Analytical and Bioanalytical Chemistry

, Volume 402, Issue 3, pp 1229–1239 | Cite as

Attribution of the discrepancy between ELISA and LC-MS/MS assay results of a PEGylated scaffold protein in post-dose monkey plasma samples due to the presence of anti-drug antibodies

  • Shujie J. Wang
  • Steven T. Wu
  • Jochem Gokemeijer
  • Aberra Fura
  • Murli Krishna
  • Paul Morin
  • Guodong Chen
  • Karen Price
  • David Wang-Iverson
  • Timothy Olah
  • Russell Weiner
  • Adrienne Tymiak
  • Mohammed Jemal
Original Paper

Abstract

High-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) methods were developed for the quantification of a PEGylated scaffold protein drug in monkey plasma samples. The LC-MS/MS method was based on the extraction of the therapeutic protein with a water-miscible organic solvent and the subsequent trypsin digestion of the extract followed by the detection of a surrogate peptide. The assay was linear over a range of 10–3,000 ng/mL. The ELISA method utilized a therapeutic target-binding format in which the recombinant target antigen was used to capture the drug in the sample, followed by detection with an anti-PEG monoclonal antibody. The assay range was 30–2,000 ng/mL. A correlation study between the two methods was performed by measuring the drug concentrations in plasma samples from a single-dose pharmacokinetic (PK) study in cynomolgus monkeys following a 5-mg/kg subcutaneous administration (n = 4). In the early time points of the PK profile, the drug concentrations obtained by the LC-MS/MS method agreed very well with those obtained by the ELISA method. However, at later time points, the drug concentrations measured by the LC-MS/MS method were consistently higher than those measured by the ELISA method. The PK parameters calculated based on the concentration data showed that the two methods gave equivalent peak exposure (Cmax) at 24–48 h. However, the LC-MS/MS results exhibited about 1.53-fold higher total exposure (AUCtot) than the ELISA results. The discrepancy between the LC-MS/MS and ELISA results was investigated by conducting immunogenicity testing, anti-drug antibody (ADA) epitope mapping, and Western blot analysis of the drug concentrations coupled with Protein G separation. The results demonstrated the presence of ADA specific to the engineered antigen-binding region of the scaffold protein drug that interfered with the ability of the drug to bind to the target antigen used in the ELISA method. In the presence of the ADAs, the ELISA method measured only the active circulating drug (target-binding), while the LC-MS/MS method measured the total circulating drug. The work presented here indicates that the bioanalysis of protein drugs may be complicated owing to the presence of drug-binding endogenous components or ADAs in the post-dose (incurred) samples. The clear understanding of the behavior of different bioanalytical techniques vis-à-vis the potentially interfering components found in incurred samples is critical in selecting bioanalytical strategies for measuring protein drugs.

Keywords

ELISA LC-MS/MS Free and total drug Immunogenicity Pharmacokinetics Therapeutic proteins 

