Immunoassay validation parameters

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. 

VALIDATION PARAMETERS AND FACTORS AFFECTING THE PERFORMANCE OF IMMUNOASSAYS... 

The first step in the validation process involves the establishment of acceptance criteria, which are assay dependent, for the various validation parameters. Assays must meet these criteria in order to receive validation status. The most relevant validation parameters are specificity, accuracy, precision, sensitivity, and robustness. In this section we describe these parameters as they apply to the evaluation of immunoassays. 

For proteins, most applications rely on immunoassays and alternatives are not readily available total recovery and possible denaturation that renders the extracted proteins undetectable by antibodies are the most critical factors. Depending on the tissue type analyzed (leaves, seeds, roots) validation needs to be performed separately on all different tissue types. For each tissue type the extraction efficiency as well as sensitivity, linearity and range of the method (amongst other parameters) need to be assessed. 

During early immunoassay development, it is important to assess the intended use of the assay. When the intended use is to support pharmacokinetics in either nonclinical PK/TK or clinical bioequivalence (BE) studies, developing a validat-able assay is of utmost importance. A well-thought-out validation plan and fully documented assay methods and results that support the validation are crucial. To withstand the scrutiny of the authorities, and to guarantee the optimal method is applied to your drug development process, the following parameters should be assessed critically, validated to specific criteria, and fully documented. 

The goal of the development phase for immunoassays is to establish a method that can consistently produce a reliable result culminating in a validation plan with established target acceptance criteria for accuracy and precision. The following recommended parameters are assessed critically during early development, evaluated in late development, and finally, confirmed during validation. 

To ensure the reliability of analytical techniques, they need to be validated. Validation provides information on the overall performance of the assay as well as on individual parameters and factors that can be used to estimate the degree of uncertainty associated with an assay (Ellison et al., 2000). An adequate validation procedure assesses, and therefore ensures, that the immunoassay performs within an acceptable range of established criteria. Parameters used to evaluate the performance of the assays may be affected by (1) factors inherent to the analytical technique, such as antibody specificity and antibody cross-reactivity, and (2) external factors such as environmental conditions (temperature) and type of sample (matrix, processed food vs. raw ingredients). A... 

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