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

Importance of Topic

High-resolution size exclusion chromatography (SEC) is essential for characterizing aggregation in viral vectors and virus-like particles (VLPs), critical quality attributes that impact the safety and efficacy of gene-therapy products. Fast, reliable separation of monomer and higher-order species underpins regulatory compliance and process control in biopharmaceutical development.

Study Objectives

This study evaluates two novel Agilent AdvanceBio SEC columns (500 Å and 1000 Å pore sizes, 2.7 µm particles) tailored to analyze adeno-associated virus (AAV) serotypes and VLP aggregates. The goals were to demonstrate high resolution, rapid runtime, reproducibility across injections and column lots, robustness over extended use, and performance versus competitor columns.

Methodology

SEC separations were conducted on Agilent 1290 and 1260 Infinity II Bio LC systems using a phosphate buffer (50 mM, 400 mM NaCl, pH 7.2). Columns tested included 4.6×300 mm and 4.6×150 mm formats of 500 Å and 1000 Å pore size. Samples comprised full-capsid AAV serotypes 1–9 and human papilloma virus type 16 (HPV-16) VLP. Forced-aggregation studies employed thermal stress at 45 °C. Performance metrics (retention time, resolution, monomer/aggregate percentage, tailing factor) were assessed over replicate injections, column production lots, and after 1000 sequential injections of matrix and AAV9 sample.

Instrumentation

• Agilent 1290 Infinity II Bio LC system with binary high-speed pump
• Agilent 1260 Infinity II fluorescence detector (λ_ex = 280 nm, λ_em = 348 nm)
• AdvanceBio SEC columns (500 Å & 1000 Å, 2.7 µm, 4.6×300 mm and 4.6×150 mm)
• Phosphate buffer mobile phase, flow rates 0.15–0.35 mL/min

Key Results and Discussion

• Fast throughput: baseline separation of AAV9 monomer and aggregate achieved in <6 min on the 150 mm column with resolution up to 2.94.
• Reproducibility: injection-to-injection RSD ≤1%, lot-to-lot consistency across four column batches, and batch-to-batch column reproducibility for retention time, resolution, and recovery (~99%).
• Robustness: after 1000 injections, monomer/aggregate resolution remained constant (3.3) and tailing factors stable, demonstrating sustained performance.
• Serotype coverage: all six tested AAV serotypes exhibited clear baseline separation (resolution 2.35–3.17) with monomer purity >95%.
• Competitive advantage: AdvanceBio columns outperformed alternative vendor columns in resolution (up to 1.63 vs. 0.97) and aggregate recovery.
• Compatibility: demonstrated low background signal with light scattering and ESI-MS detection, enabling multi-detector workflows.

Benefits and Practical Applications

The new SEC columns enable rapid, high-resolution monitoring of viral vector and VLP aggregation, facilitating quality control in gene-therapy development and manufacturing. Reduced run times and robust column lifetime support high throughput in regulated environments.

Future Trends and Opportunities

• Integration with mass spectrometry and multi-angle light scattering detectors for detailed aggregate characterization.
• Miniaturized column formats and microflow LC to decrease sample consumption and solvent use.
• Standardization of SEC methods for regulatory filings and cross-platform comparability.
• Application to emerging nanoparticle delivery systems and other biotherapeutic modalities.

Conclusion

Agilent’s AdvanceBio SEC columns deliver fast, reproducible, and robust separation of viral vector and VLP monomer versus aggregates. Their performance stability over extensive injection series and superior resolution against competitor columns position them as valuable tools for biopharmaceutical QC workflows.