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The AAPS Journal

, 21:4 | Cite as

Approaches to Resolve False Reporting in Neutralizing Antibody Assays Caused by Reagent Leaching from Affinity Capture Elution Solid Phase

  • Yuhong Xiang
  • John Kamerud
  • Jean Donley
  • Katrina Olson
  • Teresa Caiazzo
  • Dave Yeung
  • Chuenlei Parng
  • Boris GorovitsEmail author
Research Article
  • 357 Downloads

Abstract

Insufficient drug tolerance presents a major challenge in the development of neutralizing antibody (NAb) assays for biotherapeutics. Sample pre-treatment using solid-phase extraction with acid dissociation (SPEAD) is widely reported to improve drug tolerance. In this paper, a case study is presented in which SPEAD was used in conjunction with a competitive ligand binding NAb assay format. A significant degree of biotin-drug conjugate leaching was observed resulting in the reporting of both false positive and false negative results in NAb assay. Mitigation steps have been evaluated to address drug/biotin-drug conjugate leaching. These steps included assessment of the streptavidin-coated plate in conjunction with biotin-drug conjugates at various biotin molar challenge ratios (MCR). In addition, an alternative method based on covalent capture of the drug on an aldehyde-activated plate was assessed. Both approaches were compared for the degree of drug/biotin-drug conjugate leaching during the second elution step of the SPEAD procedure. Moreover, the impact of various conditions on the assay performance was assessed, including elution pH, sample incubation time, and biotin MCR. For the covalent drug capture method, capture conditions were evaluated. Optimized conditions in both streptavidin capture and covalent capture methods enabled a significant reduction of drug/biotin-drug conjugate leaching. A streptavidin high binding capacity approach using biotin-drug conjugate with a MCR of 50:1 was chosen as the optimal method yielding a NAb assay with a fit for purpose sensitivity (153 ng/mL) and a drug tolerance of up to 50 μg/mL with 500 ng/mL PC.

KEY WORDS

ACE anti-drug antibody biotin-drug conjugate/drug leaching drug tolerance immunogenicity neutralizing antibody sample pre-treatment SPEAD 

Notes

Acknowledgements

The authors thank Lee Walus, Renee Ramsey, and Glenn Miller for the preparation of critical reagents and Fengping Li for the LC-TOF analyses of the biotin-drug conjugates. The authors also thank Frederick McCush, Ying Wang, Alison Joyce, Liang Zhu, and Marcela Araya Roldan for helpful input.

Funding information

This work was funded by Pfizer.

