Journal: Vaccines

Article Title: Resources, Production Scales and Time Required for Producing RNA Vaccines for the Global Pandemic Demand

doi: 10.3390/vaccines9010003

Figure Lengend Snippet: Sensitivity analysis showing RNA vaccine drug substance manufacturing uncertainties and their impact on annual production amounts and costs per dose. Input variables which are uncertain, their uncertainty ranges and units are shown on the vertical y-axis and the outputs of drug substance annual production amounts and costs per dose and shown on the horizontal x-axis. The zero values shown on the horizontal x-axes and the corresponding vertical line indicates a baseline scenario which describes an RNA vaccine production process with a 30 L bioreactor working volume scale, a final titre in the bioreactor of 5 g/L, 44% combined losses in the downstream purification and formulation steps, 30 µg of RNA per vaccine dose, a production process failure rate of 5%, 5′ cap analogue (CleanCap) purchase price of 3000 USD/g, 1-methyl-pseudouridine (Mod-UTP) purchase price of 4700 USD/g, basic labour rate of 20 USD/hour, quality control testing (QC/QA) cost of 50% of the labour costs, and 444–471 production batches completed per year. ( A ) The impact of uncertainties and their ranges listed on the vertical y-axis on the amount of doses worth of lipid nanoparticles (LNP) formulated RNA that can be produced annually shown on the horizontal x-axis. Results are shown relative to a baseline scenario at zero on the x-axis, as described above. ( B ) The impact of uncertainties and their ranges listed on the vertical y-axis on the production cost of the LNP formulated RNA drug substance per dose shown on the horizontal x-axis. Results are shown relative to a baseline scenario at zero on the x-axis.

Article Snippet: The RNA DS cost per dose was impacted to a smaller extent by the purchase price of the 5′ cap analogue (CleanCap, TriLink Biotechnologies, Inc. San Diego, CA, USA) and purchase price of the mod-UTP.

Techniques: Purification, Formulation, Control, Produced

Journal: Nucleic Acids Research

Article Title: Ethylenediamine derivatives efficiently react with oxidized RNA 3′ ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation

doi: 10.1093/nar/gkab867

Figure Lengend Snippet: Mono- and dual labelling of IVT RNA. ( A ) Scheme of the process: NTPs mix in the presence or absence of initiating dinucleotide (ARCA, N 3 -AG or N 3 -m 7 GpppG) are incubated with gene-coding DNA template in presence of T7 RNA polymerase. After isolation from IVT mixture, RNA products are subjected to 3′ labelling (top) or simultaneous 5′+3′ dual labelling (bottom). ( B ) Representative HPLC chromatograms of crude labelling products of Cy3 3′ labelling (top) or Cy5/Cy3 5′+3′ dual labelling (bottom) performed on 35, 276 and 993 nucleotide-long RNAs (ppp-RNA 35 , N 3 -RNA 35 , N 3 -m 7 GRNA 276 and N 3 -m 7 GRNA gluc ). ( C ) Absorbance and fluorescence HPLC profiles of crude dual labelling product Cy5-m 7 GRNA gluc -Cy3 with designated retention times of collected fractions (right) and agarose electrophoresis of concentrated HPLC fractions (left); ( D ) HPLC chromatogram of crude 3′-biotinylated mRNA N 3 -m 7 GRNA gluc -Biot (right) and electrophoretic mobility shift assay (EMSA) of crude and HPLC-isolated products of 3′ biotinylation (left).

Article Snippet: Examples of such caps are commercially available anti-reverse cap analogue (ARCA), which is as dinucleotide cap 0 analogue (m 7,3 ′ - O GpppG) ( , ), or a trinucleotide cap 1 analogue (m 7,3′-O Gppp m2′-O ApG), which is used in the production of BioNTech/Pfizer mRNA COVID-19 vaccine ( ).

Techniques: Incubation, Isolation, Fluorescence, Electrophoresis, Electrophoretic Mobility Shift Assay