The AAPS Journal

, Volume 14, Issue 2, pp 316–328 | Cite as

Ligand Binding Assays in the 21st Century Laboratory: Recommendations for Characterization and Supply of Critical Reagents

  • Denise M. O’Hara
  • Valerie Theobald
  • Adrienne Clements Egan
  • Joel Usansky
  • Murli Krishna
  • Julie TerWee
  • Mauricio Maia
  • Frank P. Spriggs
  • John Kenney
  • Afshin Safavi
  • Jeannine Keefe
White Paper Theme: Ligand Binding Assays in the 21st Century Laboratory


Critical reagents are essential components of ligand binding assays (LBAs) and are utilized throughout the process of drug discovery, development, and post-marketing monitoring. Successful lifecycle management of LBA critical reagents minimizes assay performance problems caused by declining reagent activity and can mitigate the risk of delays during preclinical and clinical studies. Proactive reagent management assures adequate supply. It also assures that the quality of critical reagents is appropriate and consistent for the intended LBA use throughout all stages of the drug development process. This manuscript summarizes the key considerations for the generation, production, characterization, qualification, documentation, and management of critical reagents in LBAs, with recommendations for antibodies (monoclonal and polyclonal), engineered proteins, peptides, and their conjugates. Recommendations are given for each reagent type on basic and optional characterization profiles, expiration dates and storage temperatures, and investment in a knowledge database system. These recommendations represent a consensus among the authors and should be used to assist bioanalytical laboratories in the implementation of a best practices program for critical reagent life cycle management.

Key words

characterization critical reagents data management inventory knowledge database ligand binding assays 





Anti-drug antibody




Bovine serum albumin


Contract research organization


Dynamic light scattering




Good laboratory practices (21CFR part 58)


Host cell protein


High-performance liquid chromatography


Horseradish peroxidase


Immunoglobulin G


Investigational new drug


Keyhole limpet hemocyanin


Ligand binding assay(s)


Liquid chromatography


Laboratory information management system


Monoclonal antibody(ies)


Mass spectrometry


Neutralizing antibody(ies)


Polyclonal antibody(ies)


Positive control




Quality control(s)


Signal-to-background ratio


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis


Size exclusion chromatography


Standard operating procedure


Surface plasmon resonance



We extend our thanks to all the reviewers of this manuscript, in particular, Jean Lee, Valerie Quarmby, Marian Kelley, all departmental reviewers, and the Ligand Binding Assay Bioanalytical Focus Group (LBABFG) of the American Association of Pharmaceutical Scientists (AAPS) steering committee for their critical review of this manuscript and helpful comments. We also thank Terri Caiazzo (Pfizer), Rosemary Lawrence-Henderson (Pfizer), Brian J. Geist (Janssen R&D, LLC), Michele Frigo (Janssen R&D, LLC), Tong-Yuan Yang (Janssen R&D, LLC), and Yanhong Li (Genentech/Roche) for providing data shown in the Electronic supplementary material S1. We acknowledge the AAPS Twenty-First Century Bioanalytical Laboratory Workshop: planning committee members Jean Lee (co-chair), Valerie Quarmby (co-chair), Ago Ahene, Marian Kelley, Sheldon Leung, Chad Ray, Huifen Faye Wang, Melvin Weinswig; the programming committee (Jean Lee and Valerie Quarmby); and the steering committee (Ago Ahene, Chad Ray and Sheldon Leung). This manuscript was prepared by members of the Twenty-First Century Bioanalytical Laboratory: Reagent Subcommittee of the LBABFG of AAPS.

