Consecutive Affinity and Ion-Exchange Chromatography for AAV9 Vectors Purification.

Area of Science:

• Biotechnology
• Gene Therapy
• Virology

Background:

• Adeno-associated virus (AAV)-based gene therapy faces production challenges due to impurities like empty capsids.
• These impurities lack functional genomes, hindering therapeutic efficacy.

Purpose of the Study:

• To develop and assess a sequential affinity chromatography (AC) and anion-exchange chromatography (AEX) method for purifying AAV9 vectors.
• To separate fully genome-containing AAV vectors from byproduct empty and partially filled capsids.

Main Methods:

• AAV9 vectors encoding fLuc-YFP under the CBA promoter were purified using sequential AC and AEX chromatography.
• Three different AEX columns (two cross-linked sepharose, one monolithic) were evaluated for their separation efficiency.

Main Results:

• The dual chromatography method achieved approximately 20-80% recovery rates.
• A DNA-containing to empty capsid ratio exceeding 90% was consistently obtained.
• In vitro luciferase assays confirmed the infectivity of the purified AAV vectors.

Conclusions:

• The combined AC and AEX chromatography is an effective strategy for producing high-purity AAV9 vectors.
• This method yields rAAV9 vectors with over 90% DNA-containing capsids, crucial for clinical applications.