Andreas Kuhn, Ph.D., Vice President RNA Biochemistry & Manufacturing, BioNTech SE
The talk given by Dr. Kuhn focused on mRNA therapeutics and vaccines. He explained that every therapeutic that uses proteins can be alternately addressed by mRNA therapeutics that encode the protein of interest. mRNA synthesis is done enzymatically and takes a few hours to produce hundreds of copies of RNA. The necessary components are:
Dr. Kuhn continued his presentation by explaining the components of mRNA structure consisting of open reading frame, untranslated regions (UTR), 5’ cap and 3’ polyA tail. His team has worked on structural optimization, leading to improvements in intracellular half-life, translational efficiency, and protein functionality.
While 5’ and 3’ UTR regions increase the stability and translational efficacy, some optimizations of coding region regarding codon usage and optimized base usage improve translation efficiency. It is also possible to modify the protein coding region by adding tags or MHC trafficking sequences which help in processing and presentation of antigenic sequences or epitopes on the surface of the cells.
Dr. Kuhn underscored the importance of assessment of mRNA using an analytical quality control strategy. He shared how a target product profile (TPP) can be used to baseline parameters such as indication, mode of action, application route, dosing and storage specifications.
Critical quality attributes (CQA) are a set of properties that help in ensure the quantity of mRNA in intact form with essential components such as 5’ Cap and polyA tail, without exceeding a threshold for impurities such as residual DNA template and nucleoside triphosphate (NTP). Critical process parameters (CPP) including specification of starting materials, reaction conditions, DNA template sequence and concentration, RNA yield also influence critical quality attributes. Specifications for mRNA release regarding identity, concentration, integrity, critical residues, bioburden, and potency are important to be controlled both within the process and at the end.
Regarding the integrity parameter, mRNA needs to be in full-length with 5’ Cap and 3’ polyA tail to be translated into protein. Dr. Kuhn listed some ways of capping and polyA tailing, and testing their presence. There are two ways of 5’ capping which are using cap analogs and post-transcriptional capping. A high-degree of capping is achieved when cap analog is added to the reaction and level of Guanosine-5'-triphosphate (GTP) -- a competing nucleotide for the initiation of RNA transcription -- is kept at a low level. The other option is using a capping enzyme, usually from Vaccinia virus, after the RNA is synthesized. For polyA tailing, the sequence encoding polyA tail can be included in the DNA template or a second enzymatic reaction can be induced following iVT.
Following 5’ Cap and 3’ polyA tail addition methods, Dr. Kuhn shared options for analyzing mRNA capping. One of them is to degrade mRNA into nucleotides/nucleosides resulting in individual bases (U, C, G, A) and building blocks specific for cap structure. Quantification of nucleoside/nucleotide species are indicators for capped and uncapped mRNA (i.e. m7GPPPG dinucleotide v. GTP) when analyzed by LC-MS.
The other option is hybridizing an oligo at somewhere close to the 5’ Cap (i.e. 5’ UTR) together with the use of RNase H or ribozyme so that mRNA is cleaved at a point that it is possible to separate whether the cleaved short fragment (15-20 nt) is capped or uncapped via gel electrophoresis or chromatographic procedures, then quantify them and calculate the capping degree.
The presence of polyA tail can be assessed in two ways: The first method is the PCR amplification of reverse transcribed mRNA which has undergone G/I tailing followed by Sanger sequencing which provides nucleotide level resolution. The other method is to detect polyA fragments after hydrolysis by RNase T1 and RNaseA. Combinatory use of these two enzymes do not cut between As, so most of the RNA is degraded while the polyA tail remains intact, which is visible in chromatograms as distinct peaks.
Dr. Kuhn concluded by explaining that RNA integrity parameter uses measurement of the amount of full-length RNA by capillary electrophoresis and HPLC.
Functionally inert mRNAs are produced as a result of premature abortions during in vitro translation leading to only 5’ capped RNA structures or RNA hydrolysis resulting in shorter RNA fragments either 5’ capped or polyadenylated. Premature abortions are more common and can be detected using ion-pairing RP-HPLC graphs indicating the presence of 5’ cap and the absence of polyA tail.
Finally, he shared some data indicating that absence of either 5’cap or polyA tail resulted in no protein translation which has been demonstrated via Western blot analysis. As the last experiment, they exposed mRNAs to heat degradation to reduce integrity at certain levels.
Then, they did in vitro translation and the only band they could observe in Western blot corresponded to the size of full length protein indicating that degraded or truncated RNAs not having integrity will not produce protein.
