IMPROVING PRODUCTIVITY AND ROBUSTNESS OF ADENO-ASSOCIATED VIRAL VECTOR PRODUCTION VIA MULTIVARIATE OPTIMIZATION AND POLYPLEX STABILITY

Abstract

Adeno-associated viruses (AAVs) have emerged as promising gene delivery vectors to treat debilitating and sometimes fatal genetic disorders, with six FDA-approved AAV biologics currently available in the United States. Despite their therapeutic potential, the high cost of AAV drugs may limit their accessibility, in part due to low yields in manufacturing. This thesis addresses two key aspects of AAV production aimed at improving yield in HEK293 cells: (i) volumetric productivity, and (ii) robustness of the transfection step. To improve volumetric productivity, we sought to optimize the bioprocess and transfection conditions using a commercially available transfection reagent, FectoVIR-AAV. This created a systematic workflow that other bioprocess engineers can adapt to improve productivity and product quality. The bioreactor transfection pH, production pH, viable cell density, reagent-to-DNA ratio, and complexation time were identified as important factors for process development. To address process robustness, we showed that prolonged transfection complexation time reduces productivity due to uncontrolled polyplex growth. Once the optimal size of polyplexes was identified as 800 – 1000 nm, two techniques were developed to arrest transfection complex growth and improve operational flexibility of this unit operation: (i) RODI water addition and (ii) acid quenching. The acid quenching technique was scaled to a 50 L bioreactor and achieved ~60% productivity when polyplexes were held for 4 h prior to transfection of cell culture. Compared to what would have been <1% productivity if polyplexes were not quenched prior to a 4 h incubation, acid quenching is a promising technique for improving AAV process robustness. The findings presented in this thesis provide actionable strategies for bioprocess engineers to develop high-yield and robust AAV manufacturing processes, which can promote distribution of cost-effective gene therapies.