Supplementary material

12248_2012_9334_Fig3_ESM.jpg (70 kb)
Fig. S1

Examples of reagent characterizations and assay performance. a A large quantity of a critical reagent was requested from a vendor who had previously supplied it using a soluble expression system in CHO cells. To supply material for the large request, the vendor changed the expression system to Escherichia coli, harvested inclusion bodies, and refolded the protein reagent. Performance of the E. coli expressed protein in an established LBA showed similar maximum and minimum signal responses compared with the earlier preparation. However, three times the amount of this protein was required to coat the plate and the IC50 of the ELISA changed by twofold relative to the LBA, using the reagent from the mammalian secreted process. Characterization of the reagents, derived from the different expression systems, by SEC showed that the reagent from the E. coli process eluted earlier and had an apparent higher molecular weight than the corresponding reagent from the CHO-derived process. Expressing, harvesting, and refolding protein reagents from bacterial inclusion bodies may have resulted in misfolded and/or aggregated material that was not present in the reagent lot from the soluble mammalian expression system. Qualification of the new reagent lot relative to the soluble mammalian expressed reagent demonstrated differences in performance of the LBA, and the SEC profile of the reagent provided a means to screen new lots of this critical reagent. b The biophysical state of an LBA capture reagent can affect assay performance. The example here is of a recombinant protein that naturally exists in a dimeric state. The protein was purified as monomeric and dimeric proteins and subsequently conjugated with biotin. Under identical conditions (10% mouse plasma) and concentrations, these capture reagents were coated onto streptavidin plates to compare their ability to capture a biotherapeutic recognizing the target protein. While the assay background remained the same, the signal/noise ratio was higher, by at least tenfold with the monomeric capture reagent than the dimeric capture reagent. c Asterisk: sheep polyclonal anti-human IgG antibody spiked into cynomolgus pooled serum. A bridging assay format was used, in which assay signal derived from ADA molecules that bridged two drug molecules, one drug molecule labeled with ruthenium, the other with biotin. ECL units were measured using a commercial chemiluminescence analyzer. A therapeutic MAb was conjugated with a ruthenium-label at different molar challenge ratios. The effect of different molar challenge ratios is evident in the LBA signal, which corresponds to electrochemiluminescence (ECL) units measured in an immunogenicity assay. The ADA specific for the therapeutic MAb was detected using a bridging assay format where the assay signal is obtained from molecules that bridged two labeled drug molecules (one labeled with ruthenium, the other labeled with biotin). The assay background and the positive-sample assay signal increased with increasing challenge ratios. The signal-to-background (S/B) ratio also increased with higher molar-ratio challenges, indicating the potential for improved method sensitivity with greater ratios of ruthenium to protein. d To develop a NAb assay, a positive control (PC) with neutralizing activity is required as a critical reagent. Several animals may be initially immunized initially to generate a PAb PC, and it is essential to gather information on the sample time points (bleeds) for neutralizing activity. In this assay, a dramatic decrease in IC50 in the LBA is observed as the neutralizing activity of PAbs matures with time a dramatic decrease in IC50 in the LBA is observed. This demonstrates that bleeds collected at different times from the same animal are not always equivalent in reactivity. It is important to maximize the amount of each lot of PAb reagent, perhaps by pooling several bleeds with appropriate activity. As part of a strategic plan to maintain the assay and to assure appropriate controls, subsequent lots may require a partial validation to determine new assay acceptance criteria for that new assay reagent. e Four lots of reagent Abs were produced and purified on separate occasions from the same hybridoma cell bank. The purification procedures for all lots were similar, and no differences between lots were observed when the Abs were evaluated by SEC or by reducing and non-reducing SDS-PAGE. The purified reagent Abs were then conjugated with ruthenium for use as a detection Ab in a PK assay. A conjugated MAb from Lot #301 was initially validated at a working concentration of 1.25 μg/mL in the PK assay, and attempts were made to qualify the subsequent lots at the same concentration. A conjugated antibody made from Lot #302 was qualified in the assay at an identical concentration as Lot #301. However, a conjugate made from Lot #303 could not be qualified in the assay and a conjugate made from Lot #304 could only be qualified in the assay at twice the concentration (2.5 μg/mL) of earlier lots. The source lots of these conjugated reagent antibodies were then evaluated for intact and reduced forms by LC-MS (reversed-phase HPLC coupled to time-of-flight mass spectrometry). The LC-MS experiments revealed that Lot # 301 and 302 had nearly identical spectra for their heavy chains, while a set of larger (∼1 kD shift) peaks were in the spectra from the heavy chains of Lots 303 and 304. These experiments indicate that as the relative signal of this higher mass species (when compared with the signal of the original mass) increased, the assay performance also decreased. Therefore, the LC-MS method could be used to prescreen subsequent lots for use in the assay (JPEG 70 kb)

12248_2012_9334_MOESM1_ESM.tif (1.9 mb)
High-resolution image (TIFF 1953 kb)


