Upstream process development for Adeno-Associated Virus production in a continuous bioreactor

Recombinant adeno-associated viruses (rAAV) have been the viral vector of choice for most gene therapies. However, their large-scale production remains challenging due to insufficient yields to support the increasing clinical demand, high manufacturing costs, and limited scalability. Continuous production allows to reduce costs, minimize batch-to-batch variability, and enhance space-time yield. This thesis aimed to develop the upstream processing of rAAV production for further implementation in a continuous two-stage bioreactor system using a HeLaS3 producer cell line (PCL). Two HeLaS3-PCL clones were adapted to grow and produce rAAV in the same culture medium, eliminating the need for media exchanges between growth and production phases being practiced so far. These adapted cells showed stable growth kinetics, and the adaptation process did not adversely impact their ability to produce rAAV. Infections experiments revealed that a high cell concentration at infection (CCI) negatively affected yields due to the cell density effect (CDE), leading to the selection of a lower CCI for optimized rAAV production. While complete media exchange (M.E.) at the time-of-infection (TOI) allowed to maximize titers, its implementation in a bioreactor setting is challenging; thus, a pseudo-perfusion strategy was implemented in shake-flask experiments to simulate different perfusion rates conferring partial media exchange (pM.E.). These experiments showed that by-product accumulation may be compromising productivity in adapted cells when compared to non-adapted cells. Through an econometric analysis, the most cost-effective pM.E. strategies for both cell lines were identified. Continuous rAAV production was simulated in shake-flask via a virus-passage stability study, in which both cell lines were infected in 5 consecutive rounds for rAAV production. While adapted cells showed stable rAAV titers, non-adapted cells showed fluctuations, which could have been linked to the generation of defective interfering particles (DIPs). Despite these oscillations, a second econometric study confirmed that producing rAAV in a continuous setting using non-adapted cells remains the most cost-effective option. This study provides the foundation to establish the first proof-of-concept of continuous rAAV production in a two-stage bioreactor using a stable producer cell line (PCL), paving the way to process intensification through integration of continuous upstream and downstream manufacturing.