High-performance chromatographic immunoassay utilizing a biotin-streptavidin platform for activity-based analysis of therapeutic monoclonal antibodies

There has been rapid growth in the use of monoclonal antibodies (mAbs) as biopharmaceuticals over the last twenty years. This has led to the need for new analytical methods that can rapidly and specifically measure or characterize mAbs for research, development and quality control, including means for the assessment of a therapeutic mAb's biological activity. High-performance immunoaffinity chromatography (HPIAC) was examined in this report as an approach for such work, in which the interactions between an antibody and its antigen were used for the selective isolation and analysis of one of these components. This report describes the utilization of a biotin-streptavidin platform in HPIAC and with affinity microcolumns that were used together in a chromatographic immunoassay for the analysis of a therapeutic mAb. Various components of this method were characterized and optimized to provide a method that was reusable and that could provide results within 7 min. The final assay could measure down to 0.03 mg/mL mAb (1.5 μg) for a 50 μL sample injection. The assay precision was ±0.7–1.3 % based on peak area measurements and ± 1.0–2.4 % using peak heights. The method was then evaluated for its use with typical samples encountered for a therapeutic mAb during its development and processing. Each microcolumn in this assay could be used for more than 350–400 sample application/elution cycles. The extension of this platform and approach to other applications was also considered.