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High-Efficiency Analytical Protein A Columns for High Sensitivity Monoclonal Antibody Titer Analysis
Beatrice Muriithi1, Fabrice Gritti1, Martin Gilar1
1Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, USA.
Analytical Chemistry
|November 18, 2025
Summary
This study introduces a new analytical Protein A chromatography column using small, nonporous particles for faster and more sensitive quantification of monoclonal antibodies (mAbs). The improved column enhances bioprocessing analytics through rapid, reproducible, and sensitive measurements.
Area of Science:
- Biopharmaceutical Analysis
- Chromatography Science
- Analytical Chemistry
Background:
- Traditional Protein A HPLC columns with large, porous particles (≥20 μm) offer limited speed, sensitivity, and peak resolution for monoclonal antibody (mAb) quantification.
- Existing methods in bioprocessing analytics face challenges due to long run times and broad peaks, hindering high-throughput monitoring.
Purpose of the Study:
- To develop and evaluate a novel analytical Protein A chromatography column utilizing small, nonporous particles (3.5 μm) for enhanced mAb quantification.
- To improve the speed, sensitivity, and efficiency of mAb analysis in bioprocessing by minimizing nonspecific binding and band broadening.
Main Methods:
- Development of an analytical Protein A column packed with 3.5 μm nonporous particles in organosilica-modified hardware.
- Comparative analysis of the new column against traditional columns packed with 20 μm porous particles using experimental and theoretical approaches.
- Evaluation of peak shape, height, dynamic binding capacity, recovery, and carryover for mAb quantification.
Main Results:
- The 3.5 μm nonporous particle column produced peaks three times narrower and higher compared to 20 μm porous particle columns.
- Despite reduced surface area and Protein A density, the new column demonstrated a high dynamic binding capacity (62% at 10% breakthrough).
- Excellent recovery (>98%) and low carryover (<0.3%) were achieved, indicating high reproducibility and minimal sample loss.
Conclusions:
- The novel analytical Protein A column with 3.5 μm nonporous particles significantly enhances speed, sensitivity, and efficiency in mAb quantification.
- This advancement makes the method highly suitable for high-throughput bioprocess monitoring and analysis.
- The findings suggest that smaller, nonporous particles optimize analyte dispersion and mass transfer, overcoming limitations of conventional porous particle columns.