  1. 1.
    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-E122.Google Scholar
  2. 2.
    Cawkill D, Eaglestone SS. Evolution of cell-based reagent provision. Drug Discov Today. 2007;12(19–20):820–5.PubMedCrossRefGoogle Scholar
  3. 3.
    Mairal T, Cengiz Özalp V, Lozano Sánchez P, Mir M, Katakis I, O’Sullivan CK. Aptamers: molecular tools for analytical applications. Anal Bioanal Chem. 2008;390(4):989–1007.PubMedCrossRefGoogle Scholar
  4. 4.
    Tremblay GA, Oldfield PR. Bioanalysis of siRNA and oligonucleotide therapeutics in biological fluids and tissues. Bioanalysis. 2009;1(3):595–609.PubMedCrossRefGoogle Scholar
  5. 5.
    Lee JW, Devanarayan V, Barrett YC, Weiner R, Allinson J, Fountain S, et al. Fit-for-purpose method development and validation for successful biomarker measurement. Pharm Res. 2006;23(2):312–28.PubMedCrossRefGoogle Scholar
  6. 6.
    Lee JW, Weiner RS, Sailstad JM, Bowsher RR, Knuth DW, O’Brien PJ, et al. Method validation and measurement of biomarkers in nonclinical and clinical samples in drug development: a conference report. Pharm Res. 2005;22(4):499–511.PubMedCrossRefGoogle Scholar
  7. 7.
    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
  8. 8.
    Gupta S, Indelicato SR, Jethwa V, Kawabata T, Kelley M, Mire-Sluis AR, et al. Recommendations for the design, optimization, and qualification of cell-based assays used for the detection of neutralizing antibody responses elicited to biological therapeutics. J Immunol Methods. 2007;321(1–2):1–18.PubMedCrossRefGoogle Scholar
  9. 9.
    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
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    O’Hara DM, Xu Y, Liang Z, Reddy MP, Wu DY, Litwin V. Recommendations for the validation of flow cytometric testing during drug development: II assays. J Immunol Methods. 2011;363(2):120–34.PubMedCrossRefGoogle Scholar
  12. 12.
    Good Laboratory Practice for Nonclinical Laboratory Studies, Title 21 CFR Part 58.83 (2007).Google Scholar
  13. 13.
    Rup B, O’Hara D. Critical ligand binding reagent preparation/selection: when specificity depends on reagents. AAPS J. 2007;9(2):E148-E155.Google Scholar
  14. 14.
    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.PubMedCrossRefGoogle Scholar
  15. 15.
    Bjerner J, Børmer OP, Nustad K. The war on heterophilic antibody interference. Clin Chem. 2005;51(1):9–11.PubMedCrossRefGoogle Scholar
  16. 16.
    Ismail AAA. Interference from endogenous antibodies in automated immunoassays: what laboratorians need to know. J Clin Pathol. 2009;62(8):673–8.PubMedCrossRefGoogle Scholar
  17. 17.
    Levinson SS, Miller JJ. Towards a better understanding of heterophile (and the like) antibody interference with modern immunoassays. Clinica Chimica Acta. 2002;325(1–2):1–15.CrossRefGoogle Scholar
  18. 18.
    Altinier S, Varagnolo M, Zaninotto M, Boccagni P, Plebani M. Heterophilic antibody interference in a non-endogenous molecule assay: an apparent elevation in the tacrolimus concentration. Clinica Chimica Acta. 2009;402(1–2):193–5.CrossRefGoogle Scholar
  19. 19.
    Kahn MN, Findlay JWA, editors. Ligand binding assays: development, validation and implementation in the drug development arena. Hoboken: John Wiley & Sons; 2010.Google Scholar
  20. 20.
    DeForge LE, Loyet KM, Delarosa D, Chinn J, Zamanian F, Chuntharapai A, et al. Evaluation of heterophilic antibody blocking agents in reducing false positive interference in immunoassays for IL-17AA, IL-17FF, and IL-17AF. J Immunol Methods. 2010;362(1–2):70–81.PubMedCrossRefGoogle Scholar
  21. 21.
    Preissner CM, Dodge LA, O’Kane DJ, Singh RJ, Grebe SKG. Prevalence of heterophilic antibody interference in eight automated tumor marker immunoassays. Clin Chem. 2005;51(1):208–10.PubMedCrossRefGoogle Scholar
  22. 22.
    Spencer DV, Nolte FS, Zhu Y. Heterophilic antibody interference causing false-positive rapid human immunodeficiency virus antibody testing. Clinica Chimica Acta. 2009;399(1–2):121–2.CrossRefGoogle Scholar
  23. 23.
    Hennig C, Rink L, Fagin U, Jabs WJ, Kirchner H. The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs. J Immunol Methods. 2000;235(1–2):71–80.PubMedCrossRefGoogle Scholar
  24. 24.
    Levinson SS. Antibody multispecificity in immunoassay interference. Clin Biochem. 1992;25(2):77–87.PubMedCrossRefGoogle Scholar
  25. 25.
    Tatarewicz S, Miller JM, Swanson SJ, Moxness MS. Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics. J Immunol Methods. 2010;357(1–2):10–6.PubMedCrossRefGoogle Scholar
  26. 26.
    Lee JW, Kelley M, King LE, Yang J, Salimi-Moosavi H, Tang MT, et al. 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. 2011;13(1):99–110.PubMedCrossRefGoogle Scholar
  27. 27.
    Patton A, Mullenix MC, Swanson SJ, Koren E. An acid dissociation bridging ELISA for detection of antibodies directed against therapeutic proteins in the presence of antigen. J Immunol Methods. 2005;304(1–2):189–95.PubMedCrossRefGoogle Scholar
  28. 28.
    Wild DG, editor. The immunoassay handbook. 3rd ed. Kidlington: Elsevier Science; 2005.Google Scholar
  29. 29.
    Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256(5517):495–7.PubMedCrossRefGoogle Scholar
  30. 30.
    Milstein C. With the benefit of hindsight. Immunol Today. 2000;21(8):359–64.PubMedCrossRefGoogle Scholar
  31. 31.
    Subramanian G, editor. Antibodies: volume 2: novel technologies and therapeutic use. New York, NY: Kluwer Academic/Plenum Publishers; 2004.Google Scholar
  32. 32.
