High-throughput protein A chromatography platform for accurate antibody quantification in IV admixtures

Antibodies hold significant therapeutic potential, but accurate quantification of them in complex intravenous (IV) admixtures is critical for ensuring therapeutic efficacy and patient safety. Traditional reversed-phase liquid chromatography (RPLC) often faces challenges in resolving antibodies from non-product related impurities within IV matrices. This study first evaluates the specificity of Protein A (ProA) affinity chromatography for quantifying bispecific antibodies (BsAbs) in IV matrices. The method leverages the selective binding of ProA to the antibody Fc region to achieve clear separation of antibodies from potential interference, including human serum albumin (HSA), and extractables and leachables (E&L) from contact materials, enabling accurate quantification down to 0.1 μg/mL. The fit-for-purpose method qualification secondly demonstrates linearity, accuracy, and precision across the tested concentration ranges. The ProA method achieves rapid and complete separation in 5 min without sample preparation, significantly enhancing throughput. This platform capability is further demonstrated by its successful application to five different BsAbs in this study. These findings highlight the ProA method as a reliable, efficient, and specific approach for accurately quantifying antibodies in the presence of challenging IV admixture matrices, supporting the development and administration of low-dose antibody therapies.