High-Efficiency Analytical Protein A Columns for High Sensitivity Monoclonal Antibody Titer Analysis.

The quantification of monoclonal antibodies (mAbs) using analytical Protein A affinity chromatography HPLC columns with porous particles of 20 μm or larger diameters is a common method in bioprocessing analytics. The limitations of this method are long run times, relatively broad peaks, and low sensitivity. To address this, we developed an analytical Protein A column packed with 3.5 μm nonporous particles in organosilica-modified hardware, designed to minimize nonspecific binding of mAbs and enable rapid, reproducible, and sensitive measurements. Experimental and theoretical results show that smaller, nonporous particles reduce analyte axial dispersion and mass transfer-related band broadening. This leads to three times narrower peaks and greater peak heights compared to those obtained with 20 μm fully porous particles. Although the column with 3.5 μm nonporous particles has more than ten times less surface area and 50% less Protein A density, its dynamic binding capacity is 62% at 10% breakthrough compared to a column of the same size packed with 20 μm porous particles. This implies that not all of the available surface area or ligand capacity is accessible for binding for the fully porous particles. Both columns demonstrated greater than 98% recovery and a low carryover of less than 0.3% for the analytical scale mass load. The use of 3.5 μm nonporous particles improves the speed, sensitivity, and efficiency of analytical Protein A chromatography, making it applicable to high-throughput bioprocess monitoring.