Scalable process development for rAAV transient transfection production using computational fluid dynamics modeling.

Area of Science:

• Biotechnology and Bioprocessing
• Gene Therapy Manufacturing
• Cell and Molecular Biology

Background:

• Recombinant adeno-associated virus (rAAV) is a key vector for gene therapies.
• Current upstream development for rAAV faces challenges in productivity and consistency.
• Scale-up of rAAV production requires careful management of process parameters.

Purpose of the Study:

• To design and implement a robust scale-up process for rAAV production at a 250L scale.
• To investigate and mitigate the impact of mechanical shear forces on rAAV production in bioreactors.
• To identify critical process attributes for consistent and high-yield rAAV manufacturing.

Main Methods:

• Process development in shake flasks to optimize plasmid ratios.
• Computational fluid dynamics (CFD) modeling (M-STAR) and empirical correlations (Dynochem) to characterize bioreactor hydrodynamics.
• Determination of optimal power per unit volume (P/V) for different bioreactor scales.
• Assessment of shear protectants (Poloxamer-188) and critical attributes like pre-transfection viable cell density (VCD).

Main Results:

• Initial optimization in shake flasks improved rAAV production, but scale-up to stirred-tank bioreactors showed a significant decrease in genome titer due to shear stress.
• CFD modeling guided the selection of optimal P/V values (71 W/m³ at 250mL, 20 W/m³ at 5-50L, and 15 W/m³ at 250L) to minimize shear.
• Addition of Poloxamer-188 and control of pre-transfection VCD enhanced process robustness.
• The final 250L scale process, incorporating a 30% cell culture dilution and controlled DNA complexation, demonstrated consistent cell growth and rAAV production.

Conclusions:

• Mechanical shear stress is a critical factor limiting rAAV production during bioreactor scale-up.
• A scale-up strategy based on CFD modeling and hydrodynamic characterization enables robust rAAV manufacturing.
• Optimized process parameters, including shear stress control and critical attribute management, are essential for consistent rAAV production at the 250L scale.