Development and Optimization of Strong Cation Exchange HPLC Method for Monoclonal Antibody Charge Variant Characterization

“Strong cation exchange chromatography (SCX) coupled with high-performance liquid chromatography (HPLC) is a powerful analytical technique for the separation and characterization of charge variants in monoclonal antibodies(mABs). This study focuses on developing and optimizing an SCX-HPLC method to characterize charge heterogeneity in like Amatuximab, a monoclonal antibody with structural heterogeneity due to post-translational modifications. These charge variants can impact the efficacy, safety, and stability of the therapeutic product.
The method development involved optimizing critical parameters such as pH, salt gradient, and flow rate to achieve high-resolution separation of acidic, basic, and main peak isoforms of Amatuximab. A gradient elution strategy enabled efficient separation and precise characterization of charge heterogeneity. The optimization of particle sizes from 5 to 3 microns was performed to enhance resolution and peak capacity. The smaller particle size improved separation efficiency, allowing for more distinct resolution of closely eluting charge variants.
A proprietary coated column was explored to reduce secondary interactions with hardware, which often complicate chromatographic analysis. This coating minimized non-specific interactions between the analyte and column hardware, resulting in more reliable and reproducible separations. The proprietary coating contributed to improved peak shapes and reduced tailing, enhancing the overall analytical performance of the method.
Our results demonstrate that SCX-HPLC effectively separates acidic and basic variants of Amatuximab and other mABs, providing detailed insights into its charge heterogeneity. The analysis revealed distinct charge variant patterns, highlighting differences in post-translational modifications and stability.”