Anion Exchange Chromatography to Determine mRNA Encapsulation in Lipid Nanoparticles

Article Article scientifique Article Anion Exchange Chromatography to Determine mRNA Encapsulation in Lipid Nanoparticles eng 2025-08-22 2025-09-09 author Tsalmpouris Athanasios 0009-0008-0894-7975 School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva, Switzerland.,Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva, Switzerland. 430562 Section des sciences pharmaceutiques (FARMA) author Mahjoubi Sofiane School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva, Switzerland.,Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva, Switzerland. 943707 author Malburet Camille mRNA Center of Excellence, Analytical Sciences, Sanofi, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France. author Daher-Hassan Chamsan mRNA Center of Excellence, Analytical Sciences, Sanofi, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France. author François-Heude Marc mRNA Center of Excellence, Analytical Sciences, Sanofi, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France. author Cotte Jean-François mRNA Center of Excellence, Analytical Sciences, Sanofi, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France. author Guillarme Davy 0000-0001-7883-5823 School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva, Switzerland.,Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva, Switzerland. 623637 Section des sciences pharmaceutiques (FARMA) author Maurer Jonathan 0000-0003-1011-3431 School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva, Switzerland.,Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva, Switzerland.,mRNA Center of Excellence, Analytical Sciences, Sanofi, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France. 823216 Section des sciences pharmaceutiques (FARMA) 19275-19282 10.1021/acs.analchem.5c03299 40846315 0003-2700 PMC12424022 https://pubs.acs.org/doi/10.1021/acs.analchem.5c03299 Encapsulation efficiency (EE) of mRNA-based therapeutics and vaccines is defined as the percentage of total mRNA that is efficiently protected by the delivery vehicle from nuclease degradation. As a critical quality attribute, EE must be assessed to ensure that sufficient mRNA evades enzymatic degradation and traverses biological barriers to reach the cellular machinery for translation, without triggering unwanted immune responses caused by free mRNA. In this study, we developed a strategy based on anion exchange chromatography (AEX) to separate lipid nanoparticles (LNPs) and free mRNA based on their charge differences. Carryover issues were mitigated by using a washing step with surfactant, high pH, and high salt concentration. EE was determined by analyzing undiluted samples for free mRNA and measuring total mRNA after LNP disruption using surfactants. The method was successfully applied to the analysis of 30 different mRNA-LNP samples to determine EE. The results were compared to those obtained with the RiboGreen assay, one of the reference methods to assess EE. Our results revealed significant discrepancies between the two techniques that could be explained by the structural information obtained under AEX conditions. Indeed, while the RiboGreen assay provides information on the quantification of mRNA accessible to the fluorescent dye, AEX allows relative quantification of mRNA dissociated from LNPs and information on the presence of surface-localized mRNA as well as transmembrane mRNA. These findings establish AEX as a reliable EE assay, providing information on mRNA distribution within LNPs and advancing the fundamental understanding of LNP structure-function relationships. Based on these features, it offers critical guidance for the rational design of next-generation mRNA therapeutics. Chromatography Dyes and pigments Encapsulation Dewey 615 Swiss National Science Foundation (SNSF) IC00I0-227558 31 Section des sciences pharmaceutiques 17 false 881 Sciences analytiques Analytical chemistry 97 35 c4c8217e-10a7-404c-9454-9d2e20d5445c Article (Published version) Public tsalmpouris-et-al-2025-anion-exchan.pdf 2365429 application/pdf CC BY-4.0 b50f9c407b2849f172c4e5f6602a60317e7795f11165a3bc7c6dec8500e8293b Vignette Public images_large_ac5c03299_0008.jpeg 123799 image/png openAccess Faculté des sciences / Section des sciences pharmaceutiques

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Anion Exchange Chromatography to Determine mRNA Encapsulation in Lipid Nanoparticles	Analytical chemistry	2025	3	6

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