    Spieker-Polet H, Sethupathi P, Yam PC, Knight KL. Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit–rabbit hybridomas. Proc Natl Acad Sci U S A. 1995;92(20):9348–52.PubMedCrossRefGoogle Scholar
  33. 33.
    Bradbury ARM, Marks JD. Antibodies from phage antibody libraries. J Immunol Methods. 2004;290(1–2):29–49.PubMedCrossRefGoogle Scholar
  34. 34.
    Lipovsek D, Plückthun A. In-vitro protein evolution by ribosome display and mRNA display. J Immunol Methods. 2004;290(1–2):51–67.PubMedCrossRefGoogle Scholar
  35. 35.
    Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR. Making antibodies by phage display technology. Annu Rev Immunol. 1994;12:433–55.PubMedCrossRefGoogle Scholar
  36. 36.
    Cohen AD, Boyer JD, Weiner DB. Modulating the immune response to genetic immunization. FASEB J. 1998;12(15):1611–26.PubMedGoogle Scholar
  37. 37.
    Sundaram P, Xiao W, Brandsma JL. Particle-mediated delivery of recombinant expression vectors to rabbit skin induces high-titered polyclonal antisera (and circumvents purification of a protein immunogen). Nucleic Acids Res. 1996;24(7):1375–7.PubMedCrossRefGoogle Scholar
  38. 38.
    Tang D, DeVit M, Johnston SA. Genetic immunization is a simple method for eliciting an immune response. Nature. 1992;356(6365):152–4.PubMedCrossRefGoogle Scholar
  39. 39.
    Witkowski PT, Bourquain DR, Hohn O, Schade R, Nitsche A. Gene gun-supported DNA immunisation of chicken for straightforward production of poxvirus-specific IgY antibodies. J Immunol Methods. 2009;341(1–2):146–53.PubMedCrossRefGoogle Scholar
  40. 40.
    Englebienne P. Immune and receptor assays in theory and practice. Boca Raton: CRC Press; 2000.Google Scholar
  41. 41.
    Gaberc-Porekar V, Zore I, Podobnik B, Menart V. Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr Opin Drug Discov Dev. 2008;11(2):242–50.Google Scholar
  42. 42.
    Su YC, Chen BM, Chuang KH, Cheng TL, Roffler SR. Sensitive quantification of PEGylated compounds by second-generation anti-poly(ethylene glycol) monoclonal antibodies. Bioconjug Chem. 2010;21(7):1264–70.PubMedCrossRefGoogle Scholar
  43. 43.
    Babu S. Criteria for CMO selection. Am Pharm Outsourcing. 2009;10(5):10–5.Google Scholar
  44. 44.
    Dellva P. Managing outsourcing relationships: what to do? What not to do? Am Pharm Outsourcing. 2003;4(4):30–3.Google Scholar
  45. 45.
    Bock I, Dhayalan A, Kudithipudi S, Brandt O, Rathert P, Jeltsch A. Detailed specificity analysis of antibodies binding to modified histone tails with peptide arrays. Epigenetics. 2011;6(2):256–63.PubMedCrossRefGoogle Scholar
  46. 46.
    Hawe A, Sutter M, Jiskoot W. Extrinsic fluorescent dyes as tools for protein characterization. Pharm Res. 2008;25(7):1487–99.PubMedCrossRefGoogle Scholar
  47. 47.
    Hawe A, Friess W, Sutter M, Jiskoot W. Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation. Anal Biochem. 2008;378(2):115–22.PubMedCrossRefGoogle Scholar
  48. 48.
    Cleland JL, Powell MF, Shire SJ. The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation. Crit Rev Ther Drug Carrier Syst. 1993;10(4):307–77.PubMedGoogle Scholar
  49. 49.
    Tsai PK, Bruner MW, Irwin JI, Ip CCY, Oliver CN, Nelson RW, et al. Origin of the isoelectric heterogeneity of monoclonal immunoglobulin h1B4. Pharm Res. 1993;10(11):1580–6.PubMedCrossRefGoogle Scholar
  50. 50.
    Liu H, Caza-Bulseco G, Faldu D, Chumsae C, Sun J. Heterogeneity of monoclonal antibodies. J Pharm Sci. 2008;97(7):2426–47.PubMedCrossRefGoogle Scholar
  51. 51.
    Manningc M, Patel K, Borchardt RT. Stability of protein pharmaceuticals. Pharm Res. 1989;6(11):903–18.CrossRefGoogle Scholar
  52. 52.
    Kueltzo LA, Wang W, Randolph TW, Carpenter JF. Effects of solution conditions, processing parameters, and container materials on aggregation of a monoclonal antibody during freeze-thawing. J Pharm Sci. 2008;97(5):1801–12.PubMedCrossRefGoogle Scholar
  53. 53.
    Jiskoot W, Beuvery EC, De Koning AAM, Herron JN, Crommelin DJA. Analytical approaches to the study of monoclonal antibody stability. Pharm Res. 1990;7(12):1234–41.PubMedCrossRefGoogle Scholar
  54. 54.
    Ritter N, Wiebe M. Validating critical reagents used in CGMP analytical testing: Eensuring method integrity and reliable assay performance. BioPharm. 2001;14(5):12–21.Google Scholar
  55. 55.
    ICH. Q2b. Guidance for industry: validation of analytical procedures methodology 2001.Google Scholar
  56. 56.
    Simonet BM. Quality control in qualitative analysis. TrAC-Trends Anal Chem. 2005;24(6):525–31.CrossRefGoogle Scholar
  57. 57.
    Bowsher RR, Sailstad JM. Insights in the application of research-grade diagnostic kits for biomarker assessments in support of clinical drug development: bioanalysis of circulating concentrations of soluble receptor activator of nuclear factor κB ligand. J Pharm Biomed Anal. 2008;48(5):1282–9.PubMedCrossRefGoogle Scholar
  58. 58.
    Nowatzke W. Systematic analytical validation of commercial kits for the determination of novel biomarkers for clinical drug development. Bioanalysis. 2010;2(2):237–47.PubMedCrossRefGoogle Scholar
  59. 59.
    Crowther JR. The ELISA guidebook. 2nd ed. New York: Humana Press; 2010.Google Scholar
  60. 60.
    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.PubMedCrossRefGoogle Scholar
  61. 61.
    Ruttenberg A, Clark T, Bug W, Samwald M, Bodenreider O, Chen H, et al. Advancing translational research with the Semantic Web. BMC Bioinformatics. 2007;8(SUPPL. 3).Google Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2012

