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Analytical Characterization of Heterogeneities in mRNA-Lipid Nanoparticles Using Sucrose Density Gradient
Amita Vaidya1, Dipen Parande1, Nikita Khadse1
1mRNA Center of Excellence, Sanofi, Waltham, Massachusetts 02451, United States.
Analytical Chemistry
|March 26, 2024
Summary
Researchers developed a novel ultracentrifugation method to separate and analyze mRNA-lipid nanoparticle (LNP) subpopulations. This technique reveals how LNP density heterogeneity impacts their biological function and formulation properties.
Area of Science:
- Biotechnology
- Nanomedicine
- Analytical Chemistry
Background:
- Rational design and formulation of mRNA-lipid nanoparticles (LNPs) are crucial for effective mRNA delivery.
- Heterogeneity within mRNA-LNP formulations can significantly influence their biophysical and functional characteristics.
- Comprehensive analytical techniques are needed to understand the complexity of mRNA-LNPs.
Purpose of the Study:
- To develop and validate a robust ultracentrifugation method for density-based separation of mRNA-LNP subpopulations.
- To investigate the heterogeneity of different mRNA-LNP formulations and its impact on biological performance.
- To compare the biophysical characteristics and in vivo potency of separated LNP subpopulations.
Main Methods:
- Development of a density gradient ultracentrifugation (DGC) method for separating mRNA-LNPs.
- Synthesis of four mRNA-LNP formulations using varying ionizable and helper lipids (DOPE, DEPE) with hEPO mRNA.
- Harvesting of density-separated fractions for comprehensive analysis of biophysical properties and in vivo potency.
Main Results:
- The DGC method successfully resolved mRNA-LNPs into distinct density-based fractions.
- Formulations A-DOPE and B-DOPE showed heterogeneity, with different fractions exhibiting varying hEPO expression.
- Formulation B-DEPE displayed some heterogeneity, while A-DEPE demonstrated remarkable homogeneity in potency and composition across fractions.
Conclusions:
- Density gradient ultracentrifugation is a powerful tool for characterizing mRNA-LNP heterogeneity.
- LNP density subpopulations can exhibit differential biological performance, impacting in vivo efficacy.
- This study provides a novel approach for the head-to-head comparison of mRNA-LNP heterogeneity and function.
