Analytical Solutions for mRNA Vaccines and Therapeutics mRNA 5' capping characterization The 5' cap features * Prevents the degradation by exonucleases. * Promotes translation. * Incorporated in vitro via two methods: a two-step multi-enzymatic reaction or co-transcriptionally. Hybridize probe to transcript 7mGppp AmGG AAA GUC CAG AUA AGA GAG GAG CAA GCA GCA AUC UCA GCG... CAG GUC UAU UCU CUC CUC GUU CGU C cap UTR 5' CDS UTR Poly(A) tail AAAA 3' Accurate and sensitive characterization of capped and uncapped fragment with LC-HRAM MS MS Capped Bind to magnetic bead 7mGppp AmGG AAA GUC CAG AUA AGA GAG GAG CAA GCA GCA AUC UCA GCG... CAG GUC UAU UCU CUC CUC GUU CGU C 100 100 Cleave with RNase H then isolate cleaved product 7mGppp AmGG AAA GUC CAG AUA AGA GAG GAG CAA GCA G CAG GUC UAU UCU CUC CUC GUU CGU C . Separate oligomer from probe & analyze by LC-MS/MS 7mGppp AmGG AAA GUC CAG AUA AGA GAG GAG CAA GCA G 20 30 40 50 60 70 80 90 10 2.8 3.0 3.2 3.4 3.6 3.8 Time (min) m/z View complete application note Extracted ion chromatograms of capped and uncapped fragments are used for accurate capping efficiency measurement. HRAM MS and MS/MS spectra ensure confident sequence confirmation. 20 40 60 80 893.2 893.4 893.6 893.8 894.0 894.2 894.4 m/z Predicted Uncapped Experimental 893.6243 z=8 893.4991 z=8 893.3740 z=8 893.7495 z=8 893.8746 z=8 893.9997 z=8 894.1250 z=8 894.2502 z=8 * 120 K resolution * <1 ppm mass error Bf Thermo Scientific™ DNAPac™ RP HPLC Columns Vanquish Flex 300 32.25 H x 21.65 W Thermo Scientific™ Vanquish™ Flex UHPLC System Acclaim C18 Column Columns 34 Thermo Scientific™ Orbitrap Exploris™ 240 Mass Spectrometer Orbitrap Exploris 240 Thermo Scientific™ BioPharma Finder Software Relative abundance Relative abundance Relative abundancehttps://www.genengnews.com https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/an-001108-pb-lc-hram-ms-mrna-quantification-an001108-na-en.pdf https://www.thermofisher.com/order/catalog/product/IQLAAAGABHFAPUMBJC?SID=srch-srp-IQLAAAGABHFAPUMBJC https://www.thermofisher.com/order/catalog/product/088923 https://www.thermofisher.com/order/catalog/product/BRE725535?SID=srch-srp-BRE725535 https://www.thermofisher.com/order/catalog/product/OPTON-30986?SID=srch-srp-OPTON-30986 https://www.thermofisher.com/order/catalog/product/IQLAAAGABHFAPUMBJC?SID=srch-srp-IQLAAAGABHFAPUMBJC