Take a peek inside your AAV capsid with Stunner

Importance of the Topic

Quantification of AAV titers and determination of empty/full capsid ratios are essential for reliable gene therapy vector development and manufacturing. Rapid, accurate characterization reduces development time, conserves valuable samples, and supports regulatory compliance.

Goals and Overview of the Study

This application note presents the Stunner platform, which integrates UV/Vis spectroscopy, dynamic light scattering (DLS), and static light scattering (SLS) to deliver fast, label‐free quantification of AAV capsid titer, empty/full ratio, and aggregation from minimal sample volumes.

Methodology and Instrumentation

• Platform: Stunner – combines high-speed UV/Vis, SLS, and DLS in a microvolume format
• Sample preparation: 2 µL of AAV serotypes 2, 5, and 9, in PBS or citrate buffer, measured in quadruplicate
• UV/Vis: Protein and ssDNA concentrations calculated via molar extinction coefficients based on capsid and genome sequences
• DLS/SLS: Particle size distribution, aggregation profiles, and capsid concentration determination
• Complementary assays: ELISA for capsid titer, SYBR Gold fluorescence for encapsidated ssDNA verification

Main Results and Discussion

• Capsid titers determined by Stunner closely match ELISA across 2 × 10¹²–2 × 10¹³ cp/mL (R² > 0.98)
• Full capsid titer correlates with SYBR Gold assay results for encapsidated genomes
• Accurate empty/full ratio calculation (precision < 5% CV) by combining UV/Vis and scattering data
• Detection and quantification of free/aggregated protein and DNA impurities
• Rapid buffer and stress‐condition screening: 96‐well aggregation assessment in 1 hour
• Serotype‐specific aggregation behavior revealed under storage at 4 °C for two weeks

Benefits and Practical Applications of the Method

• High throughput: automation‐compatible 96‐well format
• Minimal sample volume: 2 µL per assay
• Fast turnaround: complete characterization in minutes
• No serotype‐specific reagents or labels required
• Supports regulatory frameworks (21 CFR Part 11, Pharmacopeia standards)

Future Trends and Applications

• Integration into process development for rapid formulation and stability screening
• Extension to other viral vectors and nanoparticle therapeutics
• Full automation with robotic handling to further increase throughput
• Application of advanced data analytics to predict formulation performance

Conclusion

Stunner offers a unified, orthogonal approach for AAV characterization, combining UV/Vis and light scattering to yield fast, accurate titer and empty/full ratio data from minimal volumes. This capability streamlines gene therapy development by enabling frequent, high‐throughput quality assessments and reducing reliance on time‐intensive assays.

Instrumentation Used

• Stunner platform: integrated UV/Vis spectrometer, DLS and SLS modules
• Fluorescence microplate reader for SYBR Gold assays

References

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