Composition Analysis of Lipid Nanoparticle Components with Agilent 1290 Infinity II ELSD

Importance of the Topic

Lipid nanoparticles are crucial for mRNA vaccine delivery because they enhance stability of mRNA in vivo, prevent rapid degradation, and improve cellular uptake and expression. Composition of LNPs directly affects stability, efficacy, and safety of biopharmaceutical formulations, making accurate analysis of their lipid components a key quality control requirement.

Objectives and Overview

This study aims to develop and validate a quantitative method for analyzing common LNP components including ionizable lipids, helper lipids, PEG-lipids, and cholesterol using HPLC coupled with evaporative light scattering detection. The method is designed to provide a broad dynamic range, high sensitivity, and the ability to detect forced degradation products under acidic, basic, and oxidative conditions.

Methodology and Instrumentation

Standard mixtures of SM-102, DMG-PEG2000, DSPC, and cholesterol were prepared at ratios reflecting mRNA vaccine formulations. Serial dilutions ranging from 5 to 1000 µM total lipid concentration were conducted in ethanol. Forced degradation tests included treatment with 0.1 M HCl, 0.1 M NaOH, or 30 % hydrogen peroxide at 60 °C for 24 hours followed by neutralization.

Used Instrumentation

• Agilent 1260 Infinity II HPLC system
• Agilent 1290 Infinity II evaporative light scattering detector (cooled)
• Poroshell CS-C18 column (2.1 × 100 mm, 2.7 µm)
• Mobile phases: 5 mM ammonium formate in water/acetonitrile/methanol (25/35/40) and in methanol/ethanol (60/40)
• Gradient elution from 15 % to 100 % organic over 30 minutes, flow rate 0.6 mL/min, column temperature 40 °C
• Detection temperatures: evaporator and nebulizer at 80 °C, gas flow 1.6 SLM

Results and Discussion

The method achieved baseline separation of all four lipid components at 1 mM concentration with clear ELSD signals despite differences in component abundance. Calibration curves fitted to a quadratic model yielded R2 values ≥0.99 for each lipid. Limits of quantitation were approximately 5 µM for SM-102, 10 µM for cholesterol and DSPC, and 100 µM for DMG-PEG2000. Repeatability tests showed interday RSD ≤2 %. Forced degradation studies revealed distinct degradation products for SM-102 and DSPC under all stress conditions, demonstrating the method’s capability to monitor lipid stability and impurities.

Benefits and Practical Applications

• High sensitivity and specificity for non-chromophoric lipid analysis
• Wide quantitative range suitable for QC of LNP formulations
• Robust repeatability supports routine method implementation
• Capability to detect and monitor degradation products for stability studies

Future Trends and Opportunities

Advancements may include integration with mass spectrometry for enhanced structural elucidation, adaptation to high-throughput workflows, and application to emerging lipid chemistries with targeting moieties. Real-time monitoring of LNP formulation processes and expansion to other therapeutic delivery systems are also potential developments.

Conclusion

An HPLC-ELSD method using Agilent 1290 Infinity II instrumentation provides reliable, sensitive, and reproducible quantification of key LNP components and their degradation products. This analytical approach supports quality control and stability assessment of mRNA vaccine formulations.

Reference

• Kelsey L et al. Lipid Nanoparticle-Mediated Delivery of mRNA Therapeutics and Vaccines. Trends Mol Med. 2021;27(6).
• Schoenmaker L et al. mRNA-Lipid Nanoparticle COVID-19 Vaccines: Structure and Stability. Int J Pharm. 2021;601.
• Meredith P et al. A Novel Mechanism for the Loss of mRNA Activity in Lipid Nanoparticle Delivery Systems. Nat Commun. 2021;12:6777.