Magnetic bead extraction with acid dissociation immunoassay for the determination of serum CD80-Fc fusion protein.

Background: To detect concentrations of subtherapeutic doses of the CD80-Fc fusion protein FPT155 in serum in Phase I studies, a highly sensitive assay was developed. Materials & methods: FPT155 was purified from human serum using magnetic beads coupled to cytotoxic T-lymphocyte-associated antigen-4. After washing away the serum components, FTP155 was released by acid dissociation and neutralization. The eluted drug was quantified in an ELISA using cytotoxic T-lymphocyte-associated antigen-4 as a capture reagent and biotinylated anti-human Fc for detection. The assay was validated with a calibration range of 5-40 ng/ml and a dilutional integrity of up to 100,000 ng/ml. Conclusion: A highly sensitive assay to determine serum concentrations of FPT155 using readily available reagents was developed. The results were in conformity with theoretical calculations.

Theoretical considerations and practical approaches to address the effect of anti-drug antibody (ADA) on quantification of biotherapeutics in circulation.

Continuous improvement in bioanalytical method development is desired in order to ensure the quality of the data and to better support pharmacokinetic (PK) and safety studies of biotherapeutics. One area that has been getting increasing attention recently is in the assessment of "free" and "total" analyte and the impact of the assay format on those assessments. To compliment these considerations, the authors provide a critical review of available literature and prospectively explore methods to mitigate the potential impact of anti-drug antibody on PK assay measurement. This challenge is of particular interest and importance since biotherapeutic drugs often elicit an immune response, and thus may have a direct impact on quantification of the drug for its PK and safety evaluations.

Application and interpretation of free and total drug measurements in the development of biologics.

Ligand-binding assays are used to determine concentration levels of biopharmaceuticals in biological matrices. The whole molecule does not serve as a basis for quantification, but subregions are captured and detected by specific binding critical reagents that have been produced for the sole purpose of bioanalysis. An assay can be designed to measure the free or the total analyte. Depending on the format of the assay, different observations and interpretations could be deemed. In the case studies presented in this article, the same serum samples were subjected to analysis in parallel by two different assay formats. In three out of the four cases presented, the results and the data interpretation were remarkably different. Therefore, it is essential for the bioanalyst to communicate to other stake-holders, such as toxicologists and pharmacokineticists, what the assay detects and measures for plausible data interpretation and implication.