
mRNA-LNP integrity profiling – Ask the Expert
This Session's Expert:
For LNP encapsulated mRNAs, what is the best sample preparation method to extract the mRNA for characterization? Does the lipid composition impact the results?
That’s a great question. We have done studies in collaboration with researchers at SINTEF and found that Triton X-100 treatment combined with heat and the addition of formamide was the most effective method to release the encapsulated mRNA and to avoid secondary structures prior to analysis with CGE-LIF. The final concentration of the Triton X-100 lysis solution used in our studies was 0.2% and samples were heated at 70 ⁰C for 10 minutes to extract the mRNA from mRNA-LNPs, followed by dilution with 80% formamide. It became evident that formamide is crucial to disrupt the formation of higher order structures and to increase the signal for the main species of mRNA. We were able to use this extraction method to successfully evaluate mRNA size, integrity and nucleic acid degradation for different nucleic acid payload concentrations and different lipid compositions. The samples we analyzed contained different ionizable lipids, commonly known as MC3 and SM-102. MC3 was used for the formulation of the first FDA-approved siRNA therapeutic, while SM-102 is in use for the COVID-19 mRNA vaccine by Moderna.