Characterization of lipid components in lipid nanoparticle (LNP) formulations

Significance of the Topic

The delivery of mRNA via lipid nanoparticles (LNPs) underpins recent advances in vaccinology and gene therapy. Accurate characterization of individual lipid components and their molar ratios is essential for formulation consistency, efficacy, and regulatory compliance.

Study Objectives and Overview

This study aimed to develop and validate a simple ultrahigh-performance liquid chromatography method coupled with a charged aerosol detector (UHPLC-CAD) for quantitative determination of lipid components in two model LNP formulations.

Methodology and Instrumentation

• Instrumentation: Thermo Scientific Vanquish Flex UHPLC system with Charged Aerosol Detector and Chromeleon CDS software.
• Column and Conditions: Accucore C30 column (3.0×100 mm, 2.6 μm) with a gradient from 100% aqueous (0.1% formic acid) to organic mobile phase (0.1% formic acid in 60% IPA/30% ACN) at 0.9 mL/min and 50 °C.
• Sample Preparation: Two formulations were assembled using ionizable or cationic lipids, DSPC, cholesterol, and PEGylated lipids with poly-A as a mock payload. Post-formulation purification employed a 3 kDa MWCO filter.
• Calibration Strategy: Mixed-standard calibrations for formulation 1 and individual lipid calibrations for formulation 2, achieving correlation coefficients >0.998 over a wide dynamic range.

Key Results and Discussion

• Gradient Optimization: Starting with 100% aqueous mobile phase prevented breakthrough of intact LNPs and improved separation of ALC-0315 and ALC-0159.
• Resolution and Sensitivity: The method provided baseline separation of key lipids and detected poly-A early eluting peaks.
• Quantification: Measured molar ratios for both formulations matched reference values within 2% deviation.
• Detector Performance: CAD exhibited high sensitivity, uniform response, and wide dynamic range, simplifying sample preparation and method development.

Practical Benefits and Applications

This UHPLC-CAD approach delivers rapid identification and accurate molar ratio assessment of lipids under a compliance-ready CDS platform, making it well suited for LNP formulation development, release testing, and quality control in biopharmaceutical settings.

Future Trends and Applications

Future work may extend this methodology to varied payload types, high-throughput screening of novel lipid chemistries, integration with mass spectrometry for structural elucidation, and online process monitoring for real-time quality assurance.

Conclusion

The presented UHPLC-CAD method offers a robust, sensitive, and accurate tool for lipid profiling in LNP formulations, facilitating consistent product quality during development and manufacturing.

References

1. Dammes N.; Peer D. Paving the road for RNA therapeutics. Trends Pharmacol. Sci. 2020, 41, 755–775.
2. Hou X. et al. Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 2021, 6, 1078–1094.
3. Schoenmaker L. et al. mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 2021, 601, 120586.