Developing robust bioanalytical assays are of utmost importance during several stages of drug discovery and development. A thorough bioanalytical assay validation ensures that the assay can satisfactorily yield accurate and reliable results. Today bioanalytical assays are constantly evolving at an unprecedented rate. Fields such as biomarker research have further increased the complexities involved in biomarker assay development. Clinical biomarkers in drug discovery and development will therefore require continued efforts in biomarker assay development and validation.
Besides, each technique is unique. Bioanalytical assays may vary from one analyte to another. Hence, bioanalytical assay validation will have specific criteria for each analyte. Moreover, the final objective may further drive the extent of method validation. The current article thus highlights five crucial parameters researchers never ignore during bioanalytical assay validation.
1. Reference standards and critical reagents
Regulatory guidance recommends that all reference standards and critical reagents must be characterized and documented appropriately. Besides, under defined conditions, researchers must store all reagents and standards, such as labeled analytes, antibodies, and matrices..
Sponsors must conduct bioanalytical assays using authenticated reference standards with known purities and identities. Ideally, the reference standards must be identical to the target analyte. Moreover, assay validation becomes crucial when there is a change in critical reagents.
2. Quality control samples
Quality control samples are necessary to demonstrate assay performance and analyte stability. Sponsors incorporate performance quality controls to assess the accuracy and precision of the bioanalytical assays. On the other hand, stability quality controls are used to evaluate analyte stability under various stress conditions. Generally, quality controls and calibration standards must be prepared from separate stock solutions. However, scientists can use the same stock solution if they can show accuracy and precision in one validation run using quality controls and calibrators from separate stock solutions.
3. Specificity and selectivity
To demonstrate specificity and sensitivity, sponsors must first show that the bioanalytical assay is free from potential interferences such as metabolites and endogenous matrix components. Generally, sponsors must evaluate blank samples from ten separate sources for ligand-binding assays. If the assay quantifies more than one analyte with different methods, sponsors must test individual analytes in each of these methods for interference..
4. Precision, accuracy, and recovery
Accuracy and precision runs must include at least six independent runs for ligand-binding assays. These runs must be conducted over several days with each run, including multiple quality controls and calibration curves analyzed in replicates. The quality controls in the accuracy and precision experiments are crucial to determine the performance of the bioanalytical assays. Besides, the recovery should be consistent and reproducible throughout the assay run.
5. Partial and cross-validation
Partial validations are conducted for modifications to an already validated assay. However, partial method validation can range from as little as a single metabolite addition to nearly full method validation. On the other hand, cross-validation is necessary when two or more bioanalytical methods are used to generate results across different studies or within the same study. Moreover, cross-validation is needed when sponsors conduct the same analysis at more than one experimental site.