PHARMSCI: Rapid Analysis and Characterization of Lipid Nanoparticle Components for mRNA Delivery

Importance of the Topic

The rapid development of COVID-19 mRNA vaccines has underscored the critical role of lipid nanoparticles (LNPs) in safeguarding nucleic acids and promoting cellular uptake. Accurate and efficient analysis of LNP components and related impurities is essential to ensure the safety, efficacy, and regulatory compliance of mRNA-based therapeutics.

Objectives and Study Overview

This study aimed to establish a fast, reliable reversed-phase liquid chromatography–mass spectrometry workflow for routine characterization of the four primary lipids in LNP formulations: cholesterol, a phospholipid DSPC, an ionizable lipid MC3, and a PEGylated lipid DMG-PEG-2000. The method employs an ACQUITY Premier CSH C18 column coupled with the BioAccord LC-MS system, integrated with UNIFI informatics for data processing.

Methodology

• Chromatography: Separation on ACQUITY Premier CSH C18 column (100 x 2.1 mm, 1.7 μm) using gradient elution between mobile phase A (ACN/Water/1 M ammonium formate 600/390/10 with 0.1% formic acid) and mobile phase B (IPA/ACN/1 M ammonium formate 900/90/10).
• Mass Spectrometry: Positive electrospray ionization with m/z range 50–2000, cone voltage 30 V, and fragmentation cone voltage ramp from 120 to 200 V.
• Data Management: Automated acquisition, new peak detection, binary comparison, and library-based screening using UNIFI Scientific Information System within waters_connect.

Instrumentation

• ACQUITY Premier LC system with Premier CSH C18 column
• BioAccord LC-MS system
• UNIFI Scientific Information System for data processing and library management

Main Results and Discussion

• Single lipid analysis achieved clear chromatographic separation and accurate mass measurement for all four lipid classes.
• Complex mixture analysis detected 14 distinct DMG-PEG-2000 variants differing in PEG repeat units (38–50) and resolved multiple charge states (+2 to +4).
• Extracted ion chromatograms and low/high energy MS spectra matched predicted in silico fragments for DSPC, confirming structural integrity.
• New peak detection and binary comparison workflows identified spiked or degraded lipid species in liver extract samples, demonstrating suitability for impurity screening.
• Method sensitivity and dynamic range supported detection limits appropriate for formulation process monitoring and raw material quality control.

Benefits and Practical Applications

• Rapid routine analysis under ten minutes per run accelerates LNP formulation development.
• High sensitivity and wide dynamic range enable detection of low-level impurities and degradation products.
• Built-in UNIFI library streamlines identification of known and newly encountered lipid species.
• Method applicable to formulation process development, raw material QC, and regulatory compliance documentation.

Future Trends and Applications

• Integration of high-throughput LC-MS workflows for large-scale screening of LNP libraries.
• Expansion of lipid libraries to include novel ionizable and functionalized lipids.
• Automation of data analysis with machine learning for predictive impurity profiling.
• Extension of this platform to other nanoparticle-based drug delivery systems beyond mRNA.

Conclusion

The developed RP LC-MS method on the ACQUITY Premier CSH C18 column and BioAccord system provides a robust, sensitive, and efficient solution for comprehensive analysis of LNP components. Coupled with UNIFI informatics, this workflow offers reliable screening, quantification, and structural confirmation of lipids, supporting critical stages of mRNA therapeutic development and quality control.

References

• Isaac G, Ranbaduge N, Dhungana S. Rapid Analysis of Lipid Nanoparticle Components Using BioAccord LC-MS System. Waters Corporation Application Note 2021.
• Vaccines. 2021;9(1):65. doi:10.3390/vaccines9010065