Rational downstream development for adeno-associated virus full/empty capsid separation - A streamlined methodology...

Area of Science:

• Biotechnology
• Gene Therapy Manufacturing
• Downstream Processing

Background:

• Recombinant adeno-associated virus (AAV) is a leading gene delivery system for genetic disorders.
• AAV manufacturing challenges include the presence of empty capsids, which are product-related impurities impacting safety and efficacy.
• Separating empty from full AAV capsids is difficult due to subtle differences in physical properties.

Purpose of the Study:

• To develop a rapid, systematic methodology for optimizing the separation of empty and full AAV capsids.
• To apply high-throughput screening and mechanistic modeling for efficient process development.
• To improve the scalability and efficiency of AAV downstream processing.

Main Methods:

• Utilized high-throughput screening in filter-plate format to study AAV binding and elution behavior on ion-exchange media.
• Employed miniaturized separation of empty and full capsids via iodixanol gradient centrifugation for individual behavior analysis.
• Integrated mechanistic modeling and in-silico process development for optimization and understanding of separation constraints.

Main Results:

• Identified optimal conditions for differential elution of AAV8 and AAV9 empty and full capsids.
• Developed and validated column models for predicting separation performance.
• Demonstrated a systematic framework accelerating the development of scalable AAV purification processes.

Conclusions:

• The presented methodology provides a robust and efficient approach for AAV full/empty capsid separation.
• This systematic process development accelerates the manufacturing of high-quality AAV vectors for clinical applications.
• The approach enhances mechanistic understanding, crucial for robust downstream process design in gene therapy.