Aggregate analysis on Adeno-Associated Virus and Virus-Like Particle Analysis using newly developed novel Size Exclusion Chromatography Column

Importance of Topic

Analytical characterization of viral vectors and virus-like particles (VLPs) is critical for ensuring safety, purity, and efficacy in gene therapy and vaccine development. High-resolution size exclusion chromatography (SEC) enables accurate quantitation of monomeric and aggregated species, a key quality attribute for regulatory compliance and consistent therapeutic performance.

Objectives and Study Overview

This work presents the development and evaluation of two novel Agilent AdvanceBio SEC columns with 2.7 µm particles and pore sizes of 500 Å and 1000 Å. The aims were to demonstrate baseline separation of adeno-associated virus (AAV) serotypes and VLP aggregates, assess reproducibility and robustness over extensive injections, and compare performance against competitor columns.

Methodology and Instrumentation

• System: Agilent 1290 Infinity II Bio LC with binary high-speed pump; detection via 1260 Infinity II fluorescence detector (ex 280 nm/em 348 nm).
• Columns:
  • AdvanceBio SEC 500 Å, 2.7 µm, 4.6 × 300 mm (p/n PL1580-5325)
  • AdvanceBio SEC 1000 Å, 2.7 µm, 4.6 × 300 mm (p/n PL1580-5302)
  • AdvanceBio SEC 500 Å, 2.7 µm, 4.6 × 150 mm (p/n PL1580-3325)
• Mobile Phase: 50 mM phosphate buffer, 400 mM NaCl, pH 7.2.
• Samples: AAV serotypes 1–9 (full capsid, 10^11–10^13 GC/mL); HPV-16 VLP (1 mg/mL, ~50 nm).
• Flow Rates: 0.15–0.35 mL/min.

Main Results and Discussion

• Fast High-Resolution Separation: Baseline resolution of monomer and aggregate peaks achieved in under 6 min on the 300 mm column; comparable performance on the 150 mm column with shorter runtime.
• Reproducibility: Injection-to-injection RSD ≤1% for retention time, peak area, and resolution; batch-to-batch consistency confirmed across four column lots.
• Robustness: After 1,000 AAV9 injections, retention times, resolution (≥3.3), tailing factors, and monomer purity (≥98.8%) remained stable.
• Comparative Performance: AdvanceBio columns outperformed competitor SEC columns (Rs up to ~3.3 vs ~2.1–2.5), with higher recovery of aggregates and monomer species.
• VLP Analysis: Thermal stress of HPV-16 VLPs produced increasing aggregate formation; clear separation of monomer and high-molecular-weight species demonstrated column suitability for forced degradation studies.

Benefits and Practical Applications

• High throughput QC analysis for gene therapy and vaccine manufacturing.
• Reliable aggregate quantification to support product safety and regulatory submission.
• Flexible method development for diverse viral vectors and VLPs.
• Compatibility with fluorescence, light scattering, and mass spectrometry detection for multi-attribute characterization.

Future Trends and Application Possibilities

Ongoing advances in column technology—smaller particles, optimized pore architectures, and integrated multi-detector platforms—will further reduce analysis time and enhance resolution. Automation and high-throughput workflows, combined with high-sensitivity detectors, are poised to streamline process development and release testing in the expanding field of viral therapeutics.

Conclusion

The new Agilent AdvanceBio SEC columns deliver robust, high-resolution separations of AAV serotypes and VLP aggregates in rapid runtimes, with excellent reproducibility and longevity. Their superior performance versus competitor products makes them ideal for analytical workflows in gene therapy and vaccine quality control.

References

Agilent Technologies HPLC 2024 Poster #278