Multivariate Process Optimization for Fixed-Bed Bioreactor-Based AAV Production Improves Total Batch Yield.

Area of Science:

• Biotechnology
• Gene Therapy
• Bioprocessing

Background:

• Adeno-associated viral vectors (AAVs) are critical for gene therapy but face production scalability challenges.
• High-quality, high-yield AAV production is essential for clinical applications.

Purpose of the Study:

• To optimize adeno-associated viral vector (AAV) production in a fixed-bed bioreactor.
• To enhance AAV yield and scalability for gene therapy applications.

Main Methods:

• Systematic optimization of cell seeding density, transfection density, and DNA-to-cell ratio in a fixed-bed bioreactor.
• Utilized transient transfection in adherent HEK-293T cells, packaging reporter and therapeutic genes into AAV9 capsids.
• Monitored key bioreactor parameters (pH, dissolved oxygen, glucose) over a 10-day production cycle.

Main Results:

• Achieved cell densities up to 3 × 10^9 cells/m^2 in the fixed-bed bioreactor.
• Increased viral yield by approximately 7.6-fold compared to the prototype batch.
• Attained an average AAV vector yield of 2.3 × 10^14 vg, with an average per-cell yield of 1.4 × 10^5 vg/cell.

Conclusions:

• Optimizing process parameters in fixed-bed bioreactors is a viable strategy for scalable AAV production.
• This approach offers a promising route for cost-effective manufacturing of AAV vectors for clinical trials.
• The optimized process supports high cell densities and significantly boosts viral yield.