Meeting regulatory needs in the characterization of lipid nanoparticles (LNPs) for RNA delivery via FFF-MALS

Significance of the Topic

Lipid nanoparticles for RNA delivery have transformed modern medicine by enabling safe and effective transport of mRNA and siRNA therapies. Analytical methods that can reliably measure critical quality attributes such as particle size, morphology and concentration are essential to ensure product safety, efficacy and regulatory compliance. Field flow fractionation combined with multi angle light scattering provides a powerful platform for the advanced characterization of such nanomedicines without the need for a stationary phase, offering tunable separation and high resolution for particles below one micrometer.

Goals and Study Overview

This work presents the development and validation of an FFF MALS method in line with ISO slash TS 21362 for lipid nanoparticles encapsulating siRNA or mRNA. The study aimed to optimize the fractionation protocol, compare different channel configurations to maximize recovery and gentleness, and demonstrate the ability of the method to measure key quality attributes under regulatory expectations.

Used Instrumentation

• Field flow fractionation system operated with isocratic pump degasser and autosampler
• Agilent UV detector measuring at 260 nm for nucleic acid and lipid signals
• DAWN multi angle light scattering detector coupled with embedded WyattQELS dynamic light scattering module
• ASTRA software for data collection and particle size concentration analysis

Main Results and Discussion

• Comparing a conventional long channel with a dispersion inlet channel showed that the dispersion setup achieved nearly complete recovery and reduced risk of sample destabilization, making it preferable for delicate LNPs.
• The method enabled simultaneous determination of average size, polydispersity and morphology via the ratio of rms radius to hydrodynamic radius. Particles with higher mRNA payload exhibited more compact dense spheres while empty or low payload formulations appeared hollow and less stable.
• FFF MALS derived number based particle size distributions matched nanoparticle tracking analysis while detecting smaller particles below 30 nm that NTA could not resolve.
• Stability studies revealed that stored LNP samples underwent Ostwald ripening with increasing aggregates and broader size distribution over time, demonstrating the method’s power to monitor physical stability and aggregation propensity.

Benefits and Practical Applications

The validated FFF MALS approach offers the pharmaceutical industry a robust tool for:

• Characterizing multiple critical quality attributes in a single injection
• Ensuring consistency and reproducibility in drug development and quality control
• Meeting regulatory guidelines for nanopharmaceutical analysis

Future Trends and Opportunities

Advancements may include integration of additional detectors for composition analysis, automated method optimization for high throughput, deeper structural insights using hyphenated techniques and harmonized regulatory frameworks to support faster approval of novel RNA therapies.

Conclusion

This study demonstrates a validated field flow fractionation coupled to multi angle light scattering method that meets regulatory requirements for lipid nanoparticle characterization. The optimized dispersion inlet protocol provides high recovery, precise size, morphology and concentration data and the capability to track stability and aggregation, supporting streamlined development and quality control of RNA based nanomedicines.

References

1 Gómez Aguado I et al Nanomedicines to Deliver mRNA State of the Art and Future Perspectives J Nanomat 10 364 2020
2 Parot J et al Physical characterization of liposomal drug formulations using multi detector asymmetrical flow field flow fractionation J Control Release 320 495 510 2020
3 Caputo F et al Asymmetric flow field flow fractionation for measuring particle size drug loading and instability of liposomal formulations J Control Release 350 2023
4 Mildner R et al Improved multidetector asymmetrical flow field flow fractionation method for particle sizing and concentration measurements of lipid based nanocarriers for RNA delivery Eur J Pharm Biopharm 163 252 265 2021