Robust Column Performance for Wide Pore Size Exclusion Columns for Adeno-Associated Virus Particles (AAVs) Analysis

Significance of the Topic

This section why AAV vectors crucial gene delivery vehicles in modern biopharma applications require stringent quality control to ensure safety and efficacy. Aggregation of capsid proteins in AAV preparations can compromise potency and safety so reliable size exclusion chromatography is key to monitor aggregate species and maintain critical quality attributes.

Objectives and Study Overview

The study assessed performance of Agilent AdvanceBio SEC columns with wide pore size of 500 A compared with competitor columns for analysis of multiple AAV serotypes. Performance metrics included resolution of monomer and aggregate peaks recovery and throughput across full and empty capsid samples.

Methodology

A 50 mM phosphate buffer with 400 mM sodium chloride at pH 7.2 served as mobile phase filtered to remove particulates. Chromatography used 2.7 micron silica particles and pore diameter 500 A in 4.6 x 300 mm and 4.6 x 150 mm column formats at 0.35 mL per minute injection volume 5 microliters run time 15 minutes or less in short column mode. Samples of AAV serotypes were thawed spun and transferred to HPLC vials prior to analysis.

Instrumentation Used

• Agilent 1290 Infinity II bio LC system with binary high speed pump
• Diode array detector and fluorescence detector with excitation 280 nm emission 348 nm
• Agilent OpenLab CDS ChemStation software for data acquisition and analysis

Main Results and Discussion

The AdvanceBio SEC 500 A column achieved baseline separation with resolution greater than 2.4 for aggregates and monomers across serotypes AAV1 AAV2 AAV5 AAV6 AAV8 and AAV9. Shorter column length enabled run times under six minutes with resolution of 1.64. Compared to competitor columns with smaller pore volumes AdvanceBio SEC demonstrated superior resolution up to Rs of 3.0 lower tailing factors and higher recovery for both full and empty capsid samples. Higher pore volume was identified as the key factor in improved differential migration and peak separation.

Benefits and Practical Applications

• High resolution detection of AAV aggregates supports critical quality attribute monitoring
• Robust performance across multiple serotypes and sample types
• High throughput capability with fast analysis times for large sample sets
• Enhanced recovery ensures accurate quantitation of capsid species

Future Trends and Applications

Advances in column chemistries with even greater pore volumes and optimized particle structures promise further improvements in separation of viral vectors and macromolecules. Integration with orthogonal detection methods and automated sample handling will accelerate biopharma analytics. Data driven QC workflows and AI algorithms may leverage high resolution SEC data to predict vector performance and stability.

Conclusion

Agilent AdvanceBio SEC columns with 500 A pore size deliver robust high resolution separation and superior recovery of AAV aggregates across serotypes compared to competitor products. These columns enable reliable quality control and high throughput analysis supporting gene therapy vector characterization.

References

1. Chromatographic Comparison of Wide Pore Size Exclusion Columns from Different Vendors Agilent Technologies Application Note 5994-7934EN 2024