Applying best practices for characterizing critical reagents

The generation of critical reagents is both cost and labor intensive. Therefore, it is important that they are thoroughly characterized and managed in order to ensure the integrity of the assays in which they are used. Below we define critical reagents and describe the best practices for their characterization. We also demonstrate how Bio-Rad applies these industry specified best practices in characterizing the critical reagents that we produce.

What are critical reagents?

Critical reagents are the indispensable components of ligand binding assays (LBAs). Their unique characteristics are essential to the appropriate performance of LBAs. The categories of critical reagents include antibodies, peptides, engineered proteins, complex biologics, chemically synthesized molecules, solid supports and matrices, as well as antibody, protein and peptide conjugates. Because they are typically generated by biological processes, critical reagents are inherently prone to lot to lot variation. Consequently, proper management and characterization of these reagents in bioanalytical laboratories is important for successful drug development.

How do bioanalytical laboratories characterize critical reagents?

Characterization of critical reagents for ligand binding assays

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The characterization and management of critical reagents varies between bioanalytical laboratories. Up until 2012, there was no consensus among these laboratories on the best practices for analyzing and managing critical reagents, which is crucial for clinical research. In an effort to standardize critical reagent management and characterization, a group of scientists from the Global Bioanalysis Consortium harmonization team outlined a set of recommendations and best practices for the life cycle management, characterization and supply of critical reagents used in LBAs (O'Hara et al. 2012). For characterizing critical reagents, the agreed best practices centered on assessing sensitivity, specificity and reproducibility. Below we provide a summary of these best practices. 

Best practices for characterizing critical reagents

• It is important to generate a basic characterization profile for each critical reagent that includes assessment of concentration (antibody titer), binding activity, aggregation, purity level and molecular weight.
• Include purification assays for protein reagents during early stages of drug development to reduce the impact of impurities, which could affect stability during long term storage.
• Verify purified reagents using methods such as SDS-PAGE and SEC to confirm purity and monodispersity prior to use in the desired LBA.
• As drug development progresses, express monoclonal antibody reagents in characterized mammalian cell lines to ensure their genetic stability and consistency across reagent lots.
• 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.

What critical reagents does Bio-Rad produce and how do we characterize them?

Bio-Rad manufactures anti-biotherapeutic antibodies for scientists involved in preclinical research, clinical trials and patient monitoring for innovator and biosimilar products. These anti-biotherapeutic antibody reagents are important for anti-drug antibody and pharmacokinetic assays. Bio-Rad takes a similar approach to characterizing these antibodies as outlined by the global bioanalysis consortium. We apply the best practices outlined for quality control and antibody batch characterization as part of the manufacturing and development of our anti-biotherapeutic antibody portfolio. We specifically assess the reproducibility and sensitivity of our anti-biotherapeutic antibodies by testing purity, consistency, stability, activity, aggregation and affinity. Every new antibody is assessed by rigorous quality control procedures to ensure a high quality antibody every time. This helps the assay developer with critical reagent life cycle management, characterization and supply, and ensures optimal assay performance over time.

To learn more, check out our technical article where we highlight our anti-biotherapeutic antibody range and provide actual data of our analyses.