Development of a One-step Magnetic Separation-based Fluorescent Sandwich Assay for Residual Protein A in Antibody Drugs

Introduction: Protein A (PA) leakage during antibody purification poses significant risks due to its immunotoxic potential, necessitating highly sensitive and efficient detection methods. Current techniques like ELISA are time-consuming and complex, highlighting the need for a rapid, costeffective alternative. This study aimed to develop a one-step sandwich fluorimetric assay for trace PA detection in antibody drugs, leveraging the specificity of monoclonal antibodies and the efficiency of magnetic separation to overcome existing limitations. Method: The assay utilized magnetic beads (MBs) functionalized with a specific monoclonal antibody (mAb) to capture PA, while fluorescein isothiocyanate (FITC)-labeled immunoglobulin G (IgG) served as the secondary recognition agent to form a sandwich complex. Magnetic separation was employed to isolate the complex from the matrix, followed by fluorimetric detection. Key parameters, including blocking agents, incubation time, IgG concentration, and MBs amount, were optimized to enhance performance. Result: The assay demonstrated a linear detection range of 5-500 pg/mL for PA, with a detection limit of 1.3 pg/mL. The entire process was completed within 50 mins, significantly faster than the enzyme-linked immunosorbent assay (180 mins). High specificity was confirmed, with negligible interference from host cell proteins (HCP) and DNA (HCD). Recovery tests in real samples (adalimumab and toripalimab injections) yielded results of 98.80% to 101.55%, validating the method's accuracy and reliability. Discussion: The present study presents a rapid one-step magnetic separation-fluorescence assay for Protein A detection, completing analysis in 50 minutes (3× faster than ELISA) with a simplified workflow. While maintaining high sensitivity, potential cross-reactivity requires further validation. This rapid, simplified approach shows strong potential for routine monitoring in biopharmaceutical quality control. Conclusion: The proposed one-step sandwich fluorimetric assay, combining magnetic separation with fluorescence detection, offers a novel, rapid, and highly sensitive approach for quantifying trace PA in antibody drugs. Its simplicity, cost-effectiveness, and reduced detection time make it a promising alternative to conventional methods.