Take a peek inside your AAV capsid with Stunner

Importance of the Topic

Gene therapy vectors based on adeno-associated virus (AAV) demand precise quantification of capsid titers and empty/full particle ratios to ensure safety and efficacy. Traditional methods such as TEM, AUC, ELISA, and qPCR can be time-intensive, require large sample volumes, and involve complex assay development. A rapid, low-volume analytical tool that simultaneously assesses capsid concentration, genome packaging, and aggregation state can accelerate process development, streamline quality control, and support regulatory compliance.

Objectives and Overview of the Study

This application note presents Stunner’s AAV Quant workflow, which integrates UV/Vis spectroscopy with static light scattering (SLS) and dynamic light scattering (DLS) in a single 2 µL measurement. The goal is to demonstrate how this platform can deliver complete AAV capsid titer, empty/full ratio, and aggregation data in minutes, using minimal sample and without the need for additional reagents or serotype-specific standards.

Methodology and Instrumentation

Monovalent AAV serotypes (AAV2, AAV5, AAV9) with known empty and genome-filled ratios were diluted in buffers at pH 4.0 and 7.0, then processed as follows:

• Samples split into heated (75 °C or 90 °C) and unheated aliquots to release encapsidated ssDNA.
• Fluorescent DNA concentration measured via SYBR Gold assay on a microplate reader.
• 2 µL samples loaded in quadruplicate on SBS plates and analyzed on the Stunner platform.
• Stunner acquires a full UV/Vis absorbance spectrum and four DLS acquisitions (5 s each), from which protein and DNA titers, particle size distribution, and scattering intensities are extracted.
• Custom molar extinction coefficients for each serotype and genome provided quantitative calibration without external standards.

Main Results and Discussion

Stunner’s total capsid titers correlated strongly with serotype-specific ELISA (R²>0.98) across 2×10¹²–2×10¹³ cp/mL. Encapsidated genome titers matched SYBR Gold results, yielding accurate % full values in dilution series and known empty/full mixtures with <5% standard deviation. DLS size distributions revealed aggregate populations, enabling detection of storage-induced aggregation differences between AAV2 and AAV9 after 2 weeks at 4 °C. Buffer stress studies showed that pH 4.0 induced near-complete aggregation of AAV9 at 45 °C, whereas pH 7.0 provided greater stability.

Benefits and Practical Applications

• Rapid 1-minute, 2 µL measurement for titer, empty/full ratio, and aggregation.
• No serotype-specific antibodies or PCR primers required.
• High-throughput compatibility with 96-well SBS plates for process development screens.
• Regulatory readiness with potential compliance to Ph. Eur., USP, and 21 CFR Part 11 environments.
• Early detection of low-yield or aggregated samples to guide downstream assays.

Future Trends and Opportunities

The integrated UV/Vis-SLS-DLS approach may expand to other viral vectors or nanoparticle platforms, supporting formulation screens, stability studies, and automated workflows. As gene therapy products diversify, rapid multi-parametric quality assessment will become essential in both academic and industrial settings. Further development could include AI-driven data analysis, integration with bioprocess control systems, and extension to impurity profiling.

Conclusion

Stunner’s AAV Quant application offers a streamlined, label-free solution for comprehensive AAV characterization. By combining UV/Vis, SLS, and DLS in a single, low-volume assay, it delivers accurate capsid titers, genome packaging ratios, and aggregation profiles in minutes, accelerating gene therapy development and supporting robust quality control.

Used Instrumentation

• Stunner platform integrating UV/Vis spectroscopy, Dynamic Light Scattering (DLS), and Static Light Scattering (SLS).
• Microplate reader for SYBR Gold fluorescence assays.
• Standard laboratory equipment (pipettes, heating block, buffers).

References

1. Dobnik D, et al. Accurate quantification and characterization of adeno-associated viral vectors. Frontiers in Microbiology. 2019;10:1570.
2. Sommer JM, et al. Quantification of adeno-associated virus particles and empty capsids by optical density measurement. Molecular Therapy. 2003;7(1):122–128.
3. Rodrigues G, et al. Pharmaceutical development of AAV-based gene therapy products for the eye. Pharmaceutical Research. 2019;36:29.