The AAPS Journal

, Volume 16, Issue 3, pp 504–515 | Cite as

Ligand Binding Assay Critical Reagents and Their Stability: Recommendations and Best Practices from the Global Bioanalysis Consortium Harmonization Team

  • Lindsay E. KingEmail author
  • Esme Farley
  • Mami Imazato
  • Jeannine Keefe
  • Masood Khan
  • Mark Ma
  • K. Susanne Pihl
  • Priya Sriraman
White Paper Theme: Best Practices for Bioanalytical Methods: Recommendations from the Global Bioanalysis Consortium
Part of the following topical collections:
  1. Theme: Best Practices for Bioanalytical Methods: Recommendations from the Global Bioanalysis Consortium


The L4 Global Harmonization Team on reagents and their stability focused on the management of critical reagents for pharmacokinetic, immunogenicity, and biomarker ligand binding assays. Regulatory guidance recognizes that reagents are important for ligand binding assays but do not address numerous aspects of critical reagent life cycle management. Reagents can be obtained from external vendors or developed internally, but regardless of their source, there are numerous considerations for their reliable long-term use. The authors have identified current best practices and provided recommendations for critical reagent lot changes, stability management, and documentation.


critical reagents ligand binding assays lot changes recommendations stability 



We would like to acknowledge and thank the many scientists who provided feedback on the draft recommendations and completed the survey questionnaire. The feedback and information we received were very important in framing our final recommendations. We would also like to thank those who critically and carefully reviewed the final recommendations and manuscript.


