Issues in the Bioanalysis of Antibodies

Pharmacokinetic data analysis requires determination of the analyte in various body fluids. In the case of therapeutic antibodies, serum is the most common matrix to be analyzed. For a critical interpretation of pharmacokinetic data the chosen bioanalytical methods must be considered. The most frequently used for mAbs include enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE)/polyacrylamide gel electrophoresis (PAGE), fluorescence-activated cell sorting (FACS), and surface plasmon resonance (SPR). The challenges and limitations of bioanalytical methods used for the analysis of mAb concentrations are discussed in detail in Chapter 6. 

A common analytical problem in immunoassays is that of cross-reactivity, which is the unspecific binding of the therapeutic antibodies determined in the assay with a second (or more) antigen(s). These interfering bindings must be kept to a minimum. Among other factors, specificity of the enzyme-labeled anti-Fc and anti-Fab antibodies used for detection is of major importance. In general, each assay design bears its special drawbacks that must be carefully evaluated for its intended use. 

Typically, the relationship in the assay between the detection signal and the concentration of the mAb is nonlinear. A simple regression analysis is, therefore, not possible. Usually, a third-to-fifth degree polynomial is used to relate the detection signal to the concentration. Because of this relationship, a typical sample dilution is not possible and careful evaluations are necessary if the concentration of the antibody is above the upper limit of quantification of the assay. As a consequence, the assay should ideally cover the whole concentration range of all samples measured. 

Compared to other bioanalytical methods such as high-performance liquid chromatography (HPLC), the methods used to quantitate mAbs often display less precision and a higher between-day variability. In choosing a bioanalytical method it must also be considered that some assays measure the unbound fraction, the bound fraction, or both. When using FACS, only the fraction of the therapeutic antibody that is bound to its antigen on the cells is counted. In contrast, ELISA measures only the unbound fraction in serum that can react with the offered antigen. 

In summary, because of the differences between assays, careful consideration of which assay has been applied is highly recommended when evaluating the concentration data of mAbs in pharmacokinetic analyses. 

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