Explore AAV buffers and stability with Big Tuna and Uncle

Significance of the Topic

Adeno-associated virus (AAV) vectors are widely used in gene therapy but are sensitive to buffer composition, pH and ionic strength. These variables can influence capsid integrity, genome retention and infectivity, making buffer selection and stability profiling essential during process development. Traditional buffer exchange and stability assays are labor-intensive, consume large sample volumes and offer limited throughput, slowing down formulation screening and optimization.

Study Objectives and Overview

This work demonstrates a combined workflow using Big Tuna, an automated high-throughput buffer exchange and concentration system, together with Uncle, a multi-modal thermal stability platform. The goals were to exchange AAV9 vectors into multiple formulation buffers in parallel, concentrate samples twofold, and characterize thermal stability (capsid unfolding, genome ejection and aggregation) across different buffer conditions.

Methodology and Instrumentation

• Big Tuna automated buffer exchange and concentration system using pressure-based ultrafiltration/diafiltration with 10 kDa Unfilter 96 plates. Up to 96 samples can be processed simultaneously, achieving 96% buffer exchange per pool and target 2× concentration.
• Uncle stability platform employing intrinsic fluorescence, static light scattering (SLS) and dynamic light scattering (DLS). Thermal ramps (15–95 °C at 0.4 °C/min) were monitored for capsid unfolding (intrinsic fluorescence), genome ejection (DNA-binding dye SYBR Gold) and aggregation (SLS 473 nm intensity and DLS size distribution).
• AAV9-CMV-GFP at 7×10^11 cp/mL in PBS with 0.001% Pluronic F68 was buffer-exchanged into five conditions: PBS (pH 7.4), phosphate (pH 6.5), Tris (pH 8.0), acetate (pH 5.0) and citrate-phosphate (pH 4.0), followed by twofold concentration.
• Capsid titers measured by ELISA in triplicate to assess recovery after exchange.

Main Results and Discussion

Big Tuna completed buffer exchange and concentration of 10 wells in under two hours. Capsid recovery by ELISA was 96–101% for four buffers, but only 71% in the pH 4.0 citrate-phosphate buffer, indicating reduced stability. Initial DLS profiles showed single 25 nm peaks for intact capsids except for citrate buffer, which exhibited a second 120 nm aggregate peak. Uncle measured capsid unfolding Tm values near 76–77 °C in four buffers and a lower Tm of 71.7 °C in citrate buffer. Genome ejection Tm and aggregation onset (Tagg) were also significantly lower in the pH 4.0 buffer, illustrating how acidic conditions promote premature genome release and capsid aggregation.

Benefits and Practical Applications

The integrated Big Tuna and Uncle workflow enables rapid, reproducible screening of AAV formulations using minimal sample volumes. Parallel processing of up to 96 conditions accelerates buffer optimization, reduces assay time and improves decision-making during gene therapy vector development.

Future Trends and Opportunities

• Extension to other AAV serotypes and viral vectors for broader process optimization.
• Integration of additional biophysical assays (e.g., electron microscopy, analytical ultracentrifugation) for comprehensive characterization.
• Automation of data analysis with machine learning to predict stability and guide formulation design.
• Scale-up of high-throughput workflows to support clinical and commercial manufacturing.

Conclusion

By combining Big Tuna’s high-capacity automated buffer exchange with Uncle’s multi-modal thermal stability assays, researchers can efficiently map AAV stability across multiple formulations. This approach addresses key bottlenecks in gene therapy vector development, enabling faster, more reliable buffer selection and process optimization.

References

1. Hsu H, et al. Nature Communications 2020;11(1):3279.
2. Bernaud J, et al. Journal of Biological Physics 2018;44(2):181–94.
3. Potter M, et al. Molecular Therapy – Methods & Clinical Development 2014;1:14034.
4. Brument N, et al. Molecular Therapy 2002;6(5):678–86.