By Dr. Catherine Jomary, Technology Lead ATMPs

Over 30 years ago, mRNA was identified as a potential vaccination approach against cancer. In spite of its sensitivity to the almost ubiquitous ribonucleases and its rapid degradation in situ, mRNA offers strong safety advantages. Being non-replicative means that it does not interact with the genome. Its relative instability and rapid metabolic clearance in vivo can be regulated through the use of various modifications. Formulation into diverse carrier molecules have been shown to induce rapid uptake and protein expression post inoculation. Current mRNA vaccines against SARS-CoV-2 have accelerated the path for similar approaches in the fight against cancer, heart disease, and other infectious diseases.

The full mRNA manufacturing process presents several challenges to be overcome. Part 1 of this review has focused on the challenges associated with the production of the starting material – the pDNA. Part 2 review focuses on defies associated with mRNA attributes, (i) its high sensitivity to the virtually omnipresent ribonucleases, (ii) its high sensitivity to shearing, and (iii) size similarity with some impurities (double-stranded RNA, or dsRNA). In general, the three steps to manufacture acellular mRNA are:

• mRNA is synthesized during an enzymatic reaction called in vitro transcription (IVT). The template - linearized pDNA - containing the target DNA sequence, nucleotides (NTPs) and enzymes are mixed together.
• The resulting transcribed mRNAs are then purified from the reaction contaminants using chromatography, tangential filtration, and sterile filtration.
• The final step is to encapsulate the mRNAs in lipids, and the final product is purified and concentrated using tangential flow filtration (TFF) and chromatography prior to sterile doses filling.

Challenges in Manufacturing mRNA

Cell-free synthesis of mRNA requires pDNA template to be linearized by a restriction enzyme that will cut once in a specific part of the supercoiled plasmid. Impurities associated with this step, such as addition of PEG 8000 to the reaction mix, presence of DNA fragments, and endotoxins, need to be removed. TFF and chromatography can efficiently remove these impurities.

Synthesis of mRNA

Then, the linearized DNA blueprint template is transcribed in vitro into mRNA. RNA polymerase and nucleotide triphosphates are added to the linearized and purified pDNA. To increase mRNA stability, some modified nucleotide triphosphates can also be used. However, these raw materials are under a licence fee and increase the cost of manufacturing. To stabilize and allow efficient mRNA transduction in cells, 3’ poly-(A) tail and 5’ cap are required. Poly-(A) tail is crucial for termination of protein transcription and translation because it prevents mRNA digestion by 3’ exonuclease enzymes. While 5’ capping stabilizes the mRNA by preventing 5′ exonucleases degradation. Two procedures can be used to cap mRNA. Capping can occur during the transcriptional step, in the same reactor mix by adding anti-reverse cap analogue (ARCA) and guanosine triphosphate (GTP). Alternatively, capping can be achieved enzymatically post IVT using a vaccinia virus-capping enzyme, but it requires purifying the mRNA first. This approach adds extra steps to the process -enzymatic reaction and mRNA purification steps. In addition, a licence needs to be purchased to use this capping method, making the process more expensive.

Purification of mRNA

Following IVT, the transcribed poly-(A) mRNA 5’ capped needs to be purified from the impurities such as endotoxins, immunogenic dsRNA, residual DNA template, RNA polymerase enzyme, truncated RNA fragments, unused nucleotide triphosphates and other impurities generated during capping reaction. Several options are available for mRNA purification.

Poly (dT) chromatography, by capturing mRNA poly-(A) tailed, efficiently removes impurities such as DNA, nucleotides, enzymes, buffer components and any other impurities not having a poly-(A) tail, but it cannot discriminate dsRNA from single stranded RNA (ssRNA). This initial affinity chromatography is usually followed by a second chromatography step, called polishing step, to purify further ssRNA, for example using anion exchange chromatography.

Alternatively, separation of mRNA from smaller impurities can be achieved using TFF and membranes with molecular weight cut-offs ranging from 30 to 300 kDa. One advantage is that mRNA purification, concentration and diafiltration can be performed within the same unit operation.

In the standard batch IVT process described above, the desired RNA product is purified from the reaction mix upon completion of the reaction. The polymerase enzyme, pDNA template, and unreacted nucleoside triphosphates, are discarded. These IVT reaction components are expensive, and the economical fed-batch transcription strategy, described over 20 years ago, has started to be used for large-scale mRNA manufacturing. In fed-batch IVT, the progress of the IVT reaction is monitored to maintain optimal reaction conditions, and the components are added to the reactor when needed as the reaction proceeds. The efficiency of NTP incorporation into full-length RNA product versus abortive transcripts / short RNA is dependent on the RNA sequence, and also needs to be optimized to improve process yield.

RNA is highly sensitive to rapid degradation by ribonucleases present everywhere. An effective system is needed to not only stabilize mRNA but also introduce mRNA into cells. Blending of mRNA with delivery systems and a combination of lipids and polymers has been shown to protect mRNA from ribonucleases degradation, enhance cells uptake, and improve mRNA translation in the target cell cytoplasm.

Encapsulation of mRNA

Lipid nanoparticles (LNP) are the most commonly used mRNA cell delivery system; each LNP consists of four different lipids allowing the mRNA to be carried in the lipid complex and as a result been protected from ribonuclease degradation. In addition, lipid polyethylene glycosylation has also been shown to avoid binding of protein to nanoparticles therefore preventing their degradation post injection by the immune system.

The first LNP manufacturing step is to dissolve lipids in a solvent (e.g. ethanol). The dissolved lipids are rapidly mixed with an aqueous buffer containing mRNA at low pH using either crossflow or microfluidic mixers. The resulting LNP mRNA complexes are diafiltered to replace the low pH buffer by a neutral buffer. This step should be rapid to avoid lipids degradation observed at low pH. Then LNPs complexed with mRNAs are concentrated by ultrafiltration.

Disadvantages of LNPs include the fact that they may require cold chain logistics. In addition, sterile filtration is not always possible with LNPs and in such case alternatives, for example gamma irradiation, heat sterilization, high-pressure sterilization or closed processing must be considered.

Currently, a wide range of single-use equipment formats and fittings that can accommodate mRNA manufacturing are available off-the-shelf from many suppliers. However, SARS-CoV-2 mRNA vaccine production has created enormous pressure on the single-use and raw materials demand that has led to long delivery lead times.

At this time, the current IVT mRNA manufacturing technologies present technical limitations and more industrialized cost-effective process needs to be defined. Developers of mRNA technology must have the end goal in mind when they embark in a new mRNA manufacturing project. A well-defined product profile will help them to understand the future manufacturing scale and challenges associated with the likelihood of manufacturing process changes throughout clinical phases up to commercialization.