  1. 1.
    DeSilva B, Smith W, Weiner R, Kelley M, Smolec J, Lee B, et al. Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm Res. 2003;20(11):1885–900.PubMedCrossRefGoogle Scholar
  2. 2.
    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.PubMedCrossRefGoogle Scholar
  3. 3.
    Nowatzke W, Woolf E. Best practices during bioanalytical method validation for the characterization of assay reagents and the evaluation of analyte stability in assay standards, quality controls, and study samples. AAPS J. 2007;9(2):E117–22.PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Rup B, O’Hara D. Critical ligand binding reagent preparation/selection: when specificity depends on reagents. AAPS J. 2007;9(1):E148–55.PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Staack RF, Stracke JO, Stubenrauch K, Vogel R, Schleypen J, Papadimitriou A. Quality requirements for critical assay reagents used in bioanalysis of therapeutic proteins: what bioanalysts should know about their reagents. Bioanalysis. 2011;3(5):523–34. doi: 10.4155/bio.11.16.PubMedCrossRefGoogle Scholar
  6. 6.
    O’Hara DM, Theobald V, Egan AC, Usansky J, Krishna M, TerWee J, et al. Ligand binding assays in the 21st century laboratory: recommendations for characterization and supply of critical reagents. AAPS J. 2012;14(2):316–28. doi: 10.1208/s12248-012-9334-9.PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine. Guidance for industry: bioanalytical method validation. Fed Regist. 2001;66(100):28526.Google Scholar
  8. 8.
    European Medicines Agency, Committee for Medicinal Products for Human Use. Guideline on bioanalytical method validation. EMEA/CHMP/EWP/192217/2009 21 Jul 2011.Google Scholar
  9. 9.
    Clinical Laboratory Standards Institute (Formerly NCCLS). Assessing the quality of immunoassay systems: radioimmunoassays and enzyme, fluorescence, and luminescence immunoassays; approved guideline. NCCLS I/LA23-A 2004;24 (16).Google Scholar
  10. 10.
    Geist BJ, Egan AC, Yang TY, Dong Y, Shankar G. Characterization of critical reagents in ligand-binding assays: enabling robust bioanalytical methods and lifecycle management. Bioanalysis. 2013;5(2):227–44. doi: 10.4155/bio.12.304.PubMedCrossRefGoogle Scholar
  11. 11.
    Polifke T. Introduction to biochemicals and chemical reagents. IVD Technology [Internet]. 2010 Oct 16(6).
  12. 12.
    Ezzelle J, Rodriguez-Chavez IR, Darden JM, Stirewalt M, Kunwar N, Hitchcock R, et al. Guidelines on good clinical laboratory practice: bridging operations between research and clinical research laboratories. J Pharm Biomed Anal. 2008;46(1):18–29.PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Khan MN, Dass PD, Leete JH, Schuman RF, Gunsior M, Sadhu C. Development of ligand-binding assays for drug development support. In: Khan MN, Findlay JW, editors. Ligand-binding assays: development, validation, and implementation in the drug development Arena. Hoboken: Wiley; 2009. p. 39–79.CrossRefGoogle Scholar
  14. 14.
    Miller WG, Erek A, Cunningham TD, Oladipo O, Scott MG, Johnson RE. Commutability limitations influence quality control results with different reagent lots. Clin Chem. 2011;57(1):76–83.PubMedCrossRefGoogle Scholar
  15. 15.
    Myler HA, Phillips KR, Dong H, Tabler E, Shaikh M, Coats V, et al. Validation and life-cycle management of a quantitative ligand-binding assay for the measurement of Nulojix®, a CTLA-4–Fc fusion protein, in renal and liver transplant patients. Bioanalysis. 2012;4(10):1215–26.PubMedCrossRefGoogle Scholar
  16. 16.
    van Amsterdam P, Companjen A, Brudny-Kloeppel M, Golob M, Luedtke S, Timmerman P. The European Bioanalysis Forum community’s evaluation, interpretation and implementation of the European Medicines Agency guideline on bioanalytical method validation. Bioanalysis. 2013;5(6):645–50.PubMedCrossRefGoogle Scholar
  17. 17.
    Anderson G, Scott M. Determination of product shelf life and activation energy for five drugs of abuse. Clin Chem. 1991;37(3):398–402.PubMedGoogle Scholar
  18. 18.
    Kirkwood TB. Predicting the stability of biological standards and products. Biometrics. 1977;33(4):736–42.PubMedCrossRefGoogle Scholar
  19. 19.
    Wang W, Singh S, Zeng DL, King K, Nema S. Antibody structure, instability, and formulation. J Pharm Sci. 2007;96(1):1–26.PubMedCrossRefGoogle Scholar
  20. 20.
    Haldar S, Mitra S, Chattopadhyay K. Role of protein stabilizers on the conformation of the unfolded state of cytochrome c and its early folding kinetics: investigation at single molecular resolution. J Biol Chem. 2010;285(33):25314–23. doi: 10.1074/jbc.M110.116673.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Falconer RJ, Chan C, Hughes K, Munro T. Stabilization of a monoclonal antibody during purification and formulation by addition of basic amino acid excipients. J Chem Technol Biotechnol. 2011;86:942–8.CrossRefGoogle Scholar
  22. 22.
    Chang BS, Yeung B. Physical stability of protein pharmaceuticals. In: Jameel F, Hershenson S, editors. Formulation and process development strategies for manufacturing biopharmaceuticals. Hoboken: Wiley; 2010. p. 69–104.CrossRefGoogle Scholar
  23. 23.
    van de Weert M, Randolph TW. Physical instability of peptide and proteins. In: Hovgaard L, Frokjaer S, van de Weert M, editors. Pharmaceutical formulation development of peptides and proteins. Boca Raton: Taylor and Francis; 2012. p. 107–30.Google Scholar
  24. 24.
    Meyer BK, Ni A, Hu B, Shi L. Antimicrobial preservative use in parenteral products: past and present. J Pharm Sci. 2007;96(12):3155–67.PubMedCrossRefGoogle Scholar
  25. 25.
    US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research. Guidance for industry: container closure systems for packaging human drugs and biologics. Chemistry, manufacturing, and controls documentation. Fed Regist. 1999;64(129):36694.Google Scholar
  26. 26.
    ICH Harmonised Tripartite Guideline. Bracketing and matrixing designs for stability testing of new drug substances and products. Q1D. US Fed Regist 16 Jan 2003;68(11) 2339. CPMP/ICH/4104/00 February 2002. MHLW PFSB/ELD Notification No. 0731004 31 Jul 2002.Google Scholar
  27. 27.
    Westgard JO, Klee GG. Quality management. In: Burtis CA, Ashwood ER, editors. Tietz textbook of clinical chemistry. 3rd ed. Philadelphia: W.B. Saunders; 1999. p. 407.Google Scholar
  28. 28.
    Krause PR. Goals of stability evaluation throughout the vaccine life cycle. Biologicals. 2009;37(6):369–78.PubMedCrossRefGoogle Scholar
  29. 29.
    US Department of Health and Human Services, Food and Drug Administration. Good laboratory practice for nonclinical laboratory studies. 21CFR 58. Fed Regist. 1976;41(225):51206 (Proposed Regulations) and Federal Register 43(247) 59986 22 December 1978 (Final Rule).Google Scholar
  30. 30.
    ICH Harmonised Tripartite Guideline. Validation of analytical procedures: text and methodology Q2(R1). US Fed Regist 19 May 1997;62(96) 27463. CPMP/ICH281/95 December 1996. MHLW PMSB/ELD Notification No. 338 October 1997.Google Scholar
  31. 31.
    Viswanathan CT, Bansal S, Booth B, DeStefano AJ, Rose MJ, Sailstad J, et al. Workshop/conference report—quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. AAPS J. 2007;9(1):E30–42.PubMedCentralCrossRefGoogle Scholar
  32. 32.
    Bunk DM, Welch MJ. Characterization of a new certified reference material for human cardiac troponin I. Clin Chem. 2006;52(2):212–9.PubMedCrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2014

Authors and Affiliations

  • Lindsay E. King
    • 1
    Email author
  • Esme Farley
    • 2
  • Mami Imazato
    • 3
  • Jeannine Keefe
    • 4
  • Masood Khan
    • 5
  • Mark Ma
    • 6
  • K. Susanne Pihl
    • 7
  • Priya Sriraman
    • 8
  1. 1.Pfizer, Inc.GrotonUSA
  2. 2.Quintiles Bioanalytical LabsA Quintiles Company and ADME LabManassasUSA
  3. 3.Novartis Pharma K.K.Minato-kuJapan
  4. 4.Biogen Idec, Inc.CambridgeUSA
  5. 5.KCAS, LLCShawneeUSA
  6. 6.Amgen Inc.Thousand OaksUSA
  7. 7.H. Lundbeck A/SValbyDenmark
  8. 8.Celgene CorporationSummitUSA

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