Abstract
The foundation of pharmacokinetics and antidrug antibodies assay robustness relies on the use of high-quality reagents. Over the past decade, there has been increasing interest within the pharmaceutical industry, as well as regulators, on defining best practices and scientific approaches for generation, characterization and handling of critical reagents. In this review, we will discuss current knowledge and practices on critical reagent workflows and state-of-the-art approaches for characterization, generation, stability and storage and how each of these steps can impact ligand-binding assay robustness.
Lay abstract
A critical part of clinical development for new biologic drugs is the use of tests known as ligand-binding assay. These assays must be able to accurately measure drug levels and to assess if the biologic drug interacts with the immune system in patients. In order to support patient efficacy and safety, scientists must use state-of-the-art approaches to develop and identify specific reagents for each new biologic drug. This review aims to cover all key steps that are needed to support the quality and performance of the unique components of ligand-binding assays from the beginning of assay development and throughout the entire life-cycle of the biologic drug.
Papers of special note have been highlighted as: • of interest; •• of considerable interest
References
- 1. . Bioanalytical Method Validation M10 draft version (2019). https://www.ema.europa.eu/en/documents/scientific-guideline/draft-ich-guideline-m10-bioanalytical-method-validation-step-2b_en.pdf
- 2. Ligand binding assay critical reagents and their stability: recommendations and best practices from the Global Bioanalysis Consortium Harmonization Team. AAPS J. 16(3), 504–515 (2014). •• Provides comprehensive overview on ligand-binding assay reagent life-cycle management. The authors present harmonized recommendations focused on the characterization, lot changes, stability, storage and documentation of critical reagents.
- 3. 2019 White Paper on recent issues in bioanalysis: FDA immunogenicity guidance, gene therapy, critical reagents, biomarkers and flow cytometry validation (Part 3 - recommendations on 2019 FDA immunogenicity guidance, gene therapy bioanalytical challenges, strategies for critical reagent management, biomarker assay validation, flow cytometry validation & CLSI H62). Bioanalysis 11(24), 2207–2244 (2019).
- 4. . Single B cell antibody technologies. N. Biotechnol. 28(5), 453–457 (2011).
- 5. High efficiency creation of human monoclonal antibody-producing hybridomas. J. Immunol. Methods 291(1–2), 109–122 (2004).
- 6. . Phage display: a molecular tool for the generation of antibodies – a review. Placenta 21, S106–S112 (2000).
- 7. . Life cycle management of critical ligand-binding reagents. Bioanalysis 5(21), 2679–2696 (2013). • Authors describe the critical importance of maintaining supply and quality of reagents potentially over several decades and steps to ensure lot-to-lot consistency.
- 8. Ligand binding assays in the 21st century laboratory: recommendations for characterization and supply of critical reagents. AAPS J. 14(2), 316–328 (2012).
- 9. . Critical ligand binding reagent preparation/selection: when specificity depends on reagents. AAPS J. 9(2), E148–E155 (2007). • Authors discuss reagents generation from assay concept through immunization and functional characterization, and highlight need to adapt reagents and monitor changes as drug programs advance into late stages.
- 10. . Quality requirements for critical assay reagents used in bioanalysis of therapeutic proteins: what bioanalysts should know about their reagents. Bioanalysis 3(5), 523–534 (2011). •• Excellent review highlighting the impact of key steps from reagent generation through storage stability and key parameters for analytical characterization.
- 11. . Characterization of critical reagents in ligand-binding assays: enabling robust bioanalytical methods and lifecycle management. Bioanalysis 5(2), 227–244 (2013). •• Excellent resource for when considering impact of biophysical properties on reagent performance and options for use of higher resolution biophysical characterization.
- 12. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150(1), 76–85 (1985).
- 13. . Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193(1), 265–275 (1951).
- 14. 2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 3–LBA, biomarkers and immunogenicity). Bioanalysis 7(24), 3107–3124 (2015).
- 15. 2018 White Paper on recent issues in bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 – LBA/cell-based assays: immunogenicity, biomarkers and PK assays). Bioanalysis 10(24), 1973–2001 (2018).
- 16. US FDA. Immunogenicity Testing of Therapeutic Protein Products – Developing and Validating Assays for Anti-Drug Antibody Detection (2019). https://www.fda.gov/regulatory-information/search-fda-guidance-documents/immunogenicity-testing-therapeutic-protein-products-developing-and-validating-assays-anti-drug
- 17. . Protein aggregation: pathways, induction factors and analysis. J. Pharm. Sci. 98(9), 2909–2934 (2009).
