VLP Characterization with the Light Scattering Toolbox

Significance of the Topic

Virus-like particles represent a versatile platform for vaccines and gene delivery due to their ability to mimic viral structure without containing infectious genetic material. Accurate characterization of VLPs is essential throughout development and production to ensure safety, efficacy, and consistency. Light scattering techniques offer noninvasive, high-resolution analysis of size, mass, charge, and interaction properties that are critical for formulation design, process optimization, and quality control.

Objectives and Overview of the Study

This white paper presents an integrated toolbox of light scattering methods for comprehensive biophysical characterization of VLPs. It describes how static, dynamic, and electrophoretic light scattering, combined with advanced fractionation and high-throughput screening, addresses challenges in VLP assembly assessment, stability testing, and batch-to-batch comparability across the development lifecycle.

Methodology

The light scattering toolbox comprises three core techniques:

• Multi-angle static light scattering measures absolute molar mass and root mean square radius
• Dynamic light scattering determines hydrodynamic radius via translational diffusion
• Electrophoretic light scattering assesses particle surface charge by electrophoretic mobility

These measurements are coupled with sample delivery and separation approaches:

• Size-exclusion chromatography and field-flow fractionation for population distribution analysis
• Composition-gradient delivery for evaluating self-assembly and antibody binding kinetics
• High-throughput plate-based screening for rapid aggregation and dissociation assays
• Electrical asymmetric-flow field-flow fractionation for two-dimensional size and charge profiling

Used Instrumentation

The implementation uses commercial systems including:

• DAWN and miniDAWN detectors for on-line static and dynamic light scattering
• Eclipse FFF system for high-resolution separation of nanoassemblies
• Calypso composition-gradient mixer for interaction studies
• DynaPro Plate Reader for automated, high-throughput DLS screening
• ZetaStar analyzer for parallel electrophoretic light scattering

Main Results and Discussion

Applying SEC-MALS and FFF-MALS enables quantification of assembled capsids, unassembled capsomeres, malformed VLPs, and aggregates from submicrogram samples. Composition-gradient MALS accurately determines antibody binding stoichiometry and affinity without labeling. High-throughput DLS identifies optimal buffer and excipient combinations by monitoring aggregation and dissociation under stress. EAF4 extends fractionation capability by resolving particles across size and zeta potential dimensions, overcoming membrane adherence issues and offering deeper biophysical insight.

Benefits and Practical Applications

The integrated light scattering toolbox provides:

• Label-free, noninvasive analysis with minimal sample consumption
• Quantitative assessment of mass, size, shape, and surface charge
• High-resolution separation of VLP subpopulations for purity and degradant profiling
• Automated high-throughput workflows for formulation screening and stability studies
• Reliable data to support process development, formulation optimization, and regulatory compliance

Future Trends and Applications

Advances in automation, microfluidics, and data integration will further increase throughput and reproducibility of VLP characterization. Emerging applications include real-time monitoring of assembly in continuous manufacturing, coupling with machine learning for predictive stability modeling, and expansion into complex gene therapy vectors requiring simultaneous analysis of capsid and nucleic acid cargo.

Conclusion

The combination of static, dynamic, and electrophoretic light scattering with advanced fractionation and high-throughput platforms constitutes a robust, flexible toolbox for comprehensive VLP characterization. Its ability to deliver detailed biophysical profiles accelerates vaccine and gene therapy development, ensuring product quality and regulatory readiness.

References

1. Y P Chuan, Y Y Fan, L H L Lua, A P J Middelberg. Virus assembly occurs following a pH or Ca2 triggered switch in the thermodynamic attraction between structural protein capsomeres. Journal of the Royal Society Interface. 2010;7:409–421.
2. D Some, S Kenrick. Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering. In: Cai J, editor. Protein Interactions. InTech; 2012.
3. V I Razinkov, M J Treuheit, G W Becker. Methods of High Throughput Biophysical Characterization in Biopharmaceutical Development. Current Drug Discovery Technologies. 2013;10(1):59–70.
4. J Mohr, Y P Chuan, Y Wu, L H L Lua, A P J Middelberg. Virus-like particle formulation optimization by miniaturized high-throughput screening. Methods. 2013;60(3):248–256.
5. Y Ding, Y P Chuan, L He, A P J Middelberg. Modeling the Competition Between Aggregation and Self-Assembly During Virus-Like Particle Processing. Biotechnology and Bioengineering. 2010;107(3):550–560.