7 Crucial Factors Affecting Your Bioanalytical Assay Validation Results

Assay development and assay validation are two most fundamental  factors responsible for the success of all bioanalytical methods. Validated assays reassure that the generated safety and efficacy results are accurate and reliable. However, several other parameters can impact assay validation results. Following are the seven crucial factors that can affect your bioanalytical and biomarker assay validation results.

Reference standards and critical reagents

Appropriately characterized and documented reference standards and critical reagents are vital in achieving robust validation results. The purity of reference standards can affect your study data, and hence , sponsors should use such reference standards whose identity and purity are characterized for preparing solutions of known concentrations. Moreover, critical reagents such as labeled analytes, antibodies, and matrices should be stored under the required storage conditions.

Calibration curve

During assay development, the assay quantitation range and concentrations of calibration standards should be based on the expected concentration range in a particular study. Moreover, additional anchor points lying outside the quantification range are needed for ligand binding assays. A calibration curve is inherently nonlinear for most ligand-binding assays. Hence, generally, they require more calibration standards to fit the data in the calibration range.

Quality control samples

Quality controls evaluate assay accuracy and precision and the stability of the study samples. Bioanalytical labs should prepare quality control samples in the same matrix as the assay samples. Quality controls also help evaluate the assay performance and sample stability during bioanalytical assay validation. Performance quality controls assess the precision and accuracy of a bioanalytical method, while stability quality controls assess the stability of study samples under different stress conditions.

Selectivity and specificity

It is necessary to confirm that the analyte measured in the assay is the intended substance. Sponsors routinely verify assay selectivity and sensitivity by evaluating blank samples from biological matrices. Demonstrating an interference-free method is necessary during bioanalytical assay validation. Interfering substances can include metabolites, endogenous matrix components, and concomitant medications. Furthermore, multiple analytes are quantified using different methods, and sponsors should evaluate interferences for each detection method.

Accuracy, precision, and recovery

Assay validation studies conducted  to estimate assay accuracy and precision should include at least three independent runs for chromatographic assays and six runs for ligand binding assays. Moreover, each run should have a calibration curve and multiple QC concentrations in replicates. Validation results also depend on an optimized analyte recovery. Such optimal recovery ensures that the extraction process is reproducible.

Stability

Assay validation studies for assessing the stability of a drug compound should cover all aspects of an analytical site before and after the receipt and analysis of sample conditions. Before the receipt means samples may reach a particular bioanalytical lab through shipment or other secondary experimental sites. Largely, stability depends on the storage conditions, storage duration, assay matrix, physicochemical properties, and container system.

Partial or cross-validation

Often some drugs may not require a complete assay validation. Assessing when a full assay validation is needed can help expedite your validation results. Partial validations are performed when sponsors modify an already validated method. This validation can vary from a single intra-assay protocol to nearly complete validation. Cross-validation is generally performed to compare parameters among different bioanalytical approaches that are used in the same or across multiple studies.