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

TL;DR

A new high-efficiency Protein A column with small nonporous particles improves speed and sensitivity for monoclonal antibody analysis in bioprocessing.

Contribution

Development of a 3.5 μm nonporous particle Protein A column with improved chromatographic performance for mAb titer analysis.

Findings

3.5 μm nonporous particles produce three times narrower peaks and higher sensitivity than 20 μm porous particles.

Dynamic binding capacity at 10% breakthrough is 62% of the 20 μm column despite lower surface area and Protein A density.

Both columns showed >98% recovery and <0.3% carryover for analytical-scale mass load.

Abstract

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.

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