Perfusion-Based Production of rAAV via an Intensified Transient Transfection Process

Area of Science:

• Biotechnology
• Gene Therapy Manufacturing
• Viral Vector Production

Background:

• Recombinant adeno-associated virus (rAAV) gene therapies require large-scale manufacturing.
• Transient transfection is common but suffers from low yields and incomplete capsid formation.
• Existing methods contribute to high costs for rAAV-based treatments.

Purpose of the Study:

• To address suboptimal yields and capsid filling in rAAV production.
• To develop an intensified manufacturing process for rAAV using mechanistic model insights.
• To reduce the cost of goods for rAAV gene therapies.

Main Methods:

• Mechanistic modeling of rAAV2/5 production to identify yield limitations.
• Experimental validation of temporal DNA replication and capsid synthesis misalignment.
• Development of an intensified process combining perfusion with high cell density re-transfection.

Main Results:

• Multiple, time-separated plasmid doses increase rAAV production.
• The intensified process boosts cell-specific and volumetric productivity.
• High cell density re-transfection improves plasmid utilization compared to single bolus methods.

Conclusions:

• A new paradigm for continuous rAAV manufacturing via transient transfection is established.
• The intensified process significantly improves productivity.
• This approach reduces overall manufacturing costs for rAAV gene therapies.