OVERCOMING mRNA PRODUCTION SCALE-UP CHALLENGES LNP component analysis by LC-MS Platform method for raw material and LNP formulation QC, suitable for different types of LNP formulations * High-resolution separation of lipid isomers using Thermo Scientific™ Accucore™ C30 UHPLC column coupled to Thermo Scientific™ Vanquish™ Horizon UHPLC system. * Confident confirmation of lipid components using HRAM MS and MS/MS on Thermo Scientific™ Orbitrap Exploris™ 120 mass spectrometer. Excellent separation and accurate mass measurement (<3ppm) of lipid components 4.53 909.5771 -0.5 ppm (n=45) 14:0 PEG 2000 8.13 369.3512 Cholesterol 9.76 634.6481 -2.4 ppm DOTMA 12.74 790.6305 DSPC 6.75 650.6440 12.98 748.5836 DSPE 2 4 6 8 min 10 12 + 7.21 -2.0 ppm 14 16 18 5.5 6.5 650.6442 7.5 8.5 min Figure 4. The upper section shows the extracted ion chromatogram of DOTMA and its MS/MS spectrum. The lower section shows the extracted ion chromatograms of DOTMA with oxidization (+16 Da) and a representative MS/MS spectrum of the oxidized DOTMA degradant eluted at 6.86 min. The very low abundant oxidized DOTMA peaks were confidently identified using both MS and MS/MS data. 9.5 10.5 11.5 + -2.0 ppm DOTMA degradants with oxidation <0.1% 6.86 NL: 1.11E6 650.6439 7.02 650.6440 MS/MS of oxidized DOTMA peak eluted at 6.86 min + + -1.0 ppm (Cholesterol - H2 O) NL: 2.19E9 MS/MS of DOTMA 10.32 634.6485 DOTMA 3+ * Sensitive detection and identification of impurities at 0.001%. * Simultaneous lipid component quantification and metabolites characterization in a single analysis. View complete application note Confident detection and characterization of low abundant metabolites Thermo Scientific™ Accucore™ C30 HPLC Columns Thermo Scientific™ Vanquish™ Horizon UHPLC System Vanquish Flex 300 32.25 H x 21.65 W Thermo Scientific™ Orbitrap Exploris™ 120 Mass Spectrometer Orbitrap Exploris 120 37https://www.thermofisher.com/us/en/home.html https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/an-000464-lc-hram-ms-lipid-nanoparticle-impurities-degradants-an000464-na-en.pdf https://www.thermofisher.com/order/catalog/product/IQLAAAGABHFAPUMZZZ?SID=srch-srp-IQLAAAGABHFAPUMZZZ https://www.thermofisher.com/order/catalog/product/BRE725531?SID=srch-srp-BRE725531 https://www.thermofisher.com/order/catalog/product/27826-152130?SID=srch-hj-27826-152130 https://www.thermofisher.com/order/catalog/product/IQLAAAGABHFAPUMZZZ?SID=srch-srp-IQLAAAGABHFAPUMZZZ https://www.thermofisher.com/order/catalog/product/BRE725531?SID=srch-srp-BRE725531