Skip to main content
SpringerLink
Log in

Accurate determination of drug-to-antibody ratio of interchain cysteine-linked antibody–drug conjugates by LC-HRMS

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Accurate determination of the drug-to-antibody ratio (DAR) of interchain cysteine-linked antibody–drug conjugates (ADCs) is challenging. High-resolution mass spectrometry (HRMS) analysis of the ADCs at the intact or subunit level provides a feasible way to measure the DAR. However, the measured DAR is usually lower than the true DAR because of the variation in ionization efficiency between different DAR species. In this work, we developed a novel standard-free HRMS method involving isotope-labeled payload conjugation, protease digestion, and liquid chromatography–HRMS (LC-HRMS) analysis for accurate determination of the DAR of the interchain cysteine-linked ADCs with cleavable or non-cleavable linkers. Isotope-labeled payload conjugations eliminated the structural and chemical differences between different DAR species and ensured that the drugs or payload-containing peptides could be separated from each other in the mass spectrometer. A papain digestion strategy for ADCs with cleavable linkers showed a DAR of 3.79, with a relative standard deviation (RSD) of 0.48 (n = 3). Similarly, the trypsin and chymotrypsin digestion strategy that is applicable to ADCs with non-cleavable linkers showed a DAR of 3.77 and an RSD of 0.86 (n = 3). The DAR determined by this method was consistent with the DAR of the ADCs that was measured by the UV/Vis method. This method will be very useful to researchers working in the field of ADC discovery and development.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Nasiri H, Valedkarimi Z, Aghebati-Maleki L, Majidi J. Antibody-drug conjugates: promising and efficient tools for targeted cancer therapy. J Cell Physiol. 2018;233(9):6441–57.

    Article  CAS  Google Scholar 

  2. Francisco JA, Cerveny CG, Meyer DL, Mixan BJ, Klussman K, Chace DF, et al. cAC10-vcMMAE, an anti-CD30–monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood. 2003;102(4):1458–65.

    Article  CAS  Google Scholar 

  3. Wang W, Wang E, Balthasar J. Monoclonal antibody pharmacokinetics and pharmacodynamics. Clin Pharmacol Ther. 2008;84(5):548–58.

    Article  CAS  Google Scholar 

  4. Perez HL, Cardarelli PM, Deshpande S, Gangwar S, Schroeder GM, Vite GD, et al. Antibody–drug conjugates: current status and future directions. Drug Discov Today. 2014;19(7):869–81.

    Article  CAS  Google Scholar 

  5. Teicher BA, Chari RV. Antibody conjugate therapeutics: challenges and potential. Clin Cancer Res. 2011;17(20):6389–97.

    Article  CAS  Google Scholar 

  6. Laguzza BC, Nichols CL, Briggs SL, Cullinan GJ, Johnson DA, Starling JJ, et al. New antitumor monoclonal antibody-vinca conjugates LY203725 and related compounds: design, preparation, and representative in vivo activity. J Med Chem. 1989;32(3):548–55.

    Article  CAS  Google Scholar 

  7. Pietersz G, Krauer K. Antibody-targeted drugs for the therapy of cancer. J Drug Target. 1994;2(3):183–215.

    Article  CAS  Google Scholar 

  8. O'Mahony D, Bishop MR. Monoclonal antibody therapy. Front Biosci. 2006;11:1620–35.

    Article  CAS  Google Scholar 

  9. Chari RV. Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc Chem Res. 2007;41(1):98–107.

    Article  Google Scholar 

  10. Jaracz S, Chen J, Kuznetsova LV, Ojima I. Recent advances in tumor-targeting anticancer drug conjugates. Bioorg Med Chem. 2005;13(17):5043–54.

    Article  CAS  Google Scholar 

  11. Sievers EL, Senter PD. Antibody-drug conjugates in cancer therapy. Annu Rev Med. 2013;64.

    Article  CAS  Google Scholar 

  12. Tsuchikama K, An Z. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein & cell. 2018;9(1):33–46.

    Article  CAS  Google Scholar 

  13. Jain N, Smith SW, Ghone S, Tomczuk B. Current ADC linker chemistry. Pharm Res. 2015;32(11):3526–40.

    Article  CAS  Google Scholar 

  14. Sanderson RJ, Hering MA, James SF, Sun MM, Doronina SO, Siadak AW, et al. In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin Cancer Res. 2005;11(2):843–52.

