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Bioanalytical Assay Validation

7 Important Tips To Remember When Conducting A Bioanalytical Assay Validation

Validated bioanalytical methods are critical for quantitating analytes in complex biological matrices. These bioanalytical assays form the foundational success for the conduct of several clinical and nonclinical drug development pathways. Validated methods ensure that the bioanalytical assay is adequate to produce reliable and accurate drug data. Bioanalytical assay validation strives to answer critical questions on clinical drug development. These questions include understanding the feasibility to measure the target analyte, discover measurement variability, estimate assay range, and answering issues related to logistics.

Assay method development and validation are necessary for generating robust bioanalytical assays. Today assay validation services employ a fit-for-purpose approach for validating bioanalytical methods. This approach focuses on validating assays according to the intended purpose of the study. Hence, the extent of validation depends on the intended goal of bioanalysis. Pivotal analyses such as safety and labeling studies that are crucial for FDA submissions will require complete method validation. On the other hand, an exploratory study may need partial method validation.

Understanding the complexity revolving around assay development and validation, we present seven tips from FDA’s bioanalytical method validation guidance. Click Here for Chemical Handling Course

Reference standards

Pure reference standards can have a positive impact on quality control samples and calibrators. Hence, researchers must use authenticated reference standards having known purities and identities while preparing known concentration solutions. Ideally, the reference standard must be similar to the analyte of interest. However, if this is not possible, researchers can use an established alternative of known purity.

Critical reagents

All critical reagents must be adequately characterized and documented. This documentation must include labeled analytes, antibodies, reference standards, and matrices. Besides, assay variation becomes crucial when critical reagents are changed, for example, switching to another reagent.

Calibration curve

The calibration must be reproducible and continuous during bioanalytical assay validation. Besides, the calibration standards must have the same biological matrix as the intended sample. Most often, study samples may consist of more than a single analyte. Hence, sponsors should have a separate calibration curve for each analyte.

Quality control samples

Quality control samples are necessary to assess sample stability, accuracy, and precision of bioanalytical assays. Hence, FDA recommends using freshly prepared quality control samples to test accuracy and precision. Moreover, performance quality controls and stability quality controls must be incorporated during method validation.

Must Read: Researcher’s Guide: A Quick Overview Of Meso Scale Discovery

Interferences

Researchers must confirm that the intended assay is free of interference. These interfering substances may include metabolites, endogenous matrix components, and anticipated concomitant medications. Besides, in the case of additional analytes in the study sample, researchers must assess interference from other analytes.

Accuracy and precision

Accuracy and precision run for assay validation must include at least six independent runs for ligand-binding assays and three for chromatographic assays. Moreover, each accuracy and precision run must have multiple quality control concentrations and calibration curves analyzed in replicates.

Recovery

Efficient recovery runs ensure robust and reproducible sample extraction. Although 100% recovery is not required, internal standards and analyte recovery must be reproducible and consistent. Furthermore, researchers must compare the results of the extracted sample against post-extraction samples spiked with blank extracts in recovery experiments.

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