mRNA is a breakthrough technology for the development of prophylactic and therapeutic vaccines or drugs against a variety of diseases including infections and cancers. The development of mRNA vaccines includes mRNA synthesis, vector delivery and other processes.
The working principle of the mRNA vaccine is to encapsulate the mRNA fragments of the virus into special lipid nanoparticle, inject them into human body to generate antigens, and then stimulate a specific immune response to achieve the effect of forming immune memory.
After obtaining the plasmid DNA (pDNA) template of the desired sequence of interest, the steps for cell-free downstream production of mRNA can be summarized as:
·      mRNA is synthesized in an enzymatic reaction called in vitro transcription (IVT), in which a template for the target DNA sequence – linearized pDNA, nucleotides (NTPs), and enzymes are mixed together.
·      The transcribed mRNA is then purified from the reaction mixture/contaminants using steps such as chromatography, tangential flow filtration, and sterile filtration.
·      The mRNA is finally encapsulated in lipid nanoparticles (LNPs) typically composed of 4 lipid components, including ionizable cationic lipids, helper phospholipids, cholesterol, and polyethylene glycol (PEG)-lipid . Encapsulation of mRNA in lipid complex protects it from ribonuclease degradation while efficiently delivering it into the cytoplasm of cells.
mRNA in vitro transcription
Cell-free synthesis of mRNA requires the pDNA template to be linearized with a restriction enzyme that cuts at a specific site of the supercoiled plasmid. Impurities associated with this step need to be removed, such as using tangential flow filtration and/or chromatography.
Then, in vitro transcription (IVT) uses a linearized plasmid as template to mimic the in vivo transcription process to generate mRNA under conditions with RNA transcriptase and NTP. The promoter usually used in the plasmid template is the T7 promoter, which has a high transcriptional intensity and is currently the most widely used promoter for prokaryotic expression. T7 RNA Polymerase is highly specific to the T7 promoter and has high activity. With the assistance of inorganic pyrophosphatase, murine RNase inhibitor, and DNaseI, it can synthesize high-yield mRNA.
mRNA purification
After IVT, the transcribed 3'poly-(A)-mRNA-5' cap product needs to be purified from the mixture of endotoxin, immunogenic dsRNA, residual DNA template, RNA polymerase, truncated RNA fragments, unused nucleotide triphosphates and other impurities produced during the capping reaction. Various options are available for the purification of mRNA, typically including one or two chromatography steps, and ultrafiltration/diafiltration steps based on tangential flow filtration.
Affinity chromatography is a commonly used high-efficiency purification strategy, such as Poly (dT) chromatography, which effectively removes DNA, nucleotides, enzymes, buffer components, etc. without poly-(A) by capturing mRNA with poly-A tails, but it cannot distinguish dsRNA from single-stranded RNA (ssRNA). After the initial affinity chromatography, there is usually a second chromatography step, called polishing, to further purify the ssRNA, for example, using anion exchange chromatography. Sometimes a combination of ion exchange and hydrophobic interaction chromatography is used.
mRNA encapsulation
RNA is highly sensitive to rapid degradation by ubiquitous ribonucleases, requiring an efficient system that not only stabilizes mRNA but also introduces it into cells. The incorporation of mRNA into the delivery system, along with selected combinations of various lipid components, has been shown to protect mRNA from ribonuclease degradation, enhance cellular uptake, and improve mRNA translation in target cells. The first step in LNP preparation is to dissolve the lipids in a solvent (e.g. ethanol), then rapidly mix the dissolved lipids with a low pH aqueous buffer containing mRNA using a cross-flow or microfluidic mixer to obtain LNP that encapsulates mRNA, the optimal control of the mixing channel and speed is the key to obtaining the required particle size, particle size distribution, and encapsulation efficiency. The resulting mRNA-LNP complexes were diafiltered to replace the low pH buffer with a neutral buffer. This step should be performed quickly to avoid lipid degradation observed at low pH. Concentration is then performed by ultrafiltration.
Disadvantages of LNPs include that they may require cold chain logistics. Furthermore, due to the particle size of LNP, it is not always possible to perform sterile filtration, in which case alternatives must be considered, such as a fully closed single-use process.
The entire mRNA production process faces multiple challenges; therefore the raw material for mRNA plays a crucial role for the production of mRNA vaccines. Real-Gene is offering “GMP grade” comprehensive range of raw material including Enzymes, Cap Analogs, Nucleosides, Lipid Nanoparticles (LNPs) for mRNA vaccine production process. 


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