Soup-up AAV & LNP sample prep with Big Tuna

Significance of the Topic

Gene therapy development relies on accurate characterization and formulation of delivery vehicles such as lipid nanoparticles (LNPs) and adeno-associated viruses (AAVs). Efficient desalting, buffer exchange, and concentration protocols are essential to preserve sample integrity and to scale analytical workflows.

Objectives and Study Overview

This application note evaluates Big Tuna, an automated pressure-based ultrafiltration/diafiltration (UF/DF) platform, for high-throughput buffer exchange and concentration of LNPs, AAVs, and nucleic acids. Key aims include:

• Automating low-volume, high-throughput sample preparation.
• Optimizing processes tailored for LNP, AAV, and DNA samples.
• Comparing performance metrics such as recovery, exchange efficiency, and processing time.

Methodology and Instrumentation

Big Tuna integrates with Unfilter 96 and Unfilter 24 filter plates, enabling simultaneous processing of up to 96 (100–450 µL) or 24 (0.45–8 mL) samples. The system cycles through filtration, volume measurement, and buffer refill under user-selected pressure settings with gentle orbital mixing. Analytical instruments included:

• SpectraMax i3 plate reader with RiboGreen assay for encapsulation efficiency.
• Stunner AAV Quant application for concentration and empty/full capsid ratio.
• Lunatic UV/Vis system for DNA concentration measurements.

Main Results and Discussion

1. LNP buffer exchange and concentration:

• Achieved 99.7 ± 0.0% buffer exchange and final concentration of 83.5 ± 2.3 µg/mL (target 78.4 µg/mL).
• Encapsulation efficiency remained high (96.5 ± 0.1% vs. 98% initial).

2. AAV buffer exchange:

• Unfilter 96: 97.0 ± 0.2% exchange, final titer 2.37E13 cp/mL, empty/full ratio shift from 23/77 to 28/72.
• Unfilter 24: 96.2 ± 0.2% exchange, final titer 3.13E13 cp/mL, empty/full ratio from 29/71 to 36/64.

3. AAV volume reduction:

• 36-fold concentration from 18 mL to 523.7 ± 4.2 µL, achieving 1.88E13 cp/mL (target 1.74E13 cp/mL).
• Empty/full capsid ratio remained stable.

4. DNA desalting:

• Unfilter 96: 97.8 ± 1.4% exchange, final 7.16 ± 0.35 mg/mL (target 6.24 mg/mL).
• Unfilter 24: 98.5 ± 0.2% exchange, final 6.71 ± 0.20 mg/mL (target 6.24 mg/mL).

Benefits and Practical Applications

Big Tuna delivers significant workflow advantages:

• High throughput capacity up to 96 samples per run.
• Customizable pressure and exchange parameters for diverse biomolecules.
• Improved reproducibility and sample integrity compared to manual dialysis or ultrafiltration.
• Reduced hands-on time and scalable processing for research and quality control.

Future Trends and Applications

Emerging needs in gene therapy and vaccine development call for even faster, fully integrated sample prep and analytics. Potential future developments include:

• Integration with inline analytics for real-time quality metrics.
• Expanded filter chemistries for broader molecular targets.
• Automation enhancements for end-to-end workflows from cell culture to characterization.

Conclusion

Big Tuna provides a versatile, high-throughput solution for buffer exchange and concentration of LNPs, AAVs, and nucleic acids, preserving sample integrity while streamlining gene therapy workflows. Its flexibility and performance support robust analytical characterization and scale-up demands.

Reference

Unchained Labs Application Note: Soup-up AAV and LNP sample prep with Big Tuna, Rev A, 2021.