes using traveling wave ion mobility mass spectrometry. Anal Chem 84(11):4703–4710

    Article  CAS  Google Scholar 

  16. Ruotolo BT, Benesch JLP, Sandercock AM, Hyung S-J, Robinson CV (2008) Ion mobility–mass spectrometry analysis of large protein complexes. Nat Protoc 3:1139

    Article  CAS  Google Scholar 

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Correspondence to Sarah Cianférani .

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Hernandez-Alba, O., Ehkirch, A., Beck, A., Cianférani, S. (2020). Analysis of ADCs by Native Mass Spectrometry. 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_13

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  • DOI: https://doi.org/10.1007/978-1-4939-9929-3_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9928-6

  • Online ISBN: 978-1-4939-9929-3

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