Key Parameters and Acceptance Criteria for Bioanalytical Assay Development and Validation

Bioanalytical assay development and validation is the key to a successful drug development process. Regulatory guidance helps sponsors of INDs, NDAs, BLAs, ANDAs, validate bioanalytical methods used in human clinical pharmacology, bioavailability (BA), and bioequivalence (BE) studies.

Such guidance applies to bioanalytical procedures like chromatographic assays (CCs) and ligand binding assays (LBAs) that determine the levels of drugs, their metabolites, and biomarkers in biological matrices.

Analytical method development involves optimizing the procedures involved in extracting and detecting the analyte. It comprises improving important bioanalytical parameters to ensure that the method is suitable for validation. Following are the key parameters and acceptance criteria needed for robust bioanalytical assay development and validation.

• Reference standards: The sponsor should use authenticated analytical reference standards with known identities and purities to prepare solutions of known concentrations. Provision of certificates of analyses (CoA) for commercially available reference standards is needed.

• Critical reagents: The sponsor should characterize and document (i.e., determine the identity, purity, and stability) the critical reagents, including but not limited to any reference standards, antibodies, labeled analytes, and matrices.

• Calibration curve: The quantitation range of the assay and the concentrations of the calibration standards should be chosen based on the concentration range expected in a particular study. For LBAs, in the calibration standards, anchor points outside the range of quantification can facilitate the fitting of the curve. For most LBAs, calibration curves are nonlinear. The calibration standards should be prepared in the same biological matrix as the samples in the intended study. When the method validation is over, the calibration curve must be continuous and reproducible.

• Quality control samples (QCs): Quality controls are used to assess the precision and accuracy of an assay and the stability of the samples. QCs must be prepared in the same matrix as the study samples to be assayed by the validated method. QCs that are freshly prepared are recommended for precision and accuracy analyses during method development because stability data are usually not available at this point.

• Selectivity and specificity: The sponsor should verify that the substance being measured is the intended analyte to minimize or avoid interference. The selectivity of the method development is demonstrated by analyzing blank samples of the appropriate biological matrix (e.g., plasma) from multiple sources. During validation, confirm that the essay is free of potentially interfering substances. Suppose the study sample contains more than one analyte, and the analytes are intended to be quantified by different methods, in that case, the sponsor should test each method for interference from the other analyte.

• Sensitivity: The LLOQ defines the sensitivity of the method, and it should be determined during method development. The method should be able to meet the essential requirements for given study samples. The LLOQ evaluation can be done as part of the precision and accuracy assessment or separately for the calibration range.

• Accuracy, Precision, Recovery: Evaluation of accuracy and precision during method development is necessary to determine whether the method is ready for validation. It involves analyzing replicate QCs at multiple concentration ranges across the assay. Evaluation of the performance at the LLOQ, low, mid, and high QCs (and the ULOQ for LBAs) is required to determine if the method is suitable to analyze study samples. Optimization of the recovery of an analyte is required to ensure that the extraction is efficient and reproducible.

• Stability of the analyte in the matrix: Determination of the chemical stability of the analyte in a given matrix is required, including the effects of sample collection, handling, and storage of the analyte. For CCs, the accuracy at each level should be ± 15%. For LBAs, the accuracy at each level should be ± 20%.

 Conclusion

 The key parameters of bioanalytical assay development and validation include reference standards, critical reagents, calibration curve, QC samples, selectivity, specificity, sensitivity, accuracy, precision, recovery, and stability of the analyte in a given matrix.