    CAS  PubMed  Google Scholar 

  15. Schrama D, Reisfeld RA, Becker JC. Antibody targeted drugs as cancer therapeutics. Nat Rev Drug Discov. 2006;5(2):147.

    Article  CAS  Google Scholar 

  16. Ouyang J (2013) Drug-to-antibody ratio (DAR) and drug load distribution by hydrophobic interaction chromatography and reversed phase high-performance liquid chromatography. In: Antibody-Drug Conjugates. Springer, pp 275–283

  17. Chen J, Yin S, Wu Y, Ouyang J. Development of a native nanoelectrospray mass spectrometry method for determination of the drug-to-antibody ratio of antibody–drug conjugates. Anal Chem. 2013;85(3):1699–704.

    Article  CAS  Google Scholar 

  18. Valliere-Douglass JF, McFee WA, Salas-Solano O. Native intact mass determination of antibodies conjugated with monomethyl Auristatin E and F at interchain cysteine residues. Anal Chem. 2012;84(6):2843–9.

    Article  CAS  Google Scholar 

  19. Fo D, Bœuf A, Wagner-Rousset E, Colas O, Ayoub D, Corvaïa N, et al. 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. 2014;86(21):10674–83.

    Article  Google Scholar 

  20. Wagh A, Song H, Zeng M, Tao L, Das TK. Challenges and new frontiers in analytical characterization of antibody-drug conjugates. In: MAbs, 2018. vol 2. Taylor & Francis, pp 222–243.

  21. Friese OV, Smith JN, Brown PW, Rouse JC. Practical approaches for overcoming challenges in heightened characterization of antibody-drug conjugates with new methodologies and ultrahigh-resolution mass spectrometry. In: MAbs, 2018. vol 3. Taylor & Francis, pp 335–345.

  22. Said N, Gahoual R, Kuhn L, Beck A, François Y-N, Leize-Wagner E. 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. 2016;918:50–9.

    Article  CAS  Google Scholar 

  23. Okeley NM, Miyamoto JB, Zhang X, Sanderson RJ, Benjamin DR, Sievers EL, et al. Intracellular activation of SGN-35, a potent anti-CD30 antibody-drug conjugate. Clin Cancer Res. 2010;16(3):888–97.

    Article  CAS  Google Scholar 

  24. Lu J, Jiang F, Lu A, Zhang G. Linkers having a crucial role in antibody–drug conjugates. Int J Mol Sci. 2016;17(4):561.

    Article  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowledge Dr. Mu Chen and Dr. Shengsheng Xu for their technical assistance and useful discussions. We also thank Dr. Erhu Lu and Dr. Li Tan for their suggestions on the synthesis of vcMMAE-d8. This study was supported by Frontage Laboratories, Inc. and the Missouri University of Science and Technology.

Author information

Authors and Affiliations

  1. Department of Chemistry and Center for Biomedical Research, Missouri University of Science and Technology, Rolla, MO, 65409, USA

    Ke Li, Honglan Shi & Yinfa Ma

  2. Department of Bioanalysis, Frontage Laboratories, Inc., Exton, PA, 19341, USA

    Zhiling Zhang & Zhongping(John) Lin

  3. Department of Chemistry, California State University, Sacramento, CA, 95819, USA

    Yinfa Ma

Authors
  1. Ke Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhiling Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhongping(John) Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Honglan Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yinfa Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yinfa Ma.

Ethics declarations

Conflict of interest

Ke Li, Dr. Yinfa Ma, and Dr. Honglan Shi were employees of Missouri University of Science and Technology at the time of this study; Dr. Zhiling Zhang and Dr. Zhongping (John) Lin were employees of Frontage Laboratories, Inc. This study was performed at Frontage Laboratories, Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 75 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, K., Zhang, Z., Lin, Z. et al. Accurate determination of drug-to-antibody ratio of interchain cysteine-linked antibody–drug conjugates by LC-HRMS. Anal Bioanal Chem 412, 833–840 (2020). https://doi.org/10.1007/s00216-019-02280-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-019-02280-5

Keywords

Search

Navigation