Immunoassay bioanalytical method validation

As for any bioanalytical method, the extent of validation for an immunoassay should be related to the intended application of the assay. Thus, if an immunoassay is intended to support rapid screening in discovery R D, the characterization of specificity and the accuracy and precision specifications may be less stringent than if the assay is used to support pre-clinical and clinical development studies. Indeed, an assay for discovery support may be designed to detect active metabolites as well as parent molecule, so that... 

As for all bioanalytical methods applied to support of drug development, validation of immunoassays is important. However, several validation issues need special attention for immunoassays. These include stability of the critical reagents, the curvilinear nature of the calibration curve, the greater variability of immunoassays, and, particularly important, the specificity of the assay. 

The optimization and validation of immunoassays for immunogenicity (ADA) testing has been described in detail in several publications [9,14,33,34]. In this section, we will describe the evaluation of relevant performance characteristics (validation parameters) that require the most effort. Some of these are different from the validation of traditional bioanalytical pharmacokinetic (PK) methods for macromolecules [35 37]. Precision, specificity, robustness, and ruggedness are determined similarly between ADA and PK methods. However, recovery/accuracy, sensitivity, stability, linearity, system suitability controls, and selectivity are treated differently between these two types of assays. 

The ideal PK immunoassay standard curve, which is nonlinear and heteroscedastic, is derived from solutions of well-characterized macromolecules added to a relatively nonreactive sample matrix. However, rarely does one encounter an ideal situation when describing bioanalytical methodology, thus developing and validating analytical methods for macromolecules, and analyzing samples from preclinical or clinical trials must include an evaluation of these variables and possibly many others. Careful consideration must be given to the topics described in this chapter to achieve the goal of accurate and reproducible quantification of biotherapeutics necessary for pharmacokinetic analysis. 

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