Separation of empty and full adeno-associated viral capsids by MCSGP: From model-based design to implementation

Area of Science:

• Biotechnology
• Bioprocessing
• Gene Therapy

Background:

• Adeno-associated viruses (AAVs) are critical vectors for gene therapy delivery.
• Separating full, gene-containing AAV particles from empty or misloaded ones remains a significant challenge.
• Current methods struggle to achieve efficient and scalable empty/full AAV separation.

Purpose of the Study:

• To systematically investigate and optimize anion exchange chromatography for AAV2 empty/full particle separation.
• To develop a model-based optimization strategy for maximizing yield and productivity.
• To evaluate the performance of multicolumn countercurrent solvent gradient purification (MCSGP) for this application.

Main Methods:

• Systematic investigation of anion exchange chromatography parameters (gradient slope, salt concentration) in single-column operations.
• Development of a model-based optimization algorithm to identify optimal separation conditions.
• Experimental validation of single-column and multicolumn countercurrent solvent gradient purification (MCSGP) processes.

Main Results:

• Single-column chromatography revealed a trade-off between yield and productivity, with maximized yield at 11.3% and productivity at 3.85E13 VP/(L_resin h).
• Multicolumn countercurrent solvent gradient purification (MCSGP) significantly improved performance, achieving 57.8% yield and 8.65E13 VP/(L_resin h) productivity at 70% purity.
• MCSGP reduced process mass intensity by a factor of 3, demonstrating enhanced efficiency.

Conclusions:

• Anion exchange chromatography is a viable method for empty/full AAV2 separation.
• Multicolumn countercurrent solvent gradient purification (MCSGP) offers a substantial advancement over single-column methods for AAV purification.
• MCSGP presents a promising, scalable solution for improving gene therapy vector production efficiency.