Abbreviations

ADA

Anti-drug antibody

BSA

Bovine serum albumin

ELISA

Enzyme-linked immunosorbent assay

HCl

Hydrochloric acid

HRP

Horseradish peroxidase

IgG

Immunoglobulin G

IS

Internal standard

LBA

Ligand binding assay

LC-MS/MS

High-performance liquid chromatography–tandem mass spectrometry

LLOQ

Low limit of quantification

mAb

Monoclonal antibody

OD

Optical density

PBS

Phosphate-buffered saline

PBST

Phosphate-buffered saline containing 0.05% Tween-20

PD

Pharmacodynamics

PEG

Polyethylene glycol

PK

Pharmacokinetics

QC

Quality control

SDS

Sodium dodecyl sulfate

SDS-PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SRM

Selective reaction monitoring

TMB

Tetramethylbenzidine

References

  1. 1.
    Viswanathan CT, Bansal S, Brian B, DeStefano AJ, Rose MJ, Sailstad J, Shah VP, Skelly JP, Swann PG, Weiner R (2007) Workshop/Conference report—quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. AAPS J 9:E30–E42CrossRefGoogle Scholar
  2. 2.
    Wang W, Wang EQ, Balthasar JP (2008) Monoclonal antibody pharmacokinetics and pharmacodynamics. Clin Pharmacol Ther 84:548–558CrossRefGoogle Scholar
  3. 3.
    Brekke OH, Sandlie I (2003) Therapeutic antibodies for human diseases at the dawn of the twenty-first century. Nature Review Drug Discovery 2:52–62CrossRefGoogle Scholar
  4. 4.
    Kelley M, DeSilva B (2007) Key elements of bioanalytical method validation for macromolecules. AAPS J 9:E156–E163CrossRefGoogle Scholar
  5. 5.
    Rosenberg AS, Worobec A (2005) A risk-based approach to immunogenicity concerns of therapeutic protein products. Part 3: effects of manufacturing changes in immunogenicity and the utility of animal immunogenicity studies. Biopharm International 18:32–36Google Scholar
  6. 6.
    Shankar G, Pendley C, Stein KE (2007) A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs. Nat Biotechnol 25:555–561CrossRefGoogle Scholar
  7. 7.
    Koren E, Smith HW, Shores E, Shankar G, Finco-Kent D, Rup B, Barrett YC, Devanarayan V, Gorovits B, Gupta S, Parish T, Quarmby V, Moxness M, Swanson SJ, Taniguchi G, Zuckerman LA, Stebbins CC, Mire-Sluis A (2008) Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products. J Immunol Methods 333:1–9CrossRefGoogle Scholar
  8. 8.
    Becher F, Pruvost A, Clement G, Tabet JC, Ezan E (2006) Quantification of small therapeutic proteins in plasma by liquid chromatography–tandem mass spectrometry: application to an elastase inhibitor EPI-hNE4. Anal Chem 78:2306–2313CrossRefGoogle Scholar
  9. 9.
    Buscher BA, Gerritsen H, van Schöll I, Cnubben NH, Brüll LP (2007) Quantitative analysis of Tenecteplase in rat plasma samples using LC-MS/MS as an alternative for ELISA. J Chromatogr B Analyt Technol Biomed Life Sci 852:631–634CrossRefGoogle Scholar
  10. 10.
    Wu ST, Ouyang Z, Olah TV, Jemal M (2011) A strategy for liquid chromatography/tandem mass spectrometry based quantitation of PEGylated protein drugs in plasma using plasma protein precipitation with water-miscible organic solvents and subsequent trypsin digestion to generate surrogate peptides for detection. Rapid Commun Mass Spectrom 25:281–290CrossRefGoogle Scholar
  11. 11.
    Liu H, Manuilov AV, Chumsae C, Babineau ML, Tarcsa E (2011) Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography–mass spectrometry. Anal Chem 414:147–153Google Scholar
  12. 12.
    Prenni JE, Shen Z, Trauger S, Chen W, Siuzdak G (2003) Protein characterization using liquid chromatography desorption ionization on silicon mass spectrometry. Spectroscopy 17:693–698Google Scholar
  13. 13.
    Lu Q, Zheng X, McIntosh T, Davis H, Nemeth JF, Pendley C, Wu SL, Hancock WS (2009) Development of different analysis platforms with LC-MS for pharmacokinetic studies of protein drugs. Anal Chem 81:8715–8723CrossRefGoogle Scholar
  14. 14.
    Heudi O, Barteau S, Zimmer D, Schmidt J, Bill K, Lehmann N, Bauer C, Kretz O (2008) Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry. Anal Chem 80:4200–4207CrossRefGoogle Scholar
  15. 15.
    Jemal M, Xia YQ (2006) LC-MS Development strategies for quantitative bioanalysis. Curr Drug Metab 7:491–502CrossRefGoogle Scholar
  16. 16.
    Jemal M, Ouyang Z, Xia YQ (2010) Systematic LC-MS/MS bioanalytical method development that incorporates plasma phospholipids risk avoidance, usage of incurred sample and well thought-out chromatography. Biomed Chromatogr 24:2–19CrossRefGoogle Scholar
  17. 17.
    Hakimi J, Chizzonite R, Luke DR, Familletti PC, Bailon P, Kondas JA, Pilson RS, Lin P, Weber DV, Spence C (1991) Reduced immunogenicity and improved pharmacokinetics of humanized anti-Tac in cynomolgus monkeys. J Immunol 147:1352–1359Google Scholar
  18. 18.
    Hwang WY, Foote J (2005) Immunogenicity of engineered antibodies. Methods 36:3–10CrossRefGoogle Scholar
  19. 19.
    van der Laken CJ, Voskuyl AE, Roos JC, Stigter van Walsum M, de Groot ER, Wolbink G, Dijkmans BA, Aarden LA (2007) Imaging and serum analysis of immune complex formation of radiolabelled infliximab and anti-infliximab in responders and non-responders to therapy for rheumatoid arthritis. Ann Rheum Dis 66:253–256CrossRefGoogle Scholar
  20. 20.
    Rehlaender BN, Cho MJ (1998) Antibodies as carrier proteins. Pharm Res 15:1652–1656CrossRefGoogle Scholar
  21. 21.
    Lee JW, Kelly M, King LE, Yang J, Salimi-Moosavi H, Tang MT, Lu JF, Kamerud J, Ahene A, Myler H, Rogers C (2011) Bioanalytical approaches to quantify “total” and “free” therapeutic antibodies and their targets: technical challenges and PK/PD applications over the course of drug development. AAPS J 13:99–110CrossRefGoogle Scholar
  22. 22.
    Walker DK (2004) The use of pharmacokinetic and pharmacodynamic data in the assessment of drug safety in early drug development. Br J Clin Pharmacol 58:601–608CrossRefGoogle Scholar
  23. 23.
    Hoos JS, Damsten MC, de Vlieger JS, Commandeur JN, Vermeulen NP, Niessen WM, Lingeman H (2007) Automated detection of covalent adducts to human serum albumin by immunoaffinity chromatography, on-line solution phase digestion and liquid chromatography–mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 859:147–156CrossRefGoogle Scholar
  24. 24.
    Dubois M, Becher F, Herbet A, Ezan E (2007) Immuno-mass spectrometry assay of EPI-HNE4, a recombinant protein inhibitor of human elastase. Rapid Commun Mass Spectrom 21:352–358CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Shujie J. Wang
    • 1
  • Steven T. Wu
    • 2
  • Jochem Gokemeijer
    • 3
  • Aberra Fura
    • 2
  • Murli Krishna
    • 2
  • Paul Morin
    • 2
  • Guodong Chen
    • 2
  • Karen Price
    • 4
  • David Wang-Iverson
    • 5
  • Timothy Olah
    • 2
  • Russell Weiner
    • 6
  • Adrienne Tymiak
    • 2
  • Mohammed Jemal
    • 2
  1. 1.Pfizer Inc.GrotonUSA
  2. 2.Bristol-Myers SquibbPrincetonUSA
  3. 3.Bristol-Myers SquibbWalthamUSA
  4. 4.Bristol-Myers SquibbNew BrunswickUSA
  5. 5.StocktonUSA
  6. 6.Merck Research LaboratoriesRahwayUSA

Personalised recommendations