Comprehensive Comparison of AAV Purification Methods: Iodixanol Gradient Centrifugation vs. Immuno-Affinity...

Area of Science:

• Gene Therapy
• Biotechnology
• Virology

Background:

• Recombinant adeno-associated viruses (AAVs) are crucial gene delivery tools for research and therapy.
• Optimizing AAV vector production is essential for enhancing safety and efficacy.
• Suspension HEK293 cells are commonly used for large-scale AAV vector manufacturing.

Purpose of the Study:

• To compare the efficacy of two distinct downstream purification methods for recombinant AAV vectors.
• To evaluate the impact of purification methods on vector purity, recovery, and infectivity.
• To assess batch-to-batch variability in AAV vector production and its influence on purification outcomes.

Main Methods:

• Comparison of iodixanol gradient ultracentrifugation and immuno-affinity chromatography (POROS™ CaptureSelect™ AAVX column).
• Purification of multiple AAV vector batches (serotypes AAV5, AAV8, AAV9) using both methods.
• Characterization of purified vectors and assessment of in vitro and in vivo transduction efficiency in mice with various transgenes.

Main Results:

• Both purification methods demonstrated advantages and disadvantages concerning purity, viral genome recovery, and empty particle content.
• Transduction efficiency differences were primarily linked to inherent batch-to-batch variability.
• Neither purification method showed a definitive superiority in yielding potent AAV vectors.

Conclusions:

• Iodixanol gradient ultracentrifugation and immuno-affinity chromatography are both effective for purifying recombinant AAV vectors.
• Batch-to-batch variability is a significant factor influencing the performance of purified AAV vectors.
• The choice of purification method may depend on specific project requirements regarding purity and recovery.