Removal of Empty AAV Capsids to Undetectable Levels Using Orthogonal Purification Steps.

Area of Science:

• Biotechnology and Bioprocessing
• Gene Therapy Manufacturing
• Viral Vector Purification

Background:

• Adeno-associated virus (AAV) gene therapy requires high-purity AAV vectors.
• Production systems yield many empty capsids, complicating purification due to similar properties to full capsids.
• Existing purification methods struggle to completely remove empty capsids.

Purpose of the Study:

• To develop and validate a robust, scalable purification process for AAV vectors.
• To achieve complete removal of empty capsids and other impurities.
• To enhance the purity of AAV for gene therapy applications.

Main Methods:

• Utilized orthogonal purification techniques: Anion Exchange Chromatography (AEX) and Zonal Ultracentrifugation (ZUC).
• Investigated empty capsid heterogeneity using AEX gradient elution and Analytical Ultracentrifugation (AUC).
• Evaluated the combined AEX-ZUC process for impurity removal and robustness.

Main Results:

• AEX and ZUC demonstrated complementary separation mechanisms (surface charge and density).
• The AEX-ZUC combination process effectively removed empty capsids to undetectable levels (by AUC).
• Significant reduction in aggregates, high molecular weight (HMW) species, and Rep protein impurities was achieved.

Conclusions:

• The AEX-ZUC combination is a highly effective and scalable method for AAV purification.
• This process significantly enhances AAV vector purity, addressing a key challenge in gene therapy manufacturing.
• The developed method ensures high-quality AAV production suitable for clinical applications.