Multi-parameter process optimization for high yield AAV2 vector production using scale-down multi-parallel bioreactor...

Area of Science:

• Biotechnology
• Gene Therapy
• Bioprocess Engineering

Background:

• Adeno-associated virus (AAV) vectors are crucial for gene therapy delivery.
• Transient production is a common but variable method for AAV manufacturing.
• Optimizing process parameters is essential for efficient AAV vector production.

Purpose of the Study:

• To evaluate critical process parameters for adeno-associated virus (AAV) production.
• To compare batch versus perfusion bioprocess modes for AAV yield.
• To investigate the impact of temperature shifts on AAV production efficiency.

Main Methods:

• Utilized a 15 mL multi-parallel scale-down bioreactor system to assess cell densities and plasmid DNA concentration.
• Employed a 250 mL bioreactor to compare batch and perfusion processes.
• Investigated temperature-shift strategies to enhance AAV yields.
• Assessed production using biolayer interferometry (BLI) for capsid titers and Incucyte® for functional titers.

Main Results:

• Perfusion processes yielded over three-fold lower functional titers than batch processes at 37°C in low cell density conditions.
• Temperature shifts increased functional AAV yields by nearly two-fold in batch and over three-fold in perfusion processes.
• Temperature shifts improved the ratio of functional to capsid titers, indicating enhanced encapsidation.

Conclusions:

• Optimized bioreactor conditions, including temperature shifts, significantly enhance functional AAV yields.
• The study demonstrates the scalability and reproducibility of the optimized AAV production process.
• High-throughput analytical techniques enable rapid assessment of AAV production quality.