Size-exclusion chromatography of adeno-associated viruses with the SurePac Bio 550 SEC MDi column

Significance of the Topic

Size-exclusion chromatography (SEC) of adeno-associated virus (AAV) is a critical step in ensuring the purity, homogeneity, and safety of viral vectors used in gene therapy. Efficient separation of monomeric capsids from aggregates and impurities supports accurate dosing, minimizes patient risk, and streamlines quality control in both research and biomanufacturing settings.

Study Objectives and Overview

The main goal of this work was to demonstrate rapid, high-resolution baseline separation of AAV monomers and high molecular weight species (HMWS) using a 4.6 × 150 mm SEC column packed with 3 µm monodisperse silica particles (550 Å pore size). The study evaluated detection by UV, fluorescence (FLD), and multi-angle light scattering (MALS), and benchmarked performance against competitor columns.

Methodology and Instrumentation

• Chromatographic system: Thermo Scientific™ Vanquish™ Flex UHPLC with quaternary pump, variable wavelength UV detector, fluorescence detector, and optional SEC-MALS configuration.
• Column: SurePac Bio 550 SEC MDi 3 µm, 4.6 × 150 mm, 550 Å pore size, hydrophilic diol-coated monodisperse silica media.
• Mobile phase: 50 mM phosphate buffer, 300 mM NaCl, pH 6.5.
• Flow rate: optimized between 0.05 mL/min (high resolution) and 0.6 mL/min (high throughput).
• Detection: UV at 260 and 280 nm to distinguish protein and nucleic acid content; FLD (Ex 280 nm, Em 330 nm) for enhanced sensitivity; SEC-MALS for absolute molecular weight and radius.
• Samples: AAV serotypes 3, 5, 8, 9 (capsid concentrations ~2 × 10¹³ vg/mL), with injection volumes from 1 μL to 50 μL.

Main Results and Discussion

• UV and FLD traces provided clear identification of monomer peaks, aggregate species (HMWS), and extracellular DNA impurities through characteristic absorbance and emission patterns.
• SEC-MALS confirmed monomer, dimer, trimer molecular weights and radii with higher resolution and half the run time compared to a 4.6 × 300 mm, 5 µm, 500 Å competitor column.
• Flow-rate study: Lower flow (0.05 mL/min) increased resolution (R≈4.7) at the expense of run time (~45 min). Higher flow (0.6 mL/min) achieved baseline separation within 4 min (R≈2.2), suitable for high throughput.
• Injection-volume study: Increasing injection volume to 40× did not compromise baseline chromatographic separation; peak broadening remained under 30%, validating large-volume capabilities for SEC-MALS workflows.
• Column comparison: SurePac Bio 550 SEC MDi delivered sharper peaks, improved resolution, and ~200 psi lower backpressure than a 2.5 µm, 450 Å competitor, enabling faster and more efficient separations.
• Lot-to-lot reproducibility: Analyses of three independent column lots showed consistent retention times, resolutions, and relative peak areas, underscoring manufacturing precision of monodisperse media.

Benefits and Practical Applications

• Rapid, high-resolution separation of AAV monomers and aggregates for gene therapy vector characterization.
• Versatile detection options (UV, FLD, MALS) yield comprehensive information on capsid composition and aggregate quantification.
• Flexible method parameters (flow rate, injection volume) accommodate both in-depth research and high-throughput QC environments.
• Robust column performance ensures consistent quantitation and quality control across production lots.

Future Trends and Potential Uses

• Integration of SEC with online mass spectrometry or advanced detectors for deeper characterization of AAV payloads.
• Automated, high-throughput SEC workflows supporting large-scale vector production and release testing.
• Development of specialized stationary phases to resolve empty/full capsid populations more effectively.
• Expansion to other viral vectors and nanoparticle analysis in biotherapeutic research.

Conclusion

The SurePac Bio 550 SEC MDi column demonstrates exceptional performance in AAV analysis, combining rapid baseline separation, flexible method optimization, and reproducible manufacturing quality. Its compatibility with UV, FLD, and SEC-MALS detection positions it as a powerful tool for gene therapy vector development and quality control.

Reference

1. Rittié L.; et al. The Landscape of Early Clinical Gene Therapies outside of Oncology. Molecular Therapy 2019, 27(10), 1706–1717.
2. Tustian A.D.; Bak H. Assessment of Quality Attributes for Adeno-associated Viral Vectors. Biotechnology & Bioengineering 2021, 118(11), 4168–4203.
3. Khatwani S.L.; Pavlova A.; Pirot M.; Zhu Y. Anion-exchange HPLC assay for separation and quantification of empty and full capsid in multiple adeno-associated virus serotypes. Molecular Therapy – Methods & Clinical Development 2021, 21, 548–558.