Soup-up AAV & LNP sample prep with Big Tuna

Význam tématu

Efficient buffer exchange and concentration of lipid nanoparticles (LNPs), adeno-associated viruses (AAVs) and nucleic acids are essential steps in gene therapy development and vaccine research. Rapid removal of solvents and salts while preserving particle integrity and payload encapsulation is critical for downstream analysis, formulation and scale-up.

Cíle a přehled studie

This application note demonstrates how the Big Tuna automated ultrafiltration/diafiltration platform addresses high-throughput, low-volume buffer exchange and concentration challenges for LNPs, AAVs and DNA. It showcases optimized presets and workflows using Unfilter 96 and Unfilter 24 plate formats for solvent removal, buffer replacement and sample concentration.

Použitá metodika a instrumentace

The Big Tuna platform employs pressure-based UF/DF combined with gentle orbital mixing to prevent membrane fouling and ensure uniform flow. Two plate formats were used:

• Unfilter 96: 100–450 µL per well, up to 96 samples
• Unfilter 24: 0.45–8 mL per well, up to 24 samples

Key analytical tools:

• Big Tuna Client software: experimental control and data logging
• RiboGreen assay on SpectraMax i3: LNP encapsulation efficiency
• Stunner AAV Quant application: AAV concentration and empty/full capsid ratio
• Lunatic UV/Vis: DNA concentration measurement

Hlavní výsledky a diskuse

LNP buffer exchange:

• 99.7 % buffer exchange achieved with 100 kDa Unfilter 24
• Final volume 436.5 ± 12 µL and target concentration 78.4 µg/mL met
• Encapsulation remained high (96.5 % vs. 98 % initial)

AAV buffer exchange and concentration:

• 96–97 % buffer exchange in both Unfilter 96 and 24
• Final volumes and concentrations matched targets (2.37×10^13 cp/mL and 3.13×10^13 cp/mL)
• Capsid empty/full ratio remained stable within ±5 %
• 36-fold concentration achieved from 18 mL to ~524 µL

DNA desalting:

• 96–98.5 % exchange into nuclease-free water using both plate formats
• Final DNA concentrations exceeded targets at ~6.7 mg/mL

Přínosy a praktické využití metody

The Big Tuna workflow provides:

• High-throughput, automated buffer exchange and concentration
• Customizable pressure and exchange parameters optimized per sample type
• Minimal manual intervention and rapid processing times
• High recovery rates and preservation of particle integrity

Budoucí trendy a možnosti využití

Future developments may include:

• Expanded presets for additional biomolecules (proteins, peptides, virus-like particles)
• Integration with upstream formulation and downstream analytics
• Automation of multi-step purification workflows
• Scale-up for manufacturing and QA/QC environments

Závěr

Big Tuna offers a versatile, high-throughput UF/DF solution for gene therapy and vaccine sample preparation. By combining pressure-based filtration with gentle mixing and optimized presets, it achieves rapid buffer exchange and concentration while maintaining sample integrity and high recovery.

Reference

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