Quantifying quality attributes of AAV gene therapy vectors by SEC-UV-MALS-dRI

Significance of the Topic

The accurate quantification of adeno-associated virus (AAV) vectors is critical for ensuring the safety and efficacy of gene therapies. AAVs combine low immunogenicity with efficient delivery of genetic payloads, making them a leading platform for treating genetic diseases. Robust characterization methods support process development, quality control, and regulatory compliance throughout AAV manufacturing.

Objectives and Study Overview

This study presents a streamlined size-exclusion chromatography (SEC) method coupled with UV, multi-angle light scattering (MALS), and differential refractive index (dRI) detection to assess key quality attributes (QAs) of AAV vectors: total particle concentration, ratio of empty to full capsids, and aggregation levels. By evaluating empty and full AAV9 samples—alone and in defined mixtures—the method’s accuracy, reproducibility, and suitability for rapid release testing were demonstrated.

Methodology

SEC separations were performed on an Agilent 1260 Infinity II HPLC system using a Wyatt WTC-050S5 column with phosphate-buffered saline at 0.5 mL/min. Samples (30 μL injections) comprised purified empty and full AAV9 (single‐stranded DNA payload) and mixtures at ratios from 1:1 to 1:10. Pre‐analysis screening with a DynaPro Plate Reader removed large aggregates.

Instrumentation

• Agilent 1260 Infinity II HPLC with UV‐Vis detector (260 and 280 nm)
• Wyatt DAWN® MALS with WyattQELS® dynamic light scattering module
• Wyatt Optilab® dRI detector
• ASTRA® software for data analysis and protein conjugate modeling

Key Results and Discussion

The SEC-UV-MALS-dRI method achieved baseline separation of monomeric AAV from aggregates and fragments, with excellent retention time and peak area reproducibility. Molar mass analysis yielded capsid masses of ~3.77 MDa and DNA payload masses of ~1.16 MDa for full particles, consistent across injections. Extinction coefficients at 260 nm were established (protein: 1.3 mL/(mg·cm), DNA: 25 mL/(mg·cm)), enabling precise mass calculations.

Using combined signals, total particle concentrations were determined as 8.9×10^13 mL^–1 (empty) and 4.0×10^13 mL^–1 (full). Validation across mixed samples exhibited linear correlation between measured and expected capsid/DNA ratios (Cp/Vg). Aggregation levels were quantified: monomer content of ~92% in empty and ~99% in full preparations. The approach requires no prior structural assumptions and can be extended to other serotypes.

Benefits and Practical Applications

• Rapid analysis in under 30 minutes per sample supports high-throughput process monitoring.
• Direct quantitation of particle titer, empty/full ratio, and aggregation aids release testing and in-process control.
• High reproducibility and linearity facilitate regulatory filings and comparability studies.

Future Trends and Potential Applications

Integration with orthogonal techniques such as field-flow fractionation (FFF) can further enhance aggregate characterization without stationary-phase interactions. Adaptation to emerging engineered capsids and payloads will require updated extinction coefficients and method validation. Automated, online SEC-MALS-dRI platforms may accelerate real-time release assays in commercial manufacturing.

Conclusion

The SEC-UV-MALS-dRI workflow provides a robust, accurate, and efficient platform for comprehensive AAV vector characterization. By delivering reproducible measurements of particle concentration, capsid content, and aggregation, it addresses critical needs in gene therapy development and quality control.

References

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