Biophysical Characterization of Enveloped VLPs Using a Waters GTxResolve™ 2000 Å SEC Column, MaxPeak™ Premier 3 μm

Importance of the Topic

Enveloped virus-like particles (eVLPs) mimic native viruses by displaying membrane-bound antigens and serve as versatile carriers for nucleic acids, proteins, or small-molecule drugs in gene therapy and vaccine development. Accurate biophysical characterization of eVLPs is essential to ensure structural integrity, batch consistency, safety, and therapeutic efficacy in industrial and research settings.

Objectives and Study Overview

This application note describes an optimized analytical workflow combining size-exclusion chromatography with multi-angle light scattering (SEC-MALS) and batch dynamic light scattering (DLS) to characterize eVLP size distribution, particle concentration, and homogeneity. Key goals include mobile phase optimization, evaluation of a GTxResolve™ 2000 Å SEC Column with MaxPeak™ Premier 3 µm packing for high recovery separations, and comparison of DLS versus SEC-MALS results.

Methods and Instrumentation

The model sample was an HEK293-derived enveloped capsid VLP (estimated Rh 50–150 nm) formulated in PBS with trehalose. Batch DLS measurements were performed on a DynaPro™ NanoStar™ instrument after dilution with the mobile phase. SEC-MALS analysis used an ACQUITY™ Arc™ Premier System (Quaternary Solvent Manager, Flow Through Needle Sample Manager) with a GTxResolve™ 2000 Å SEC Column (4.6 × 150 mm and 7.8 × 150 mm, MaxPeak™ Premier 3 µm). The optimized mobile phase comprised 20 mM phosphate, 276 mM NaCl, 5.4 mM KCl, 0.006% polysorbate 80, and 25% sucrose (pH 7.4). Isocratic separation was performed at 0.30 mL/min, 40 °C, injection volume 80 µL. Detection modules included UV (ACQUITY 2489 TUV, 280 nm and 220 nm), MALS (Wyatt DAWN™), online DLS (WyattQELS™), and differential refractive index (Wyatt Optilab™). Data processing was carried out in ASTRA™ 8.3 software.

Key Results and Discussion

• Optimal ionic strength of 400 mM (KCl or NaCl) was required for quantitative eVLP recovery; buffer concentration (1–100 mM) had negligible effect when ionic strength was held constant.
• Inclusion of 60 ppm polysorbate 80 and 25% sucrose improved peak shape and recovery of membrane-bound particles.
• Batch DLS yielded an average hydrodynamic radius of 59.9 ± 0.2 nm, PDI 0.10, and total concentration of 1.2 × 10^11 VLP/mL, with no evidence of large aggregates or small fragments.
• SEC-MALS resolved two particle populations: Region 1 (larger species with Rh 78–88 nm) and Region 2 (singlet VLPs with Rh 60.9 nm), corroborating DLS monomer size.
• Detailed size distribution by MALS indicated 85% of particles with Rh < 62 nm, 14% between 62–70 nm, and < 2% above 70 nm.
• Total recovery by SEC-MALS was ~50% of the batch DLS count, suggesting potential column interactions or sample loss and highlighting the need for orthogonal methods (e.g., field-flow fractionation-MALS) to fully assess recovery.

Benefits and Practical Applications

• The combined SEC-MALS and DLS approach delivers high-resolution, absolute measurements of eVLP size, concentration, and homogeneity, supporting rigorous quality control in vaccine and gene therapy production.
• GTxResolve™ 2000 Å SEC Column with MaxPeak™ Premier technology minimizes secondary interactions and maximizes recovery of large biomolecular assemblies.
• Complementary data from DLS (rapid screening) and SEC-MALS (detailed distribution and absolute quantitation) enable a comprehensive understanding of eVLP formulations.

Future Trends and Potential Applications

• Integration of field-flow fractionation coupled with MALS to validate recovery and eliminate potential adsorption effects inherent to stationary phases.
• Development of advanced packing materials and surface chemistries to further improve analyte recovery across a broader size range of viral vectors and nanoparticles.
• Implementation of real-time process analytical technologies (PAT) for in-line monitoring of eVLP manufacturing.
• Extension of this analytical platform to other complex biotherapeutics, including exosomes and enveloped viral vectors used in emerging gene therapies.

Conclusion

The optimized SEC-MALS method leveraging a GTxResolve™ 2000 Å SEC Column, MaxPeak™ Premier 3 µm, combined with batch DLS, constitutes a robust platform for detailed biophysical characterization of enveloped VLPs. This approach enhances recovery, resolution, and quantitative accuracy, addressing critical quality attributes for advanced biotherapeutic development.

References

1. He J et al. Viruses. 2022;14:1905.
2. Tariq H et al. Front Microbiol. 2022;12.
3. Zhang L et al. Biotechnol Bioprocess Eng. 2023;28:1–16.
4. Wetzel D et al. J Biotechnol. 2019;306:203–212.
5. Redecker AS et al. iScience. 2025;28.
6. Camacho KJ et al. J Sep Sci. 2024;47:e202400541.
7. Kulyabina EV et al. Reference Materials in Measurement and Technology. 2024;23–30.
8. Finny AS et al. Waters Application Notes. 2025.
9. Some D. WP9003. Wyatt application note. 2019.
10. Challener CA. Biopharm Int. 2025;28:46–49.
11. Sripada SA et al. Anal Chem. 2024;96:9593–9600.
12. Striegel AM et al. Nat Rev Methods Primers. 2025;5:40.
13. Wyatt PJ. Anal Chim Acta. 1993;272:1–40.
14. Wyatt PJ, Weida MJ. US Patent. 2004;13.
15. Wyatt PJ. Anal Chem. 2014;86:7171–7183.
16. Bousse T et al. J Virol Methods. 2013;193:589–596.
17. Wei Z et al. J Virol Methods. 2007;144:122–132.
18. Deng JZ et al. Mol Ther Oncolytics. 2022;24:139–147.
19. Padilla MS et al. Nat Biotechnol. 2025.