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Updated: Jun 13, 2025
Production and Titering of Recombinant Adeno-associated Viral Vectors
Published on: November 27, 2011
Intensification of rAAV Production Based on HEK293 Cell Transient Transfection
Ye Zhang1, Emil Sundäng Peters1, Olalekan Daramola2
1Cell Technology Group (CETEG), Dept. of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, AdBIOPRO, VINNOVA Competence Centre for Advanced Bioproduction by Continuous Processing, KTH Royal Institute of Technology, Stockholm, Sweden.
Manufacturing recombinant adeno-associated virus (rAAV) vectors for gene therapies is challenging. This study introduces high cell density perfusion culture to significantly boost rAAV production capacity and meet growing demand.
Area of Science:
- Biotechnology
- Bioprocessing
- Gene Therapy Manufacturing
Background:
- Recombinant adeno-associated virus (rAAV) vectors are crucial for gene therapies.
- Current manufacturing capacity is a bottleneck due to increasing global demand.
- Novel strategies are required to enhance rAAV production yields.
Purpose of the Study:
- To develop an intensified rAAV production process using high cell density (HCD) stirred tank perfusion culture.
- To optimize transfection and production parameters for rAAV vectors at HCD.
- To demonstrate the scalability and efficiency of this HCD perfusion approach.
Main Methods:
- Utilized suspension HEK293 cells for triple-plasmid transfection of rAAV1 and rAAV9 vectors (GFP cargo).
- Employed a 5 mL pseudo-perfusion spin tube system for initial optimization at HCD (50 million cells/mL).
- Scaled up the process to a 200 mL stirred tank bioreactor (STR) in perfusion mode, maintaining cell density ≥ 30 million cells/mL.
Main Results:
- Optimized transfection and production parameters at HCD in a screening system.
- Achieved rAAV9 production levels per cell comparable to conventional methods (1 million cells/mL shake flasks).
- Demonstrated successful proof-of-concept by scaling up to a 200 mL perfusion STR.
Conclusions:
- High cell density stirred tank perfusion culture is a viable strategy for intensified rAAV production.
- This approach significantly enhances volumetric production capacity.
- It offers a promising solution to address the manufacturing bottleneck for gene therapies.
