Validation of immunoassays for macromolecules from biotechnology

Guidelines have been proposed for the validation of bioanalytical methods applied to the support of clinical and preclinical pharmacokinetic studies. These guidelines deal primarily with accuracy, precision, sensitivity, and establishment of the calibration curve, and are generally applicable to quantitative immunoassays for recombinant proteins. Definition of specificity of immunoassays for macromolecules is more difficult than for small molecule immunoassays since the metabolism and degradation of proteins is not usually well defined and comparator assays are normally not available. For multifunctional molecules, however, the use of antibodies raised against the individual functional components of the macromolecule for capture and detection functions in the assay can confer enhanced specificity. Immunoassays (most commonly enzyme-linked immunosorbent assays) for the detection of antibodies elicited by treatment with macromolecules are needed to evaluate the immune response to the macromolecule when administered in vivo. The precision of these antibody detection assays can be characterized, whereas accuracy is more difficult to define without a specific immunoglobulin standard. Evaluation of the clinical significance of such antibodies must take into account the immunoassay results, assessment of their neutralizing activity in in vitro tests and their effects, if any, on clinical efficacy and safety parameters in vivo.