Ligand binding assays require specific biomolecular interactions between the critical reagents and the analyte to identify and quantify the analyte. Used throughout a drug life cycle, from discovery and development to post-marketing surveillance, ligand binding assays give us bioanalytical data that supports in vitro screening, product development and release, biomarker, PK and immunogenicity analyses.
Critical reagents used in these assays include antibodies, peptides, engineered proteins, complex biologics, chemically synthesized molecules, solid supports and matrices, as well as antibody, protein and peptide conjugates. Developing critical reagents is both a cost and time intensive process to make sure that any reagents selected for development show sufficient robustness and reproducibility to the required assay conditions, particularly in the presence of biological matrices. As they are generated by biological processes, critical reagents are inherently prone to batch-to-batch variation. This makes thorough characterisation of these reagents essential to ensure the unique characteristics that drive assay performance are selected for and maintained over the drug development life cycle.
Regulatory guidance recognises the importance of these reagents in ligand binding assays, but no official regulations exist for the control of the critical reagents. Successful management of critical reagents for ligand binding assays minimises the risk of assay performance problems that stem from declining reagent activity or the loss of reagent supply, resulting in less disruption and delay during preclinical and clinical studies. Proper characterisation, maintenance, long-term storage and inventory of critical reagents enables the smooth transition of any ligand binding assay between different laboratories and the timely collection of data.
Harmonisation guidelines for the characterisation and supply of critical reagents (link is external) were developed by the Global Bioanalysis Consortium, following the 2009 American Association of Pharmaceutical Scientists workshop on the Twenty-First Century Bioanalytical Laboratory,1 sponsored by the Ligand Binding Assay Bioanalytical Focus Group. The purpose of these guidelines is to build alignment across the industry on the expectations and operations of a bioanalytical lab.
Best Practice Recommendations for Ligand Binding Assay Critical Reagent Characterisation
To ensure optimal performance of the ligand binding assay over time, in terms of specificity, sensitivity and reproducibility, it is recommended that critical reagents are characterised according to the following points:
- Characterisation of any critical reagent according to the physico-chemical factors of reagent concentration, binding activity, aggregation levels, purity levels and molecular weight, with other factors of cross-reactivity and stability to be assessed as needed according to assay conditions.
- SDS-PAGE or SEC can be employed to confirm purity and monodispersity of the reagent prior to its use in the ligand binding assay.
- Building purification assays for critical protein reagents into the early stages of drug development may reduce the impact of impurities, which can affect stability during long term storage.
- Evaluation of conjugate ratios to ensure optimal conditions are selected and excess reactants removed from the product to assure reproducibility between critical reagent batches.
- Where appropriate, consider assessing optional characterization parameters such as determining protein A levels in a protein purified using a protein A column, bovine IgG levels from tissue culture expansion, and residual host cell protein levels.
- Final release criteria for any batch of critical reagent may need to include a functional assessment of the reagent, as physico-chemical characterisation alone may not accurately predict performance.
Monitoring these important characteristics in this way allows bioanalytical scientists to guide the generation of reagents and screen different lots that may be qualified for use in a ligand binding assay, identify the cause of any assay performance problems and determine when a reagent is deteriorating and must be replaced.
Applying critical reagent standards to Affimer processes
Avacta develop catalogue and custom anti-idiotypic reagents for use across all stages of drug development and patient therapeutic drug monitoring for innovator and biosimilar products. Our anti-idiotypic Affimer reagents are rapid to develop and show a high level of specificity and sensitivity when incorporated into PK assays. Manufactured using a recombinant bacterial production process, batch-to-batch consistency for Affimer reagents is assured throughout the drug development process. As part of the Affimer development and production processes we apply best practices for critical reagent quality control through analysis of purity, concentration and binding characteristics, with the potential to perform SEC, SPR, pH stability testing, mass spectrometry and ITC to ensure the consistent reproducibility of each batch of Affimer reagent produced. Functionalisation of any Affimer reagent is performed, according to optimised protocols for ideal Affimer-conjugate ratios, at an early stage in the development process to confirm the reagent’s performance, stability and expression in an assay-ready format. Each new batch of Affimer reagent is assessed according to our rigorous quality control procedures to ensure optimal performance of your key critical reagents throughout the life cycle of the ligand binding assay.
1. O’Hara DM et al. (2012). Ligand binding assay critical reagents and their stability: Recommendations and best practices from the global bioanalysis consortium harmonization team. AAPS J 16, 504-515.