test tubesCritical reagents are instrumental to large molecule drug development. They constitute a key element in the bioanalytical assay for the quantitative determination of the test compound. Companies often find it challenging to produce and manage lots of consistent quality, even though such consistency governs the accuracy, precision and robustness of the assay.

Critical reagents and their role in bioanalytical studies

ICH M10, draft guidance from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), states that critical reagents and binding agents, i.e., binding proteins, aptamers, antibodies or conjugated antibodies, directly impact assay results, thus ensuring their quality is paramount. According to the guidelines, “Critical reagents bind the analyte and, upon interaction, lead to an instrument signal corresponding to the analyte concentration.” 1

Ligand-bind assays (LBAs) are often used to capture and signal the molecule’s detection to understand how it performs, which is where critical reagents come into play. The insights LBAs provide help support consistent dosing and ensure acceptable performance. Laboratory facilities will often need multiple lots of critical reagents due to the long duration of the drug development process.

Researchers must closely monitor the reagent’s stability throughout the drug development process to achieve optimal quality that meets regulatory requirements. Consistent reagent quality and make-up are essential to safeguard the reliability of LBA results. Proper management ensures that factors such as affinity and potency do not vary across lots.

The importance of a critical reagent management strategy

It is imperative to the overall program that development teams implement a robust management strategy. Drug development can take more than ten years — ample time for critical reagents to expire or become exhausted. The inevitable lot changes could lead to management challenges large and small as the biologic nature of critical reagents renders them susceptible to variability.

Unlike major lot changes, a minor lot change, such as the source of a reagent, is less likely to influence assay performance. Conversely, major changes may include altering the production method of antibodies or using a new supplier for monoclonal antibody production. Drug developers should prepare validation methods to assess the variation between lots. As stated explicitly in the M10 guidance, “A critical reagent lifecycle management procedure is necessary to ensure consistency between the original and new batches of critical reagents.”

Best practices for critical reagent management

The U.S. FDA and the American Association of Pharmaceutical Scientists (AAPS) provide management best practices. Their primary directive to developers is to prioritize characterization and documentation. Specifically, the FDA Bioanalytical Method Validation (BMV) guidance says, “Assay validation is important when there are changes to the critical reagents, such as lot-to-lot changes or switches to another reagent.”

The guidance also says if there are changes to the labeled analytes, detector reagents, or antibodies, the sponsor should:

  • Evaluate binding and re-optimize assays.
  • Verify performance with a standard curve and QCs.
  • Evaluate cross-reactivities.

The AAPS further details processes for managing critical reagents, including designing the reagent, assigning stability and changing procedures for minor and major changes. The steps used to respond to minor changes can also act as baseline procedures for responding to major changes.3

These foundational management steps aim to test the functionality of the assay. For PK/biomarkers, regulatory expectations require three levels of quality control. If multiple reagents need to be changed, developers should perform multiple runs. Laboratories can begin documentation only after they monitor instrument response and compare results to the original lot. Researchers can also use these steps as a framework for major lot changes.

At a minimum, developers need to conduct three runs to document assay performance. Labeling can help identify changes between lots and determine whether to apply a full or partial validation.

During lot changes, bridging testing is crucial. Inconsistent performance can signal a variation that could ultimately compromise test results. Bridging testing helps determine whether the assay or the critical reagent caused the deviation. In cases where assay performance is altered, or acceptance criteria have not been met, developers may be able to validate the lot as long as the assay is still fit for the purpose. However, understanding how this new lot impacts assay parameters is necessary to protect data integrity.

Final thoughts

Critical reagents support a developer’s understanding of molecule performance as it progresses through the development life cycle. A critical reagent management strategy should be in place early in the process to ensure reliable data from the start.

Both drug developers and testing laboratories need to implement reliable safeguards to ensure consistent quality and adequate quantity of critical reagents. For example, such safeguards could include setting up robust manufacturing processes to reduce the variability between lots and monitoring the stability annually. Collaboration between drug developers and laboratory testing partners can help reduce barriers, execute best practices and successfully bring a molecule to the next stage of development.

Xuesong Chen, PhDXuesong Chen joined WuXi AppTec in 2020 as a technical director supporting client engagement and technical discussions for preclinical and clinical large molecule bioanalytical services.

Before joining WuXi AppTec, Chen worked in the pharmaceutical field of high-throughput screening for novel lead compounds using automation systems and led a successful team specializing in MD/MV for biologics, pharmacokinetics, anti-drug antibodies (ADA) and biomarkers for regulated studies. He has more than a decade of hands-on experience with Ligand Binding Assay (LBA) using different platforms for LM, including MSD, ELISA, ELLA, HTRF, AlphaLISA and Quanterix Simoa. He also has strong experience in cell-based functional assays using confocal microscopy and tissue/cultures staining assays based on immunohistochemistry. Chen recently developed two successful companion diagnostic kits for two different clinical trials under Clinical Laboratory Improvement Amendments (CLIA) regulations.

Chen received his Ph.D. in Pharmacognosy from Peking Union Medical College’s Chinese Academy of Medical Sciences and did his post-doctoral work in physiology at the University of Texas Southwestern Medical Center in Dallas.


1. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), n.d. BIOANALYTICAL METHOD VALIDATION M10. ICH. https://database.ich.org/sites/default/files/M10_EWG_Draft_Guideline.pdf.

2. U.S. Department of Health and Human Services Food and Drug Administration, Bioanalytical Method Validation Guidance for Industry. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry.

3. The American Association of Pharmaceutical Scientists (AAPS) Journal, Ligand Binding Assay Critical Reagents and Their Stability: Recommendations and Best Practices from the Global Bioanalysis Consortium Harmonization. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4012044/#.