Stunner AAV & AdV Characterization

Significance of the Topic

The accurate characterization of viral vectors like adeno-associated virus (AAV) and adenovirus (AdV) underpins the development, manufacture, and quality control of gene therapies and vaccines. Key parameters such as total capsid titer, empty/full capsid ratio, aggregation state, and biomolecular composition directly impact product safety, efficacy, and batch-to-batch consistency.

Objectives and Study Overview

This work presents the Stunner platform, designed to consolidate UV/Vis concentration measurements with dynamic and static light scattering data in a single 2 µL assay. It targets rapid determination of AAV and AdV attributes—capsid counts, empty versus full ratios, aggregate presence, and molecular sizing—without extensive sample preparation.

Methodology and Instrumentation

The Stunner system employs a microfluidic 96-well plate with dual fixed pathlengths (0.1 mm and 0.7 mm) covering 0.03–275 OD. UV/Vis concentration analysis uses a xenon flash lamp and polychromatic spectrophotometer across 230–750 nm, with precision of ±0.005 OD below 1 OD and ±0.5% CV at 1–2 OD. Particle sizing and aggregation are assessed by dynamic light scattering (DLS) and static light scattering (SLS) for AAV, and rotating angle DLS (RADLS) with multi-angle light scattering (MALS) for larger AdV particles. Data unmixing algorithms separate overlapping protein and nucleic acid spectra, eliminating the need for labels, dyes or external standards.

Main Results and Discussion

For AAV quantification, Stunner achieves sensitivity down to 1×10¹² vg/mL, reporting total capsid titer, full/empty ratio and residual free protein or ssDNA within one minute per sample. A full 96-well plate is processed in approximately one hour, and the system supports robotic integration for higher throughput. DLS/SLS profiling distinguishes monodisperse capsids from aggregates, maintaining sizing accuracy within ±2% over 0.3–1000 nm.
For AdV, the platform extends detection to 1×10⁹ cp/mL, providing total, full, and empty capsid counts plus protein and dsDNA titers in ~2 h 15 min per plate. Preloaded serotype calibrations or user-defined inputs ensure broad applicability across AAV and AdV variants.

Benefits and Practical Applications

• Minimal sample volume (2 µL) with no dilution or prep steps.
• Integrated, label-free workflow combining UV/Vis, DLS, SLS/RADLS/MALS.
• High throughput: 96 measurements per hour for AAV; ~2 h 15 min for AdV.
• Direct readout of critical quality attributes: titer, empty/full ratio, aggregation, sizing, composition.
• Automation-friendly design enabling seamless integration into QC and process development pipelines.

Instrumentation Used

• Stunner system with xenon flash UV/Vis spectrophotometer (230–750 nm).
• DLS: 660 nm laser diodes, avalanche photodiode detector.
• RADLS/MALS: 5–30 detection angles for AdV analysis.
• 96-well microfluidic plate, dual pathlength design (0.1 mm & 0.7 mm).
• Windows 11–controlled PC via USB or TCP/IP.

Future Trends and Opportunities

Emerging demands for faster, more precise viral vector analytics will drive integration of additional orthogonal methods (e.g., fluorescence, mass photometry) and advanced data analytics, including machine learning–based quality assessment. Adoption of fully automated workflows with real-time monitoring will accelerate process optimization and release testing in biomanufacturing.

Conclusion

The Stunner platform offers a unified, high-throughput solution for comprehensive AAV and AdV characterization, meeting critical needs in gene therapy development and quality control. By combining UV/Vis concentration analysis with multi-angle light scattering in a label- and standard-free format, it delivers rapid, robust and reproducible data from minimal sample volumes, streamlining viral vector analytics across research and manufacturing settings.