Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development

Area of Science:

• Biotechnology
• Gene Therapy
• Biopharmaceutical Development

Background:

• Recombinant adeno-associated virus (rAAV) gene therapy is rapidly advancing.
• A key challenge is separating empty capsids from full ones for effective purification.
• Accurate quantification of the empty-to-full capsid ratio is crucial for process development.

Purpose of the Study:

• To develop a novel, scalable analytical method for determining the empty-to-full capsid ratio in rAAV samples.
• To compare the new method with existing techniques like transmission electron microscopy and analytical ultracentrifugation.

Main Methods:

• Development of a novel anion-exchange high-performance liquid chromatography (HPLC) assay.
• Quantification of the empty-to-full capsid ratio using the developed HPLC method.
• Comparison of HPLC results with transmission electron microscopy (TEM) and analytical ultracentrifugation (AUC).

Main Results:

• The developed anion-exchange HPLC assay accurately determines the empty-to-full capsid ratio.
• The method shows good comparability with established techniques (TEM, AUC).
• The HPLC assay offers significantly higher throughput and a lower minimum sample concentration requirement (2.7E11 viral genomes/mL).

Conclusions:

• A novel, high-throughput anion-exchange HPLC method has been established for rAAV empty-to-full capsid ratio quantification.
• This method supports rAAV gene therapy process development by providing a scalable and sensitive analytical tool.
• The assay's improved performance facilitates better purification strategies for viral gene therapy products.