Aggregate Analysis of Adeno-Associated Viruses and Virus-Like Particles in Biopharma by Liquid Chromatography

Importance of the Topic

Adeno-associated viruses (AAVs) and virus-like particles (VLPs) are key biotherapeutic platforms in gene therapy and vaccine development. Aggregate formation can compromise product safety, efficacy, and regulatory compliance, making robust monitoring of aggregate species essential for quality control in biopharma workflows.

Objectives and Overview of the Study

This application guide presents an optimized size exclusion chromatography (SEC) approach for detecting and quantifying aggregates and fragments in AAVs and VLPs. It reviews column chemistries, pore sizes, and operating parameters to achieve clear separation between monomeric virus particles and higher-order species.

Methodology and Instrumentation

SEC exploits differences in hydrodynamic size to separate particles. Method highlights include:

• Column chemistries: Agilent Bio SEC-5 columns in 500 Å, 1000 Å, and 2000 Å pore sizes with 4.6 mm or 7.8 mm internal diameters.
• Pore-size selection: Rule of thumb—pore diameter ≈ three times the analyte diameter. A 1000 Å column suits 20–25 nm AAVs, while 2000 Å is optimal for ~40 nm VLPs.
• Mobile phase: 50 mM phosphate buffer with 400 mM NaCl at pH 7.4.
• Flow rate: 0.4–0.6 mL/min; ambient temperature.
• Detection: UV at 220 nm or fluorescence (λex 280 nm, λem 340 nm).
• Instrumentation: Agilent 1260 Infinity II LC system with quaternary pump, inline filters, and quick-connect fittings.

Key Results and Discussion

Empirical evaluations demonstrated:

• AAV analysis: A 1000 Å Bio SEC-5 column provided baseline separation of monomer and aggregates with resolution ~1.28 and retention times between 7 and 9 minutes.
• VLP separation: For a ~40 nm VLP, the 2000 Å column improved monomer–aggregate resolution (~1.51) versus the 1000 Å column.
• Best practices: Use freshly prepared, filtered mobile phase; ramp flow rates gradually; set column pressure limits; minimize injection volumes (<1% column volume); and perform regular system performance checks.

Benefits and Practical Applications

SEC offers a rapid, reproducible, and cost-effective method for aggregate profiling of viral vectors and VLPs. It supports critical quality attribute monitoring to meet regulatory requirements and can be combined with orthogonal techniques—analytical ultracentrifugation, electron microscopy, or field-flow fractionation—for comprehensive characterization.

Future Trends and Applications

• Development of specialized column chemistries to address diverse VLP architectures.
• Integration of SEC with mass spectrometry and multi-angle light scattering for concurrent size and compositional analysis.
• Miniaturization and high-throughput platforms for rapid screening during process development.
• Automation and AI-driven data analysis to detect low-abundance aggregates.

Conclusion

Optimized SEC methods using Agilent Bio SEC-5 columns deliver reliable separation and quantitation of AAV and VLP aggregates and fragments. By selecting appropriate pore sizes, controlling method parameters, and following best practices, biopharma laboratories can ensure robust quality control and regulatory compliance.

References

1. Liau B, Blackwell A, Turner ML. Resolution of Adeno-Associated Viral Vector Aggregates and Fragments with Agilent Bio SEC-5. Agilent Technologies Application Brief 5994-4270EN, 2021.
2. Mi J. Agilent Bio SEC-5 for Analysis of Virus-Like Particles (VLP). Agilent Technologies Application Brief 5994-4227EN, 2021.
3. Agilent Technologies. Agilent Bio SEC-5 Columns User Guide, Data Sheet 5973-1743, 2021.