Anion Exchange Chromatography to Determine mRNA Encapsulation in Lipid Nanoparticles

Area of Science:

• Biotechnology
• Analytical Chemistry
• Pharmaceutical Sciences

Background:

• Encapsulation efficiency (EE) is crucial for mRNA therapeutics and vaccines, ensuring mRNA protection from degradation and effective delivery.
• Current methods for EE assessment may not fully capture the complexities of mRNA-lipid nanoparticle (LNP) interactions.
• Understanding mRNA distribution within LNPs is vital for optimizing therapeutic efficacy and safety.

Purpose of the Study:

• To develop and validate a novel anion exchange chromatography (AEX) based assay for determining mRNA EE in LNPs.
• To compare the AEX method with a reference method (RiboGreen assay) for EE determination.
• To elucidate the structural information provided by AEX regarding mRNA within LNPs.

Main Methods:

• Development of an AEX strategy to separate LNPs from free mRNA based on charge differences.
• Implementation of a washing step with surfactant, high pH, and high salt to mitigate carryover issues.
• Determination of EE by quantifying free and total mRNA before and after LNP disruption.

Main Results:

• The AEX method was successfully applied to analyze 30 different mRNA-LNP samples.
• Significant discrepancies were observed between AEX and the RiboGreen assay.
• AEX provided detailed structural information, including surface-localized and transmembrane mRNA, beyond simple quantification.

Conclusions:

• AEX is established as a reliable assay for mRNA EE determination, offering superior insights into mRNA-LNP interactions.
• The method provides critical information on mRNA distribution within LNPs, advancing understanding of LNP structure-function relationships.
• This technique can guide the rational design of next-generation mRNA therapeutics and vaccines.