What’s the most reliable method for measuring the AAV full-to-total ratio?

The USP, NIST, and five leading biotech companies participated in a study to compare commonly used techniques to determine the full-to-total (FFT) ratio. They discovered problematic variability with the widely used PCR-ELISA method and found that some techniques performed better in repeatability across different labs.

Recombinant adeno-associated viruses (AAVs) are the most well-studied viral vectors for gene therapy, but the field is still relatively new. Despite five FDA-approved AAV-based therapies and hundreds of ongoing clinical trials, the standard methods for analyzing the quality of AAV particles are still under development.

PCR-ELISA and AUC

During testing, it was revealed that PCR-ELISA showed poor reproducibility. Between the different labs, results could vary by more than 75%, making results unlikely to be replicated reliably between labs. This is concerning as PCR-ELISA is widely used for AAVs titer measurement.

AUC (Analytical Ultracentrifugation), a highly regarded method for detecting partially filled particles, demonstrated good repeatability within individual labs. However, the complexity of analyzing mixed samples made it harder to reproduce results consistently between labs, resulting in approximately 25% standard deviation across sites.

SEC-MALS: The Most Reliable Method

The most consistent method tested was size exclusion chromatography with multi-angle light scattering (SEC-MALS) combined with UV/Vis and refractive index detection. SEC-MALS showed the best agreement between laboratories, with less than 10% variability—significantly better than AUC and PCR-ELISA. In addition, SEC-MALS also has the added benefit of providing information on size and aggregation within the same run, something that a plain UV/Vis measurement is unable to provide. Researchers suggest that, “SEC-MALS could be implemented as a general method, with [SV-AUC] being used for more complete analysis as necessary.”

Wyatt SEC-MALS Solution

Wyatt's SEC-MALS solution provides the most reliable method for measuring the full-to-total ratio of AAV particles:

• DAWN™ MALS instrument: State-of-the-art detector offering the highest sensitivity and range for AAV analysis.
• Dedicated analysis module: Simplifies data processing and is specifically designed for AAV characterization.
• Implementation training: Helps your team get started quickly and achieve consistent results between runs and across sites.
• Regulatory compliance: Uses 21 CFR Part 11-compliant software and can be implemented in a cGMP environment.