AAV Aggregate Quantitation and Identification with the Aura System

Significance of the Topic

Adeno-associated viruses (AAVs) are central to modern gene therapy because they efficiently deliver therapeutic genes to target cells. Monitoring subvisible particle formation and capsid stability is critical for ensuring safety, efficacy, and regulatory compliance, as aggregates can induce immune responses and affect shelf life.

Objectives and Study Overview

This application note aimed to:

• Quantify subvisible aggregates in AAV2, AAV5, and AAV8 under various stress conditions using the Aura System.
• Compare aggregate formation to thermal stability predictions from differential scanning fluorimetry (DSF).
• Characterize particle morphology with combined brightfield and fluorescence membrane microscopy (FMM).

Methodology and Instrumentation

Viral samples (AAV2, AAV5, AAV8) were formulated in PBS with 0.01% pluronic F68. Stress conditions included:

• Control at 4 °C
• Thermal stress at 73 °C for 2 hours
• Rotational stress (vortex, rotate, vortex)
• Two freeze–thaw cycles
• Surface contact with ETFE plastic mimics
• Water-for-injection control with Thioflavin-T (ThT) membrane pre-treatment

Samples (5–30 µL) were analyzed on the Aura platform, which integrates ultra-low-volume flow imaging and fluorescence membrane microscopy. Brightfield imaging captured particle morphology, while ThT fluorescence identified proteinaceous capsid aggregates.

Main Results and Discussion

• Unstressed samples showed serotype-dependent baseline aggregation: AAV2 < AAV5 < AAV8 both in count and membrane coverage.
• Thermal stress increased aggregation for all serotypes, yielding distinct morphologies: granular (AAV2), fibrillar (AAV5), and film-like matrix (AAV8).
• Quantitative counts (≥2 µm) and membrane coverage aligned across stress conditions, with thermal stress producing up to 2×10⁶ particles/mL and ~16% coverage.
• Rotational and freeze–thaw stresses yielded moderate aggregation, with rotational stress generating more large particles (≥25 µm).
• Fluorescence microscopy with ThT confirmed that >90% of detected particles were protein capsid aggregates; ETFE mimics exhibited scattering but no fluorescence.
• Comparison with DSF melting temperatures (AAV2: 67 °C, AAV8: 73 °C, AAV5: 89 °C) revealed no direct correlation between T_m and subvisible aggregation, highlighting that thermal unfolding does not predict particle formation.

Benefits and Practical Applications

The Aura System offers:

• Ultra-low sample volume (as little as 5–10 µL) compared to conventional flow imaging and light obscuration.
• Rapid analysis (<1 min per sample) of subvisible particles with morphological and fluorescence characterization.
• Enhanced insight into aggregation mechanisms for formulation development and quality control.
• Compliance support for regulatory submissions by providing robust subvisible particle data.

Future Trends and Applications

Emerging directions include:

• Extending low-volume subvisible analysis to other viral vectors and protein therapeutics.
• Integrating artificial intelligence for automated particle classification and mechanistic insights.
• Combining subvisible particle data with other biophysical assays for holistic stability profiling.
• Adapting fluorescence staining panels to detect diverse aggregate types and excipient interactions.

Conclusion

The Aura platform enables comprehensive, low-volume quantitation and identification of AAV subvisible aggregates under various stress conditions. Its combined brightfield and fluorescence capabilities deliver actionable data on particle count, morphology, and composition, outperforming conventional methods in speed, sample consumption, and mechanistic insight.

References

• Wang D, Tai PW, Gao G. Adeno-Associated Virus Vector as a Platform for Gene Therapy Delivery. Nat Rev Drug Discov. 2019;18(5):358–78.
• Test for Particulate Contamination: Subvisible Particles General Chapter Guidance for Industry. U.S. FDA. 2017.
• Bennett A, Patel S, Mietzsch M, et al. Thermal Stability as a Determinant of AAV Serotype Identity. Mol Ther Methods Clin Dev. 2017;6:171–182.