- 18. . Role of analytical ultracentrifugation in assessing the aggregation of protein biopharmaceuticals. AAPS J. 8(3), E590–E605 (2006).
- 19. . Poisson statistical analysis of repetitive subcloning by the limiting dilution technique as a way of assessing hybridoma monoclonality. Methods Enzymol. 121, 412–417 (1986).
- 20. . How to make a hybridoma. Yale J. Biol. Med. 54(5), 387–402 (1981).
- 21. . Mass spectrometry for structural characterization of therapeutic antibodies. Mass Spectrom. Rev. 28(1), 147–176 (2009).
- 22. . Epitope binning of murine monoclonal antibodies by a multiplexed pairing assay. J. Immunol. Methods 365(1–2), 118–125 (2011).
- 23. . Biolayer interferometry predicts ELISA performance of monoclonal antibody pairs for Plasmodium falciparum histidine-rich protein 2. Anal. Biochem. 534, 10–13 (2017).
- 24. . The emerging role of biosensor-based epitope binning and mapping in antibody-based drug discovery. Expert Opin. Drug Discov. 11(10), 925–937 (2016).
- 25. High-throughput epitope binning assays on label-free array-based biosensors can yield exquisite epitope discrimination that facilitates the selection of monoclonal antibodies with functional activity. PLoS ONE 9(3), e92451 (2014).
- 26. . Measuring protein-protein and protein-nucleic acid interactions by biolayer interferometry. Curr. Protoc. Protein Sci. 79, 19.25.11–19.25.26 (2015).
- 27. Using multiple platforms for critical reagents selection process to support PK ligand-binding assay development. Bioanalysis 13(10), 769–769 (2021).
- 28. Antibodies targeting closely adjacent or minimally overlapping epitopes can displace one another. PLoS ONE 12(1), e0169535 (2017).
- 29. . New insights on critical reagent optimization for antidrug antibody assays. Bioanalysis 11(9), 815–823 (2019).
- 30. . Chapter 2 – functional targets for bioconjugation. In: Bioconjugate Techniques (3rd Edition). Academic Press (Ed.). Elsevier, MA, USA, 127–228 (2013).
- 31. . Chapter 18 – PEGylation and synthetic polymer modification. In: Bioconjugate Techniques (3rd Edition). Academic Press (Ed.). Elsevier, MA, USA, 787–838 (2013).
- 32. . Sortase-tag expressed protein ligation: combining protein purification and site-specific bioconjugation into a single step. Anal. Chem. 85(22), 11090–11097 (2013).
- 33. . Chapter 17 – chemoselective ligation; bioorthogonal reagents. In: Bioconjugate Techniques (3rd Edition). Academic Press (Ed.). Elsevier, MA, USA, 757–785 (2013).
- 34. . Physicochemical stability of monoclonal antibodies: a review. J. Pharm. Sci. 109(1), 169–190 (2020).
- 35. . A comparison of biophysical characterization techniques in predicting monoclonal antibody stability. MAbs 8(6), 1088–1097 (2016).
- 36. . Hydrogen ion buffers. Methods Enzymol. 24, 53–68 (1972).
- 37. . How sugars protect proteins in the solid state and during drying (review): mechanisms of stabilization in relation to stress conditions. Eur. J. Pharm. Biopharm. 114, 288–295 (2017).
- 38. . Stability testing of pharmaceutical products. J.Appl. Pharm. Sci. 2(3), 129–138 (2012).
- 39. . Determination of product shelf life and activation energy for five drugs of abuse. Clin. Chem. 37(3), 398–402 (1991).
- 40. . The theory and practice of industrial pharmacy. Second Ed. Lachman L, Lieberman HA, Kanig JL. (Eds). Lea & Febiger, 600 Washington Square, Philadelphia, PA 19106, 1976. 787pp. 18.5 × 26.5 cm. Price $38.50. J. Pharm. Sci. 65(8), 1267 (1976).
- 41. EBF recommendation on practical management of critical reagents for PK ligand-binding assays. Bioanalysis 10(19), 1557–1565 (2018).
- 42. EBF recommendation on practical management of critical reagents for antidrug antibody ligand-binding assays. Bioanalysis 11(19), 1787–1798 (2019).
- 43. . The decennial index of the White Papers in Bioanalysis: ‘a decade of recommendations (2007–2016)’. Bioanalysis 9(21), 1681–1702 (2017).