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 and vaccine development, but their stability and yield are highly sensitive to buffer composition, pH and ionic strength. Robust, high-throughput methods for buffer exchange and stability profiling are therefore essential to accelerate process development and ensure product quality.

Objectives and Study Overview

This study combines two automated platforms, Big Tuna and Uncle, to streamline AAV buffer screening and thermal stability analysis. Big Tuna conducts high-throughput buffer exchange and concentration of AAV9 samples, while Uncle evaluates capsid integrity, genome ejection and aggregation in various formulations.

Materials and Methods

• AAV9-CMV-GFP at 7×10^11 capsids/mL in PBS with 0.001% Pluronic F68 was loaded in duplicate into a 96-well ultrafiltration plate on Big Tuna for buffer exchange into five distinct formulations, followed by two-fold concentration.
• Buffers included PBS control (pH 7.4), phosphate, Tris, acetate and citrate–phosphate buffers, with the latter serving as a destabilizing positive control.
• Big Tuna performed diafiltration with 96% exchange per cycle, 33% volume removal and orbital mixing, achieving complete processing in under two hours.
• Post-exchange capsid titers were measured by ELISA to determine sample recovery.
• Uncle assays used 9 µL samples in multi-well quartz cuvettes to monitor intrinsic fluorescence (capsid unfolding), SYBR Gold fluorescence (genome ejection) and light scattering (aggregation) during a 15–95 °C thermal ramp at 0.4 °C/min.
• Dynamic light scattering (DLS) assessed particle size distribution pre- and post-ramp.

Instrumentation

• Big Tuna automated ultrafiltration/diafiltration system for high-throughput buffer exchange and concentration.
• Uncle Multi-Mode Stability Platform equipped with full-spectrum fluorescence, static and dynamic light scattering modules.

Main Results and Discussion

• Big Tuna achieved 90–100% recovery in four buffers, while citrate–phosphate pH 4.0 showed reduced recovery (71 ± 7.4%), consistent with aggregation effects.
• Initial DLS profiles indicated intact capsids ~25 nm in PBS, phosphate, Tris and acetate buffers; citrate–phosphate samples exhibited a secondary ~120 nm peak, reflecting pre-existing aggregates.
• Citrate–phosphate buffer significantly lowered capsid unfolding (Tm = 71.7 °C) compared to other buffers (Tm ≈ 76–77 °C).
• Genome ejection Tm dropped by 11.9 °C in citrate–phosphate versus PBS, while aggregation onset (Tagg) aligned closely with genome release, highlighting formulation-dependent stability interplay.
• PBS samples exhibited aggregation temperatures nearer to capsid unfolding, underscoring buffer-specific aggregation dynamics.

Practical Applications and Benefits

• High-throughput buffer screening accelerates formulation optimization and reduces sample requirements to microliter scale.
• Combined buffer exchange and multi-modal stability analysis identifies optimal AAV conditions in a single workflow, improving quality control and process consistency.
• Reduced hands-on time and operator variability enhances reproducibility across large sample sets.

Future Trends and Opportunities

• Integration of automated buffer screening with real-time stability analytics could support adaptive process development and quality by design (QbD) frameworks.
• Expansion to additional viral vectors and nanoparticle delivery systems may broaden the platform’s impact in biotherapeutics.
• Advanced data analytics and machine learning could correlate buffer parameters with stability outcomes, enabling predictive formulation design.

Conclusion

The synergy of Big Tuna and Uncle provides an efficient, scalable solution for evaluation of AAV buffer compatibility and thermal stability. This approach facilitates rapid identification of robust formulations, ensuring high viral yield and functionality while streamlining workflow for gene therapy development.

References

• Hsu H et al. Structural characterization of a novel human adeno-associated virus capsid with neurotropic properties. Nat Commun. 2020;11(1):3279.
• Bernaud J et al. Characterization of AAV vector particle stability at the single capsid level. J Biol Phys. 2018;44(2):181–194.
• Potter M et al. A simplified purification protocol for recombinant adeno-associated virus vectors. Mol Ther Methods Clin Dev. 2014;1:14034.
• Brument N et al. A versatile and scalable two-step ion-exchange chromatography process for the purification of recombinant adeno-associated virus serotypes-2 and -5. Mol Ther. 2002;6(5):678–86.