A mechanistic model for recombinant adeno‐associated virus production dynamics in a helper virus‐assisted producer cell line

Recombinant adeno-associated virus (rAAV) is the most widely used viral vector for in vivo gene therapy, with over 200 clinical trials currently underway worldwide. Achieving a manufacturing process that is sufficiently productive with the requisite product quality is critically important for commercialization success. Mammalian producer cell lines (PCLs) are stably transfected to integrate AAV structural and non-structural genes and are frequently combined with infection by wild-type adenovirus (Ad5) to provide helper elements. Within the PCL, both Ad5 amplification and rAAV production occur. During AAV manufacturing with PCLs, the upstream process targets the generation of high rAAV titers and full-capsid percentages. This is followed by a downstream process designed to effectively clear Ad5 and other process-related impurities, while further enriching full rAAV capsids. To explore potential improvements at the cellular level, we developed a mechanistic model based on empirical cell culture data, capturing intracellular dynamics of rAAV assembly and Ad5 amplification. Sensitivity analysis using this model identified cellular processes associated with rAAV yield and full-particle percentage. Notably, the analysis confirmed the critical influence of Ad5 amplification on both rAAV yield and quality. Further analysis, including nonlinear optimization, identified additional targets for cell line and helper virus engineering to evaluate process performance.