An oncolytic strain of Coxsackievirus A21 (CVA21) is being evaluated in PhI/II clinical trials as an immunotherapy for the treatment of several different tumor types. Early clinical batches used gradient ultracentrifugation in the purification process to clear non-infectious, empty capsids and residual cell culture impurities including host-cell DNA, host-cell protein, and bovine serum albumin. However, this step is labor intensive and has limited throughput. To improve process scalability and robustness, we sought to replace ultracentrifugation with chromatographic methods while maintaining analytical comparability to the early clinical drug substance. We developed a highly selective CVA21 affinity chromatography method by repurposing commercially available GSH-immobilized resin originally intended for GST-tagged protein purification. Clarified CVA21 cell culture lysate is directly loaded to a GSH-resin packed column, washed with increased ionic strength buffer, and eluted at neutral pH with free GSH in the mobile phase by competitive displacement. The affinity chromatography step was optimized to clear cell culture impurities with high infectious V937 particle binding capacity and elution yield. Furthermore, we discovered that GSH resin selectively cleared empty capsids as a function of the cell culture conditions. The purification process using GSH affinity as the capture step was demonstrated at large scale and generated drug substance that met or exceeded analytical comparability expectations of the ultracentrifugation-based process. The established affinity chromatography process may be utilized for the purification of other enteroviruses.

Off the shelf to the manufacturing floor: Development of a novel Coxsackievirus A21 affinity chromatography step using repurposed immobilized glutathione resin

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.