Title:Development of a One-step Magnetic Separation-based Fluorescent
Sandwich Assay for Residual Protein A in Antibody Drugs
Volume: 22
Issue: 7
Author(s): Jie Xiong, Xuejun Yang and Jingxin Mao*
Affiliation:
- Technology Industry Development Center, Chongqing Medical and Pharmaceutical College, Chongqing, China
Keywords:
Protein A, magnetic beads, antibody drug, fluorescence, immunoglobulin G (IgG), magnetic separation, sandwich immunoassay, process impurities.
Abstract:
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.
