Journal of pharmaceutical sciencesOct 18, 2025

Using a new chemical method to track mRNA delivery particles during production

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 7, 2026

Abstract

The encapsulation efficiency of mRNA in lipid nanoparticles (LNPs) showed a strong correlation with increasing particle size as measured by anion-exchange high-performance liquid chromatography (AEX-HPLC).

LNP formation dynamics are not fully understood, particularly regarding mRNA loading processes.
AEX-HPLC analysis indicated that mRNA encapsulation occurred during the concentration process of LNPs.
The RiboGreen assay did not demonstrate the same correlation between encapsulation efficiency and particle size as observed with AEX-HPLC.
Mass photometry analysis supported the findings related to mRNA and lipid ratios in the fractionated LNPs.
The concentration process had a limited effect on the biological activity of LNPs across the studied range.

Simplified

Lipid nanoparticles (LNPs) have emerged as versatile delivery systems for nucleic acids, and have played a crucial role in the development of mRNA therapeutics. However, despite the significance of the buffer exchange process in loading mRNA into LNPs and forming their lipid layer, the underlying LNPs formation remain unclear. In this study, we investigated the process of mRNA encapsulation in LNPs using a tangential flow filtration system. The encapsulation efficiency was evaluated using anion-exchange high-performance liquid chromatography (AEX-HPLC) and RiboGreen assays. The encapsulation efficiency evaluated using the AEX-HPLC method showed a strong correlation with increasing particle size of the LNPs. However, this trend was not observed with the RiboGreen assay. Therefore, AEX-HPLC was used to assess LNP maturation, revealing that mRNA encapsulation also occurred during the concentration process. This observation was supported by mass photometry analysis and the mRNA/lipid ratio of the fractionated LNPs. Finally, the protein expression levels were examined using LNPs with and without the concentration process. Within the range used in this study, the concentration process had a limited effect on biological activity. These findings offer new insights into LNP formation dynamics and may contribute to the development of a more robust and scalable LNP manufacturing platforms.

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Using a new chemical method to track mRNA delivery particles during production

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