Evaluation of Purification Methods for Minimizing Transgene Expression Background During Viral Manufacturing

Gene therapy has emerged as a promising therapeutic avenue, offering targeted treatments for various diseases. Purification of viral vectors presents a pivotal challenge, demanding the removal of impurities while preserving integrity and potency. During manufacturing, producer cells in transfection systems can be transiently transfected or retro-infected by the viral vectors they have just produced-a process referred to as "retro-transduction"-leading them to express the transgenes of interest. This can be a significant source of contamination in the viral solution pool, particularly when the transgenes encode extracellular, secreted proteins, resulting in cytotoxicity and reduced viral potency. Herein, we aimed to evaluate the efficiency of different viral purification systems commonly used in academic and industry settings in removing the transgene background from viral solutions. The efficiency of each system was assessed based on the levels of the secreted transgene <i>Gaussia Luciferase</i> (GLuc), which can be quickly detected in a solution and served as a readout for transgene background contamination in the viral pool during downstream processing. Through a systematic evaluation of purification methods, we identified the most effective approaches for producing pure viral batches with minimal transgene background, all while preserving viral potency and functionality. Our study revealed superior performance of batches that underwent purification via tangential flow filtration, which yielded over 90% reduction in GLuc background and the highest transduction efficiency rates. This work provides significant insights for advancing gene therapy applications that rely on the production of viral vectors encoding secreted transgenes.