Characterization of lipid nanoparticle (LNP) composition using UHPLC-CAD

Significance of the topic

Lipid nanoparticles (LNPs) have become essential carriers for mRNA vaccines and oligonucleotide therapies due to their ability to encapsulate and deliver genetic material safely and effectively. Accurate characterization of the individual lipid components—cholesterol, phospholipids, PEGylated lipids, and ionizable cationic lipids—is critical for ensuring product efficacy, stability, and regulatory compliance. Because most lipids lack chromophores, universal detection methods are required for routine quality control.

Objectives and study overview

The study aimed to develop and validate a simple, rapid Ultra High Performance Liquid Chromatography (UHPLC) method paired with a Charged Aerosol Detector (CAD) for the compositional analysis of LNP formulations. Two gradient approaches—using formic acid (FA) and trifluoroacetic acid (TFA)—were compared to achieve baseline separation, low detection limits, and a wide linear range for key lipids.

Methodology

Sample Preparation:

• All lipids and LNPs were dissolved in 100% ethanol and handled with glassware to prevent adsorption.
• Two distinct LNP formulations were tested to assess method robustness.

Chromatography Conditions:

• Column: Thermo Scientific Accucore C30, 3.0 × 100 mm, 2.6 μm.
• Flow rate: 0.9 mL/min; column temperature: 50 °C; autosampler temperature: 15 °C; post-column cooler: 40 °C.
• FA method mobile phases: A = 0.1% formic acid in 50% ACN/50% water, B = 0.1% FA in 60% IPA/30% ACN/10% water; 12-min gradient from 25% A to 100% B.
• TFA method mobile phases: A = 0.1% TFA in 50% ACN/50% water, B = 0.1% TFA in 70% IPA/25% ACN/5% water; similar gradient program.

Used Instrumentation

• Vanquish Flex Binary UHPLC system with metal-free flow path.
• Charged Aerosol Detector (CAD) with evaporator temperature 35 °C, power function 1.0.
• Thermo Scientific Chromeleon CDS software v7.2.10 for data acquisition and analysis.

Main results and discussion

Under optimized conditions, four primary lipids (DHA, mPEG-DTA-2K, cholesterol, DSPC) were baseline-separated within 10 minutes using the FA method for formulation #1 and within 5 minutes using the TFA method for formulation #2. Limits of detection were approximately 10 μg/mL (cholesterol as low as 2.6 μg/mL). Calibration curves over 10–220 μg/mL exhibited quadratic fits with coefficients of determination above 0.999 for all lipids. TFA improved sensitivity but FA provided slightly sharper resolution for certain components. Carryover was negligible under the described workflow.

Benefits and practical applications of the method

• Rapid, robust separation of diverse lipid classes in LNP formulations.
• Universal CAD response enables quantitation of non-chromophoric lipids and trace impurities.
• Low detection limits and wide dynamic range support stringent QA/QC requirements.
• Applicability to multiple formulation types with minimal method adjustment.

Future trends and potential applications

Advances may include integration with mass spectrometry for structural confirmation, application to emerging ionizable lipid chemistries, and high-throughput adaptation for large-scale vaccine production. Further method refinement could target improved resolution of accessory lipids, rapid screening of degradation products, and automated workflows for regulatory release testing.

Conclusion

The developed UHPLC-CAD workflows using Accucore C30 columns deliver fast, sensitive, and reproducible characterization of LNP lipid compositions. The dual FA and TFA gradient approaches accommodate formulation-specific needs, providing a versatile platform for quality control in lipid nanoparticle development.

References

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