PL9006: Key publications on the characterization of vaccines with multi-angle and dynamic light scattering

Significance of Topic

This collection of publications demonstrates the critical role of multi‐angle and dynamic light scattering techniques, often combined with field‐flow fractionation, in the characterization of modern vaccine components and nanoparticle‐based delivery systems. Detailed particle sizing, concentration measurement and stability assessment are essential for ensuring vaccine efficacy, safety and batch‐to‐batch consistency. These analytical tools support formulation development, process monitoring and quality control across a broad range of viral, protein, polysaccharide and nucleic acid‐based vaccines.

Objectives and Study Overview

The primary goal of the documented studies is to highlight key applications of asymmetrical flow field‐flow fractionation (AF4/FFF) coupled with multi‐angle light scattering (MALS) and dynamic light scattering (DLS) to:

• Quantify particle size, distribution and concentration of viruses, virus‐like particles (VLPs), lipid nanoparticles and conjugate vaccines
• Evaluate formulation stability under various conditions
• Optimize vaccine candidates through high‐throughput screening and process analytical technology approaches
• Correlate physicochemical properties with immunogenicity and biological functionality

Methodology and Instrumentation Used

The featured publications employ combinations of the following techniques:

• Asymmetrical flow field‐flow fractionation (AF4) or field‐flow fractionation (FFF)
• Multi‐angle light scattering (MALS) detectors for absolute molar mass and radius measurements
• Dynamic light scattering (DLS) for hydrodynamic size and aggregation monitoring
• Size‐exclusion high‐performance liquid chromatography (SEC-HPLC)
• Differential scanning calorimetry (DSC)
• Transmission electron microscopy (TEM) and electrospray differential mobility analysis (ES-DMA)
• UV absorbance, refractive index and fluorescence detectors in series with FFF
• Miniaturized high‐throughput fractionation platforms for formulation screening

Main Results and Discussion

Viruses and VLPs:
Studies demonstrated accurate quantitation and sizing of influenza A and hepatitis B VLPs, highlighted the stabilization effects of microneedle coatings and salt formulations, and correlated particle counts with infectivity and antigenicity. Chikungunya VLP formulations were optimized for long‐term stability using polyanion screening. High‐throughput AF4‐MALS enabled rapid formulation optimization.

RNA/DNA Vectors and Lipid Nanoparticles:
Enhanced AF4‐MALS methods quantified size and concentration of lipid‐based carriers for mRNA/DNA delivery. Data revealed that lipid nanoparticle diameter directly influences in vivo immunogenicity, guiding design of potent mRNA vaccines.

Polysaccharides and Conjugates:
SEC‐MALS and DSC assessed molecular weight alignment and thermal stability of polysaccharide vaccines (e.g. PNEUMOVAX®23) under various storage conditions. Conjugate vaccines with higher carrier‐to‐polysaccharide mass ratios displayed improved antigen presentation and immunogenicity.

Protein and Peptide Antigens:
Structural‐level characterization via DLS, AF4‐UV‐MALS and chromatography tracked folding, aggregation and oligomeric state of protein vaccines (e.g. tetanus toxoid‐conjugates, HIV antigens, influenza nucleoprotein). Dynamic light scattering was successfully implemented as a process analytical technology to monitor protein quality during manufacturing.

Benefits and Practical Applications

The integration of FFF with light scattering provides non‐destructive, absolute measurement of particle size and molar mass without calibration standards. This enables:

• Robust quality control for complex biologics and nanoparticle formulations
• Rapid screening of buffer and excipient conditions to enhance stability
• Correlation of physicochemical attributes with biological performance
• Process analytical technology (PAT) applications for real‐time monitoring of critical quality attributes

Future Trends and Opportunities

Emerging directions include:

• Miniaturized and automated high‐throughput fractionation platforms for early‐stage formulation screening
• Integration of light scattering data with machine learning for pattern recognition and predictive stability models
• On‐line coupling of FFF‐MALS with mass spectrometry to elucidate composition at each size fraction
• Expansion of PAT frameworks to include real‐time DLS and MALS in manufacturing lines
• Characterization of next‐generation vaccine modalities such as self‐amplifying RNA and exosome‐based carriers

Conclusion

Multi‐angle and dynamic light scattering techniques, especially when paired with field‐flow fractionation, have become indispensable tools for the detailed physicochemical characterization of vaccine particles and nanoparticle delivery systems. They facilitate informed formulation design, robust quality control and enhanced understanding of structure‐function relationships, thereby accelerating the development of safe and effective vaccines.

References

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2. Kim, Y.C. et al. (2010) Formulation and coating of microneedles with inactivated influenza virus to improve vaccine stability and immunogenicity. Journal of Controlled Release. 142(2):187‒195. doi:10.1016/j.jconrel.2009.10.013
3. Wie, Z. et al. (2007) Biophysical characterization of influenza virus subpopulations using field flow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity. Journal of Virological Methods. 144(1-2):122‒132. doi:10.1016/j.jviromet.2007.04.008
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