Systematic Studies on Stabilization of AAV Vector Formulations by Lyophilization.

Area of Science:

• Gene Therapy
• Biotechnology
• Biopharmaceutical Formulation

Background:

• Adeno-associated virus (AAV) vectors are crucial for gene therapy delivery.
• Current deep-freezing storage (-60°C) presents logistical and cost challenges.
• Developing stable, room-temperature or refrigerated formulations is essential for widespread AAV application.

Purpose of the Study:

• To evaluate freeze-drying (lyophilization) conditions for AAV vectors.
• To identify optimal excipient formulations for stable, long-term storage of AAV vectors at 2-8°C.
• To reduce capsid stress during the lyophilization process.

Main Methods:

• Investigated various freeze-drying cycles for AAV8 vectors.
• Optimized secondary drying temperature and duration.
• Assessed residual moisture content (1.5-2%).
• Formulated AAV vectors with different salt concentrations and excipients (trehalose, hydroxyectoine, poloxamer).

Main Results:

• Reduced secondary drying temperature and duration significantly decreased AAV instability.
• A formulation containing 10 mM phosphate buffer, 5.67% trehalose, 5% hydroxyectoine, and 0.1% poloxamer with ~1.5% residual moisture demonstrated stability.
• The optimized formulation allowed for stable storage at 2-8°C and for at least 4 weeks at 25°C.

Conclusions:

• Optimized lyophilization parameters, particularly secondary drying conditions, are critical for AAV vector stability.
• Specific excipient combinations can enhance the stability of freeze-dried AAV vectors.
• This research provides a foundation for developing shelf-stable AAV gene therapy products, improving accessibility and reducing storage burdens.