Heightened Characterization of AAVs by SEC-MALS with XBridge Premier GTx BEH SEC 450 Å 2.5 μm Columns

Significance of the Topic

Adeno-associated viruses (AAVs) are central to modern gene therapies, requiring precise measurement of particle size, aggregation state, and genome packaging to ensure safety and efficacy. SEC-MALS offers direct, multiattribute data without reliance on calibration standards, making it indispensable for process development and regulatory submissions.

Objectives and Study Overview

This study assessed XBridge Premier GTx BEH SEC 450 Å 2.5 µm columns for high-resolution size exclusion chromatography coupled to multiangle light scattering (SEC-MALS). Key aims included characterization of AAV2 and AAV9 serotypes, quantifying monomer, aggregates, and empty/full capsid ratios under various column formats.

Methodology and Instrumentation

Sample preparation involved standard proteins (BSA, bovine thyroglobulin) and purified AAV2/AAV9 capsids at defined concentrations and empty/full ratios. Chromatographic conditions were isocratic in 2× PBS at 0.20 mL/min, 30 °C column temperature, and sample cooled to 6 °C. Detection combined UV absorbance (280 nm), refractive index (RI), and MALS (18 angles) with QELS for hydrodynamic radius. Data processing used Empower Pro 3 and ASTRA 8 software.

Instrumentation Used

• ACQUITY UPLC H-Class Bio system
• ACQUITY Tunable UV Detector (Titanium flow cell, 5 mm, 1500 nL)
• 2414 Refractive Index Detector
• WYATT DAWN™ MALS with QELS module
• Columns: XBridge Premier GTx BEH SEC 450 Å 2.5 µm (4.6×150 mm, 4.6×300 mm, 7.8×300 mm)

Main Results and Discussion

SEC-MALS resolved AAV monomers, dimers, and higher aggregates with minimal MALS noise (<30 µV). AAV2 monomer eluted at 12.2 min with molar mass ~3.7 MDa (Rg 11.6 nm, Rh 13.9 nm), while dimers and trimers measured 6.4 MDa and 9.1 MDa respectively. BTG dimer overlapped the AAV monomer, illustrating MALS utility for accurate mass determination regardless of retention time. AAV9 showed increased aggregation compared to AAV2, with monomeric mass ~3.8 MDa and well-resolved dimer at 8.2 MDa. Column diameter comparison (4.6 mm vs 7.8 mm) revealed slightly improved resolution on the wider format.

Empty/full capsid mixtures coeluted but ASTRA deconvoluted protein and nucleic acid masses for linear empty/full ratio determination (R² 0.9998), confirming capability for genome titration without qPCR or ELISA.

Benefits and Practical Applications

• Optimized pore size (450 Å) and 2.5 µm packing for AAV fractionation
• Low MALS noise and high sensitivity for low-volume samples
• Robust separation of monomers vs aggregates for accurate quantitation
• Simultaneous determination of molar mass, size, and empty/full capsid ratio

Future Trends and Applications

• Integration of high-throughput SEC-MALS workflows for process monitoring
• Extension to additional viral vectors and biotherapeutic assemblies
• Real-time feedback loops in manufacturing via inline MALS detectors
• Advanced software analytics for automated multiattribute profiling

Conclusion

XBridge Premier GTx BEH SEC 450 Å 2.5 µm columns deliver high-efficiency AAV separation and reliable multiangle light scattering for comprehensive size, mass, and empty/full ratio analysis. Both narrow and wide bore formats are effective, supporting streamlined characterization workflows critical for gene therapy development and regulatory compliance.

References

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