How to Perform Successful Assay Validation for Bioanalysis?

A fully characterized and validated bioanalytical method is crucial for reliable and accurate results. Today bioanalytical methods are constantly undergoing rapid changes and advancements. Increasing pressure on developing novel drug products is the primary driving factor behind innovative modifications and improvements.

Whether it’s liquid chromatography or ligand binding in vitro assay development, each bioanalytical method is unique. Hence the extent of assay qualification and validation will depend on the individual characteristics of the analyte of interest. Each bioanalytical assay will have a specific set of assay validation criteria. Besides, the final objective depends on the intended application of a technique. Considering such unique challenges faced by assay development services, the current article discusses strategies for successful assay validation during bioanalysis. 

Successful assay validation for bioanalysis

During drug development studies, a particular bioanalytical method undergoes several modifications. This change requires a different extent of assay validation to demonstrate its utility and performance. Depending on these levels of method validation, there are three specific types of assay validation; full, partial, and cross-validation. Let us understand each of these validation types. 

Full assay validation is required when developing a new bioanalytical assay for a new drug entity. Besides, adding newer metabolites to an existing validated assay will require full assay validation of the revised method. 

Partial assay validations are done when full revalidations are not necessary. However, partial assay validation can range from one assay parameter, such as precision or accuracy, to nearly full assay validation. 

Finally, cross-validation compares two bioanalytical assays. This type of validation is required when sponsors use two different bioanalytical assays within the same study. 

The US FDA guidelines recommend assessing different parameters during assay validation. Each parameter has unique requirements. The following section discusses these parameters to ensure a successful assay validation initiative. 

Selectivity and specificity

Blank samples from appropriate biological matrices should be analyzed from at least six sources. Besides, blank samples should be tested for selectivity and interference. 

Accuracy

A minimum of six readings per concentration should be measured in accuracy testing. Besides, the mean value should be around ±50% of the exact value except at the lower limit of quantification, where the values should not exceed ±20%. 

Precision

Researchers must measure precision by employing a minimum of 5 values per concentration. Besides, the mean precision values should not vary more than 15% of the CV except for the lower level of qualification, where it should not deviate more than 20% of the CV. 

Recovery

Sponsors must perform recovery experiments at low, medium, and high concentrations, representing 100% recovery. 

Calibration curves

The calibration curve should consist of a blank sample, a zero sample, and 6 to 8 standards, including the lower limit of quantification. 

Quality control samples

Quality control samples should be duplicates at three different concentration levels. All these quality samples should be incorporated in each experimental run. Besides, at least four quality control samples  out of six should lie within 15% of the nominal value. 

Stability

Moreover, analyte stability should be determined for short-term, long-term and freeze-thaw stability.