(M0930-09-52) Characterization of Lipid Nanoparticle (LNP) Composition UHPLC-CAD

Purpose: The identification, ratio, and purity of the lipid components in LNP formulations are regarded as critical quality attributes that need to be well characterized and routinely monitored during development, manufacturing and QC release testing to ensure safety and efficacy.

Methods: Here we mainly describe a few UHPLC-based chromatography methods for the characterization of lipids used for LNP, both at the raw material and formulated LNP level. To avoid interaction with metal-chelating phosphate groups presented on some of the lipids in the formulation, the biocompatible UHPLC system was used. Combining the superior shape and selectivity of C30 column for lipid separation, and high sensitivity and dynamic range of CAD detection

Results: Two fast, robust methods for the characterization of the lipid components in the lipid nanoparticles used in oligonucleotide therapeutics have been described. Formulation #1 has better separation using the FA method, whereas formulation #2 has better separation using the TFA method. Separation of several components from each LNP class was achieved on the Accucore C30 column in 10 minutes.
The CAD has low limits of detection for all the components, including cholesterol. The high sensitivity of the CAD allows any trace impurities to be monitored.
The calibration curves for all lipids show a calibration coefficient of better than 0.999.

Conclusion: The chromatographic methods were developed and optimized to provide robust, high resolution separation, high sensitivity detection and reproducible quantification of lipid components and their low level impurities. At the raw material level, two methods are validated for the linearity, selectivity and recovery of LNPs used in mRNA vaccine. Besides the characterization of LNPs composition, it is also important for the associated impurities and degradants to be confirmed¹. In this work, an efficient method is described for profiling the impurity of the raw materials from different vendors, and comparing against purity label claims.

References: 1. Kinsey, C.; Lu, T.; Deiss, A.; Vuolo, K.; Klein, L.; Rustandi, R.R.; Loughney, J.W. Determination of lipid content and stability in lipid nanoparticles using ultra high‐performance liquid chromatography in combination with a Corona Charged Aerosol Detector, Electrophoresis 2021, 1–24.
2. Tracy, M.; Hillbeck, D. Thermo Scientific Application Note 20663 – Comparative analysis of cooking oils using a solid core HPLC column.
https://assets.thermofisher. com/TFS-Assets/CMD/Application-Notes/AN20663-Comparative-Cooking-Oils- HPLC-EN.pdf
3. Criscuolo, A.; Zeller, M.; Cook, K.; Angelidou, G.; Fedorova, M. Rational selection of reverse phase columns for high throughput LC–MS.
Chemistry and Physics of Lipids 2019, 221, 120-127.

Acknowledgements: Acknowledge to my colleagues Jung Hyang Lee and Emily Hyekyung Choi.