LC/MS Analysis of Lipid Composition in an mRNA LNP Formulation: A Stability Study

Importance of the topic

Lipid nanoparticles (LNPs) have become the leading delivery vehicles for mRNA‐based biopharmaceuticals, including vaccines and gene therapies. The accurate identification and quantification of lipid components and their molar ratios are critical for product efficacy, stability and safety. Monitoring lipid composition under various storage conditions supports robust quality control and ensures consistent performance of therapeutic LNP formulations.

Objectives and Study Overview

This study aimed to apply a liquid chromatography/mass spectrometry (LC/MS) method to (1) separate and quantify four key lipids in an mRNA‐LNP formulation (ionizable lipid SM‐102, DMG-PEG 2K, DSPC and cholesterol) and (2) evaluate LNP stability under different storage and lyophilization conditions over time.

Methodology and Instrumentation

The analytical method utilized an Agilent 1290 Infinity II LC system coupled to an Agilent 6545XT AdvanceBio LC/Q-TOF operated in positive ESI mode. Key details included:

• Column: InfinityLab Poroshell 120 Phenyl-Hexyl (2.1 × 50 mm, 1.9 μm)
• Mobile phases: A = 90% MeOH/10 mM ammonium acetate; B = 90% ACN/10 mM ammonium acetate
• Gradient: 0–2 min at 100% A, 2–7 min to 100% B; flow rate 0.4 mL/min; column temperature 55 °C
• Q-TOF settings: m/z 110–1700 (2 GHz) and 350–3200 (extended range), reference mass 922.0098
• Calibration: serial dilutions of SM-102, DMG-PEG 2K, DSPC and cholesterol standards (0.1 fmol to 1000 pmol)
• LNP preparation: microfluidic mixing (N:P ratio 5.67:1), buffer exchange, lyophilization with 8% (w/v) cryoprotectants (trehalose, sucrose, mannitol)

Main Results and Discussion

Calibration curves for all four lipids exhibited excellent linearity (R2 ≥ 0.99) across three orders of magnitude. Extracted ion chromatogram overlays confirmed complete separation and clean mass spectra for each lipid.

Nonlyophilized LNPs stored at –70 °C maintained target molar ratios (50:1.5:10:38.5), whereas samples at –20 °C and 4 °C showed significant deviations after 1–4 weeks. Lyophilized LNPs with sucrose or mannitol preserved lipid ratios at –70, –20 and 4 °C for up to 12 weeks. A minor decrease of DMG-PEG 2K was observed at 4 °C after 4 weeks but remained within acceptable limits. Overall, lyophilization with appropriate cryoprotectants greatly enhanced lipid stability compared to aqueous storage.

Benefits and Practical Applications

The described LC/MS approach provides a rapid, sensitive and stability‐indicating assay for LNP lipid profiling. It supports routine quality control during formulation development, manufacturing and shelf‐life studies. Implementation of this method can accelerate regulatory approval by demonstrating robust control of critical quality attributes.

Future Trends and Opportunities

Advances in ultra‐high‐throughput LC/MS platforms, automation of data processing and integration with real‐time stability monitoring will further streamline lipid analysis workflows. Exploration of novel cryoprotectant combinations, alternative drying protocols and next‐generation mass analyzers (e.g., TIMS‐TOF) could improve sensitivity and reduce analysis time. In silico modeling of LNP degradation pathways may guide formulation optimization.

Conclusion

An Agilent 1290 Infinity II LC coupled to a 6545XT AdvanceBio LC/Q-TOF effectively separated and quantified key lipids in an mRNA LNP formulation. Lyophilized samples with sucrose or mannitol retained expected lipid ratios for at least 12 weeks under varied temperature conditions, demonstrating a robust stability‐indicating method suitable for biopharmaceutical quality control.

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