References

  1. 1.
    Beck A, Wagner-Rousset E, Bussat MC, Lokteff M, Klinguer-Hamour C, Haeuw JF, et al. Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins. Curr Pharm Biotechnol. 2008;9(6):482–501.CrossRefGoogle Scholar
  2. 2.
    Beck J, Urnovitz HB, Mitchell WM, Schutz E. Next generation sequencing of serum circulating nucleic acids from patients with invasive ductal breast cancer reveals differences to healthy and nonmalignant controls. Mol Cancer Res : MCR. 2010;8(3):335–42.CrossRefGoogle Scholar
  3. 3.
    Carter PJ. Introduction to current and future protein therapeutics: a protein engineering perspective. Exp Cell Res. 2011;317(9):1261–9.CrossRefGoogle Scholar
  4. 4.
    FDA. Guidance for industry. In: Immunogenicity assessment for therapeutic protein products; 2014.Google Scholar
  5. 5.
    Buttel IC, Chamberlain P, Chowers Y, Ehmann F, Greinacher A, Jefferis R, et al. Taking immunogenicity assessment of therapeutic proteins to the next level. Biologicals. 2011;39(2):100–9.CrossRefGoogle Scholar
  6. 6.
    Koren E, Smith HW, Shores E, Shankar G, Finco-Kent D, Rup B, et al. Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products. J Immunol Methods. 2008;333(1–2):1–9.CrossRefGoogle Scholar
  7. 7.
    Mire-Sluis AR, Barrett YC, Devanarayan V, Koren E, Liu H, Maia M, et al. Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. J Immunol Methods. 2004;289(1–2):1–16.CrossRefGoogle Scholar
  8. 8.
    Shankar G, Devanarayan V, Amaravadi L, Barrett YC, Bowsher R, Finco-Kent D, et al. Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. J Pharm Biomed Anal. 2008;48(5):1267–81.CrossRefGoogle Scholar
  9. 9.
    Shankar G, Pendley C, Stein KE. A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs. Nat Biotechnol 2007;25(5):555–561.Google Scholar
  10. 10.
    Rosenberg AS. Immunogenicity of biological therapeutics: a hierarchy of concerns. Dev Biol. 2003;112:15–21.Google Scholar
  11. 11.
    Schellekens H, Casadevall N. Immunogenicity of recombinant human proteins: causes and consequences. J Neurol. 2004;251(Suppl 2):II4–9.PubMedGoogle Scholar
  12. 12.
    Casadevall N, Nataf J, Viron B, Kolta A, Kiladjian JJ, Martin-Dupont P, et al. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N Engl J Med. 2002;346(7):469–75.CrossRefGoogle Scholar
  13. 13.
    Green D. Spontaneous inhibitors to coagulation factors. Clin Lab Haematol 2000;22 Suppl 1:21–25; discussion 30-2.Google Scholar
  14. 14.
    Li J, Yang C, Xia Y, Bertino A, Glaspy J, Roberts M, et al. Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood. 2001;98(12):3241–8.CrossRefGoogle Scholar
  15. 15.
    Hu J, Gupta S, Swanson SJ, Zhuang Y. A bioactive drug quantitation based approach for the detection of anti-drug neutralizing antibodies in human serum. J Immunol Methods 2009;345(1–2):70–79.Google Scholar
  16. 16.
    Bourdage JS, Cook CA, Farrington DL, Chain JS, Konrad RJ. An Affinity Capture Elution (ACE) assay for detection of anti-drug antibody to monoclonal antibody therapeutics in the presence of high levels of drug. J Immunol Methods. 2007;327(1–2):10–7.CrossRefGoogle Scholar
  17. 17.
    Smith HW, Butterfield A, Sun D. Detection of antibodies against therapeutic proteins in the presence of residual therapeutic protein using a solid-phase extraction with acid dissociation (SPEAD) sample treatment prior to ELISA. Regul Toxicol Pharmacol : RTP. 2007;49(3):230–7.CrossRefGoogle Scholar
  18. 18.
    Lofgren JA, Wala I, Koren E, Swanson SJ, Jing S. Detection of neutralizing anti-therapeutic protein antibodies in serum or plasma samples containing high levels of the therapeutic protein. J Immunol Methods. 2006;308(1–2):101–8.CrossRefGoogle Scholar
  19. 19.
    Xu W, Jiang H, Titsch C, Haulenbeek JR, Pillutla RC, Aubry AF, et al. Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays. J Immunol Methods. 2015;416:94–104.CrossRefGoogle Scholar
  20. 20.
    Jiang H, Xu W, Titsch CA, Furlong MT, Dodge R, Voronin K, et al. Innovative use of LC-MS/MS for simultaneous quantitation of neutralizing antibody, residual drug, and human immunoglobulin G in immunogenicity assay development. Anal Chem. 2014;86(5):2673–80.CrossRefGoogle Scholar
  21. 21.
    Gupta S, Devanarayan V, Finco D, Gunn GR 3rd, Kirshner S, Richards S, et al. Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics. J Pharm Biomed Anal. 2011;55(5):878–88.CrossRefGoogle Scholar
  22. 22.
    EMA. Guideline on Immunogenicity assessment of 6 biotechnology-derived therapeutic proteins. 2015.Google Scholar
  23. 23.
    Diamandis EP, Christopoulos TK. The biotin-(strept)avidin system: principles and applications in biotechnology. Clin Chem. 1991;37(5):625–36.PubMedGoogle Scholar
  24. 24.
    Heitzmann H RF (1974) Use of the avidin-biotin complex for specific staining of biological membranes in electron microscopy. Proceedings of the National Academy of Sciences USA 71(9):3537–41.Google Scholar
  25. 25.
    Kohler M, Karner A, Leitner M, Hytonen VP, Kulomaa M, Hinterdorfer P, et al. pH-dependent deformations of the energy landscape of avidin-like proteins investigated by single molecule force spectroscopy. Molecules. 2014;19(8):12531–46.CrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  • Yuhong Xiang
    • 1
    • 2
  • John Kamerud
    • 1
  • Jean Donley
    • 1
  • Katrina Olson
    • 1
  • Teresa Caiazzo
    • 1
  • Dave Yeung
    • 3
  • Chuenlei Parng
    • 1
  • Boris Gorovits
    • 1
    Email author
  1. 1.Pfizer Worldwide Research & Development, Biomedicine DesignMassachusettsUSA
  2. 2.Jounce Therapeutics, Inc.CambridgeUSA
  3. 3.Takeda Pharmaceutical Company LimitedCambridgeUSA

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