Microfluidic AAV Purity Characterization: New Insights into Serotype and Sample Treatment Variability.

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Adeno-associated virus (AAV) is a leading gene delivery platform due to low immunogenicity and high efficiency.
• Manufacturing high-quality AAV requires rapid analytical techniques, especially for assessing full capsids.
• Current methods for full capsid assessment are often slow and lack high-throughput capabilities across diverse AAV serotypes.

Purpose of the Study:

• To develop and validate a rapid, high-throughput analytical technique for assessing adeno-associated virus (AAV) full capsids.
• To establish a method compatible with multiple AAV serotypes without extensive optimization.
• To provide a robust tool for quality control in AAV manufacturing.

Main Methods:

• Utilized microfluidic electrophoresis for rapid assessment of AAV full capsids.
• Developed a mathematical formulation combining capsid protein and single-stranded DNA (ssDNA) profiles using a reference standard.
• Evaluated the use of a single serotype (AAV8) as a reference standard for other serotypes within a specific melting temperature range.

Main Results:

• The microfluidic electrophoresis method demonstrated compatibility with AAV1, AAV2, AAV6, AAV8, and AAV9, with some constraints for AAV5.
• The assay achieved characterization of samples with an accuracy of ±6.1%.
• The analytical turnaround time was less than 5 minutes per sample, requiring only 10 μL at a concentration of 2.5 × 10^12 VG/mL.

Conclusions:

• Microfluidic electrophoresis offers a rapid and high-throughput solution for assessing AAV full capsids.
• This technique supports the quality control of AAV manufacturing across various serotypes.
• The developed method addresses a critical bottleneck in viral vector production for gene therapy.