Presented by Julia Schlereth, Director RNA Analytics, BioNTech
In 2020, BNT162b2, a COVID-19 vaccine using mRNA technology was developed by BioNtech in cooperation with Pfizer. Julia Schlereth from BioNTech shared that now that mRNA vaccines have been established as a new class of vaccines, they are likely to be applied beyond the limits of infectious disease, and could have an impact on cancer and autoimmune diseases in the future.
Schlereth detailed the four mRNA subforms used for vaccines: Uridine mRNA (uRNA), neucleotide modified mRNA (modRNA), self-amplifying mRNA (saRNA), and trans-amplifying mRNA (taRNA). The building blocks common to all mRNA include: 5’CAP, 2 UTRs and the polyA tail.
Uridine mRNA has no additional modifications and offers a strong T cells response upon repeated administration. Neucleotide modified mRNA, is similar to uRNA but neucleotide modifications (e.g. N1-methyl-pseudouridine , m1Ψ) provide the advantage of lowered immunogenicity and induction of strong antibody responses. Self-amplifying mRNA and trans-amplifying mRNA are more complex as they include a replicase for self-amplification, generating an improved expression profile, they can also be used at lower doses. Delivery of mRNA to the cells is accomplished using one of three formulations: liposomes, lipid nanoparticles (LNPs), or polyplexes. The ultimate choice of formulation is dependent on route of administration, the target cell, and therapeutic indication.
The central principle of using mRNA as therapeutics is that it allows for introduction directly into the cells of the genetic information of the protein of interest, and that the therapeutic protein is translated using the cell’s own translation machinery. For each of the main building blocks of mRNA there is a body of literature on optimization of the sequences. BioNTech focused on the 5’CAP and poly-A components of mRNA seeking to ensure efficiency of translation in cells and maintaining stability of the mRNA. The 5’ and 3’UTRs and the encoding region were also optimized but mainly for regulatory concerns. Codons in the encoded region were also optimized to improve translation efficiency. mRNA Vaccine Manufacture
The process to manufacture mRNA vaccines includes template and mRNA production, purification, LNP formulation, and sterilization. It may be completed in one to two weeks but quality control and release processes take an additional four to five weeks. mRNA synthesis is accomplished by in vitro transcription of the template DNA, where more than 500 copies of mRNA are made per copy of DNA template. The template is removed by hydrolysis using DNAse I.
Purification of the final product is complicated by the occurrence of both process- and product-related impurities. The highly charged nature and instability of mRNA and relatedness of product to impurities bring challenges to purification. There are also difficulties in scaling up the purification process.
The process control strategy includes generic assays for composition, complex bioassays for safety concerns (e.g. bioburden, endotoxins), and RNA specific parameters (strength/concentration, identity, and purity). The analytical methods used include UV spectroscopy, HPLC, PCR sequencing, capillary electrophoresis, immunoblots, and ELISA.
To conclude, Schlereth discussed the complex issue of RNA integrity. and highlighted some areas to be worked on. Following manufacture the product not only contains full length mRNA but also shorter fragments. The fragments are due to both abortive transcription and degradation processes. It is a parameter related to the “intactness” of the RNA and thus is considered a key quality in release or subsequent stability studies. RNA integrity is analysed using capillary gel electrophoresis in which shorter fragments with fewer charges will elute faster than longer molecules. The limitations of gel electrophoresis include incomplete resolution and difficulties in integration of the data. Since individual peaks may contain more than one species, such analysis of RNA integrity cannot be used as a stand-alone parameter, it is suggested that it also be linked to a functional readout.
Julia Schlereth is Director, RNA Analytics at BioNTech.