Authors and Affiliations

  • Denise M. O’Hara
    • 1
  • Valerie Theobald
    • 2
  • Adrienne Clements Egan
    • 3
  • Joel Usansky
    • 4
  • Murli Krishna
    • 5
  • Julie TerWee
    • 6
  • Mauricio Maia
    • 7
  • Frank P. Spriggs
    • 8
  • John Kenney
    • 9
  • Afshin Safavi
    • 10
  • Jeannine Keefe
    • 11
  1. 1.Department of Pharmacokinetics, Dynamics and MetabolismPfizer Inc (formerly Wyeth)AndoverUSA
  2. 2.Clinical Assay DevelopmentGenzyme, A Sanofi CompanyFraminghamUSA
  3. 3.Department of Biologics Clinical PharmacologyJanssen Research and Development, LLCRadnorUSA
  4. 4.Product Management DepartmentThermo Fisher ScientificPhiladelphiaUSA
  5. 5.ABD-BioAnalytical Sciences, BiologicsBristol-Myers Squibb CompanyPrincetonUSA
  6. 6.Quality ControlAmgen, IncLongmontUSA
  7. 7.BioAnalytical Sciences DepartmentGenentech/RocheSouth San FranciscoUSA
  8. 8.Department of Pharmacokinetics, Dynamics, and MetabolismPfizer, IncAndoverUSA
  9. 9.Antibody SolutionsSunnyvaleUSA
  10. 10.BioAnalytical OperationsBioAgilytix LabsDurhamUSA
  11. 11.Preclinical and Clinical Development SciencesBiogen Idec IncCambridgeUSA

Personalised recommendations