Stunner AAV & AdV Characterization

Importance of the Topic

The accurate and rapid quantification of viral vectors such as adeno-associated virus (AAV) and adenovirus (AdV) is essential for gene therapy development and manufacturing. Key parameters include total capsid titer, full versus empty capsid ratio, aggregation state, and biomolecular purity. Traditional assays often require large sample volumes, labels or standards, and lengthy workflows. The Stunner platform integrates multiple analytical techniques on minimal sample volumes to streamline vector characterization.

Objectives and Overview

This study presents a unified workflow for comprehensive AAV and AdV analysis using the Stunner system. By combining UV/Vis concentration measurements with dynamic and static light scattering (DLS, SLS), rotating angle DLS (RADLS) and multi-angle light scattering (MALS), the platform aims to deliver rapid, label-free quantification of total and genome-containing capsids, while assessing aggregation and size distribution across diverse vector serotypes.

Methodology and Used Instrumentation

Samples of AAV or AdV (2 µL) are loaded directly onto a 96-well microfluidic plate featuring fixed 0.1 mm and 0.7 mm pathlength circuits for broad absorbance range (0.03–275 OD). No dilution, labeling or external standards are required. Automated UV/Vis spectrophotometry unmixes protein and nucleic acid signals. DLS and SLS characterize particle size distribution and detect aggregates for AAV, while RADLS and MALS extend these measurements to larger AdV particles. Integrated software calculates capsid titers and empty/full ratios.

• UV/Vis: Xenon flash lamp, 230–750 nm, spectral resolution <2 nm, absorbance precision <±0.01 OD.
• DLS: Two 660 nm laser diodes, avalanche photodiode, size range 0.3–1000 nm, <±2 % size accuracy.
• RADLS/MALS: Multi-angle detection for high-mass particles (molecular weight 1 kDa–10 GDa).
• Microfluidic Plate: 96 samples, 2 µL volume, retention up to 2 h, automated robotic integration.

Main Results and Discussion

For AAV, the platform quantifies total capsid titer down to ~10¹² vg/mL and empty/full ratios in under one minute per sample. DLS/SLS identifies monodisperse preparations and aggregates. Preloaded serotype methods enable rapid titration without additional calibration. In AdV assays, spectral unmixing of protein and dsDNA absorbance yields total, full and empty capsid titers down to 10⁹ cp/mL, outperforming conventional A260 measurements.

Benefits and Practical Applications

• Minimal sample consumption (2 µL) with no dilution or labeling.
• High throughput: up to 96 AAV samples per hour, ~2 h 15 min for full AdV plate.
• Automated data processing reduces user intervention and error.
• Comprehensive profiling: capsid titer, empty/full ratio, aggregation and size distribution.
• Adaptable to multiple AAV serotypes and AdV vectors.

Future Trends and Potential Applications

Advancements in scattering algorithms and spectral unmixing will further improve sensitivity and accuracy. Integration with robotic liquid-handling and laboratory information management systems will support fully automated end-to-end workflows in vector production. Extending the platform to other viral or nanoparticle systems can broaden its impact in biopharmaceutical R&D and quality control.

Conclusion

The Stunner system offers a robust, label-free approach for rapid, high-throughput viral vector characterization. By unifying UV/Vis spectroscopy with multi-angle light scattering in a microfluidic format, it delivers critical information on capsid titers, empty/full ratios, and aggregation states, addressing key analytical needs in gene therapy development and manufacturing.