Characterization and quantification of lipid nanoparticle components and their impurities/degradants using an LC-HRAM MS platform

Significance of the Topic

The development and quality control of lipid nanoparticles (LNPs) are critical for the safe and effective delivery of mRNA therapeutics and vaccines. Precise characterization and quantification of lipid components and their impurities influence formulation stability, biodistribution, and safety profiles.

Objectives and Study Overview

This study aimed to establish a robust LC-HRAM MS platform method capable of separating, identifying, and quantifying LNP lipid components and their degradants in both pure standards and complex biological matrices in a single LC–MS/MS analysis.

Methodology and Used Instrumentation

• Sample Preparation: Pure lipid standards, DOTMA solution, and bovine liver total lipid extract spiked with five common LNP lipids across a nine-point dilution series.
• Chromatography: Thermo Scientific Vanquish Horizon UHPLC with Accucore C30 column (2.1 × 150 mm, 2.6 µm); 22 min gradient, column at 50 °C, 2 µL injection.
• Mass Spectrometry: Thermo Scientific Orbitrap Exploris 120 HRAM MS; data-dependent MS/MS for untargeted profiling and targeted MS/MS (tMS/MS) for quantification; full MS at 120 000 resolution, dd-MS/MS at 30 000 resolution; stepped HCD energies.

Main Results and Discussion

• Separation and Mass Accuracy: All five lipid components resolved with < 3 ppm mass error; DSPE fatty acyl chains confirmed by MS/MS fragment ions.
• Degradant Detection: Oxidized DOTMA species (< 0.1% abundance) detected and characterized by MS and MS/MS.
• Sensitivity Improvement: tMS/MS enhanced LOD for PEG-lipids to 0.5 pg on column; calibration for DOTMA linear across four orders of magnitude (R2 = 0.9995).
• Matrix Analysis: Identification of multiple lipid classes in bovine liver extract; quantification of endogenous 18:0 Lyso PC varied consistently with injection volume.
• Metabolite Profiling: Compound Discoverer workflow automatically annotated oxidized DOTMA metabolites in complex matrix with < 1 ppm error and fragment ion scoring.

Benefits and Practical Applications of the Method

• Platform versatility supporting early formulation development and late-stage QC/QA of LNPs.
• Simultaneous untargeted profiling and targeted quantification in a single run reduces sample consumption and analysis time.
• High resolution and sensitivity enable confident detection of low-level impurities and degradants.

Future Trends and Opportunities

• Integration of real-time monitoring and multi-omics data fusion for comprehensive in vivo lipid metabolism studies.
• Advancements in HRAM MS instrumentation and AI-driven data analysis to streamline lipidomic workflows.
• Development of tailored columns and gradients for improved separation of complex LNP formulations.

Conclusion

The optimized LC–HRAM MS/MS workflow using an Orbitrap Exploris 120 and Vanquish Horizon UHPLC provides a powerful, sensitive, and versatile platform for comprehensive characterization and quantification of LNP lipid components, impurities, and metabolites, facilitating accelerated formulation development and quality control in pharmaceutical applications.

References

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8. Hassett KJ et al. mRNA Vaccine Lipid Nanoparticles. Mol Ther Nucleic Acids 2019