Improving AAV Production Yield and Quality for Different Serotypes Using Distinct Processing Methods

Area of Science:

• Gene Therapy
• Biotechnology
• Viral Vector Production

Background:

• Adeno-associated viruses (AAVs) are key gene therapy vectors with potential limited by production yields.
• Current lab-scale AAV yields range from 10^12-10^14 vgc/L, impacting cost and accessibility.
• Optimizing upstream and downstream processes is essential for increasing AAV production efficiency.

Purpose of the Study:

• To compare two purification methods (CsCl ultracentrifugation and affinity column chromatography) for adeno-associated virus (AAV) recovery.
• To optimize AAV yields and assess the impact on product quality and transduction efficiency.
• To enhance the overall production capacity and reduce the cost of AAV-based gene therapies.

Main Methods:

• Utilized an adherent cell manufacturing process for AAV production.
• Compared purification of AAV from cell lysate (CsCl ultracentrifugation) and media supernatant (affinity chromatography).
• Quantified purified AAV yields for serotypes AAV6, AAV8, and AAV9 using both methods.

Main Results:

• Achieved combined yields of 2.25 × 10^13 (AAV6), 1.11 × 10^14 (AAV8), and 7.32 × 10^13 (AAV9) vg/L.
• Affinity chromatography yielded higher percentage recoveries (AAV6: 88.35%, AAV8: 96.67%, AAV9: 93.54%) from media supernatant.
• AAV9 production showed a ~1.5-fold increase compared to previous reports; chromatography also improved transduction rates.

Conclusions:

• Affinity chromatography is a superior method for AAV purification, offering higher recovery and improved product quality.
• Optimized purification strategies demonstrate potential for significantly expanding AAV production capacity.
• Further research into serotype-specific elution pH can further refine AAV purification processes.