IVT in mRNA production
As a potential new therapeutic drug solution, mRNA has been planned or developed for the research of many intractable diseases such as oncology, epidemiology, vaccine immunology and so on. Due to the limited capacity of chemical synthesis, it is common to use plasmid DNA to achieve mass production of mRNA by in vitro transcription (IVT).
We have mentioned in the previous article: mRNA Vaccine Manufacturing Strategy 1--Plasmid DNA Production. After bacterial harvest, lysis, clarification and purification of Escherichia coli, a high-purity supercoiled plasmid DNA vector are obtained. Plasmid DNA is the key starting material for mRNA vaccine products. Based on the structural design of the mRNA sequence, it needs to include a promoter, an open reading frame (ORF) encoding the target protein, and DNA sequence for transcription into non-coding regions and poly-A tails, etc.
The circular plasmid DNA needs to be linearized by restriction endonuclease, and then purified as a DNA template, and T7 polymerase is used to perform IVT reaction to synthesize mRNA sequences based on complementary DNA templates and free nucleotide bases.
After IVT, DNase should be used to remove the residual plasmid DNA, and the mRNA should be purified and sterilized by magnetic bead method, precipitation method or chromatography to obtain the mRNA stock solution (see Figure 1).
Figure 1-Process of mRNA-IVT production
Synthesis mRNA
The main components of the mRNA sequence are: 5'Cap, untranslated region (UTR), open reading frame (ORF) and poly (A) tail. As shown in Figure 2, the 5'Cap of mRNA can cooperate with the poly (A) tail structure to enhance the stability of the mRNA structure. The poly (A) tail first binds to a large number of poly (A) binding proteins (PABP), and then recruits eukaryotic translation initiation factor 4E (eIF4G), increasing the affinity of poly (A) to the 5'Cap structure, and finally the poly (A) tail and the 5'Cap structure meet end-to-end to form a circular mRNA.
The circular mRNA structure is conducive to the recruitment of ribosomes, and its special structure can also protect mRNA from degradation. ORF is the region containing the RNA sequence encoding the target protein. The UTRs are located on either side of the ORF and are called the 5' UTR and 3' UTR, respectively. UTRs form secondary structures that ultimately affect protein expression, 5' UTRs play an important role in translation initiation, while 3' UTRs help to maintain mRNA stability.
5’ Capping
Usually, during the IVT process, we also need to cap the mRNA sequence to improve its stability and translation efficiency, while also reducing its inherent immunogenicity. There are two common methods for capping reactions (as shown in Figure 3).
One is to use one-step Co-transcriptional Reaction with capping analogs, adding capping analogs to bind to the 5' end of mRNA in the IVT reaction. This capping method is relatively simple, but the key issue is the capping efficiency. The efficiency of some cap analogs is low, such as the capping efficiency of anti-reverse cap analog (ARCA) is 60-80%, and the new cap analogs such as Clean cap can achieve capping efficiencies of 90-99%, making co-transcriptional capping a more viable option. Additionally, enzymatic digestion after IVT can be used to degrade uncapped mRNA. Moreover, cap analogs are quite expensive, but co-transcriptional capping can save time, this method can be used for rapid production of vaccines in the case of a pandemic, when production efficiency is crucial.
The second way of capping is Enzymatic Reaction, which occurs after IVT. First, Cap0 is obtained by IVT, and then under the catalysis of vaccinia capping enzyme and 2’-O-methyltransferase, the cap structure Cap 1 or Cap 2 is formed, thus increasing the one-step reaction of the whole process. While these post-transcriptional enzymatic reactions are an inexpensive alternative with a high-yield production rate, but they are generally less efficient.
Adding Poly(A) Tail
The 3' end of most eukaryotic mRNAs has a Poly(A) tail formed by polymerization of 100-200 adenine nucleotides. The Ploy (A) tail is located at the 3' end of the mRNA, it marks the end of translation, and it can be incorporated into in vitro transcribed mRNA in two main ways. First, the Ploy(A) tail can be designed into the DNA template sequence, so the Ploy(A) tail will be incorporated into the mRNA by T7 RNA polymerase during the IVT process. This tailing allows precise control of the length of the Poly(A) tail, which affects the translation efficiency of the target protein. At the same time, poly(A) tails can also be added under the cooperation of transcription without additional steps after transcription.
The other method of adding a poly(A) tail is to add it after transcription through an enzymatic reaction. Using poly (A) polymerase to add poly (A) of corresponding length to mRNA after IVT is straightforward, but the disadvantage is that it adds an extra step to the whole process. Moreover, the length of poly (A) is much more difficult to precise control. However, the length of the poly (A) tail is a critical factor affecting the efficiency of mRNA translation. Therefore, it is more advantageous to use the first method, incorporating the poly(A) tail into the DNA template sequence design in terms of efficiency and length control.
The IVT reaction is a batch process. Using the single-use products of the WAVE bioreactor system can greatly reduce the difficulty of cleaning, verification and the problem of cross-contamination. According to the selection of different specifications, the working volume that meets our IVT reaction requirements can be processed, and conditions such as 37°C and pH 8.1 can be adjusted for the reaction.
Purification
During the IVT reaction process, many process-related impurities will be introduced, such as dsRNA, DNAse, phosphatase, plasmid DNA, free NTP, Triton-X-100, T7 RNA polymerase, cap analogs, magnesium chloride, Tris buffer, sub sperm Amines and dithiothreitol, etc. Therefore, in the IVT and purification process, the solution needs to be diafiltered to remove impurities. The tangential flow ultrafiltration system is the first choice for the diafiltration step. The type of product is selected according to the corresponding processing scale, which can be used for both research and large-scale production.
According to the size of the mRNA, it can be diafiltration with a molecular weight cut-off of 100kda. Relevant impurities will be introduced into the purified solution again, and it is necessary to diafiltration the solution again, or directly exchange it with the mRNA acidic buffer(required for downstream LNP preparation). Finally, perform sterile filtration to obtain the mRNA stock solution (as shown in Figure 4).
Figure 4-mRNA IVT 200L production process
Cobetter TFF products
For multiple TTF steps involved in the mRNA IVT process. Cobetter offers ultrafiltration solutions including cassettes and hollow fiber modules. The cassette membrane is made of RC or PES, and the hollow fibers are made of mPES. Choose between these two or conduct a comparative test according to the production process and product characteristics.
For relatively sensitive to shear force solutions or relatively high viscosity/solid content of solutions, hollow fibers are preferred for testing. For conventional solutions, choose ultrafiltration methods according to the actual scale-up required and the existing hardware system. After targeted optimization of the filtration process, a high concentration and filtration effect can be achieved. Choose the right TFF products by following the specifications in Figure 5.
Figure 5- Cobetter TFF Cassette&Hollow Fiber Specifications
Quality Management
ATMP is a foreword therapeutic drug, in terms of production quality management, we can refer to "Guidelines on Good Manufacturing Practice Specific to Advanced Therapy Medicinal Products" and "Annex 1-Manufacture of Sterile Medicinal Products" promulgated by the European Union. The quality risks associated with ATMP largely depend on the biological properties and origin of the cells, the biological properties of the vector (e.g., replication competence or reverse transcription), the expression level and properties of target protein, and the properties of other noncellular components ( raw materials, substrates) and production processes. In determining the most appropriate control/mitigation measures in each case, we shall evaluate all potential risks associated with production and manufacturing and other risks to human health or the environment.
RNase contamination
Since mRNA is very sensitive to RNase, it is necessary to strictly avoid possible RNase contamination in the environment, equipment, containers, and raw and auxiliary materials that are in direct contact with the product during the production process. Among them, for the process water and raw material buffer, RNase A can be removed by adding a 10Kd ultrafiltration system, which can effectively avoid the introduction of related RNase removers and achieve the prevention and control of RNase contamination.
Single-use products in mRNA vaccines production
For the containers, gamma-irradiated and RNase-free single-use products are preferred in the process. At the same time, the optimized structural design of mRNA sequence and the effective removal of impurity dsRNA can greatly reduce the immune effects and other related problems.
Based on the quality management of the entire life cycle of mRNA vaccines, the application of single-use products and closed systems can greatly reduce the difficulty of cleaning, avoid cross-contamination and environmental pollution, thereby improving the efficiency of process development and production applications, and has advantages such as multi-products and multi-scale collinear application.
Cobetter's single-use products have been tested and verified by a third-party testing agency, it has been confirmed that there are no DNase and RNase residues (reports in Figure 6 and Figure 7), they can be used for mRNA vaccines or other products in the field of nucleic acid and cell gene therapy.
Figure 6-Cobetter single-use products DNase-free verification
Figure 7-Cobetter single-use products RNase-free verification
Summary
In the IVT production of mRNA. Cobetter's solutions can be applied to TFF concentration, buffer exchange, nuclease removal filtration and sterile filtration.
Cobetter offers TFF cassettes, hollow fiber modules and their supporting hardware systems, integrity tester and related verification services. At the same time, Cobetter has a DNase-free and RNase-free single-use solution with products such as storage bags, mixing bags, custom single-use manifolds ,etc.
Reference
1. Guidelines on Good Manufacturing Practice Specific to Advanced Therapy Medicinal Products
2. Manufacturing Strategy for the Production of 200 Million Sterile Doses of an mRNA Vaccine for COVID-19