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Currently, the mRNA drug production process is gradually maturing. The preparation and production of mRNA drugs need to consider three core issues:
- Optimization of mRNA molecular sequences.
- Preparation of mRNA stock solution.
- Development of mRNA delivery systems.
Schematic diagram of mRNA vaccines.
* Related services from BOC RNA.
mRNA Sequence Optimization
The mRNA molecular sequence is one of the key factors influencing protein expression efficiency and mRNA stability. For example, the SARS-CoV-2 spike protein mRNA sequence widely used in COVID-19 mRNA vaccines is a double-proline mutant S-2P mRNA sequence. This sequence utilizes two proline sequence mutations to stabilize the prefusion conformation of the S protein, resulting in higher levels of neutralizing antibodies induced by the vaccine and stronger immune protection. Additionally, studies by Courel and colleagues in mammalian cells have shown that the GC content of genes affects mRNA degradation in human cells, with mRNAs rich in AU and GC undergoing degradation through different pathways. Moreover, compared to an increase in GC content, an increase in GC3 content (the content of codons with G or C in the third position) significantly increases mRNA stability and translation efficiency. In summary, it is necessary to optimize mRNA sequences. The sequence of mRNA consists of several elements, including the 5' untranslated region (5' UTR), protein coding region, 3' UTR, and polyadenylation (PolyA) signal.
- Optimization of UTR Regions
The untranslated regions (UTRs) of mRNA contain multiple regulatory elements that are crucial for both mRNA stability and protein translation efficiency. For example, RNA elements on the 5' UTR may alter its secondary structure, thereby affecting the binding of ribosomes near the start codon. The 3' UTR is one of the key regulatory factors for the intracellular dynamics of mRNA, and it has an optimal length range. Currently, understanding the relationship between UTR sequences and related protein expression levels is limited, making it difficult to design UTRs from scratch. Natural UTRs are often used. Additionally, machine learning of massive UTRs through computational genetic algorithms to construct new synthetic UTRs or combine several UTR components for better effects is a promising direction.
- Optimization of the Protein Coding Region of mRNA
The sequence of the protein coding region not only affects translation efficiency and protein folding but also mRNA abundance. Therefore, when optimizing the sequence, multiple parameters need to be considered, such as the GC content in the sequence. Although sequences rich in GC may affect the formation of mRNA secondary structure, the translation efficiency of sequences rich in GC can be several times higher than that of sequences rich in AT. Additionally, the rate of translation elongation depends on the availability of the corresponding tRNA for codons, so avoiding the use of rare codons is a major focus of codon optimization.
mRNA Stock Solution Preparation
The preparation process of mRNA stock solution mainly consists of two major steps: the preparation of plasmid DNA stock solution and mRNA synthesis and purification.
Preparation of Plasmid DNA Stock Solution
Using plasmid DNA as a template, enzymatic transcription for mRNA production is currently a mature method for synthesizing mRNA. Plasmid DNA is typically obtained during recombinant Escherichia coli fermentation, with a well-established technical process including bacterial fermentation (plasmid amplification), cell lysis, and plasmid purification. Unlike in scientific experiments, industrial production requires more attention to key process steps, which directly affect the final yield and quality of plasmids:
- Oxygen supply for bacterial fermentation: Inadequate oxygen supply can lead to abnormal bacterial metabolism, decreased plasmid stability, and increased levels of open-loop plasmids and plasmid aggregation. In high-density bacterial fermentation cultures (which favor increased plasmid yield per unit volume of culture system), the efficiency of oxygen supply is particularly important. In large-scale plasmid fermentation systems, improving or optimizing oxygen supply efficiency is significant for increasing plasmid yield. Therefore, the availability of an efficient and flexible oxygen supply system is one of the important considerations when selecting suitable bioreactors.
- Alkaline lysis: In the current large-scale plasmid purification process, alkaline lysis is widely used to lyse bacterial cells, serving as a key step in plasmid purification and often prone to problems. For example, poor pH control can lead to excessively high local alkalinity, resulting in irreversible damage to plasmids. One method to prevent excessive local alkalinity is through efficient mixing, but excessive mixing should be avoided as high shear forces can cause bacterial genomic DNA to break into small fragments, contaminating the purified plasmids, and causing damage to the plasmids. Moreover, the lysis time is also crucial; too short a lysis time may result in incomplete bacterial cell lysis or insufficient denaturation of bacterial genomic DNA, increasing the difficulty and uncertainty in subsequent steps of plasmid purification, while too long a lysis time may lead to irreversible damage to plasmids.
- Plasmid purification: As a commonly used method for large-scale plasmid purification, chromatography purification mainly targets proteins rather than plasmid DNA in most commercialized column fillers. Since plasmid DNA is larger in size than proteins and cannot enter the majority of filler pores, the adsorption performance of fillers on plasmids decreases. Additionally, the purity of plasmids is one of the major challenges faced in large-scale production. Typically, the purity requirement for plasmid DNA obtained by chromatography purification is a purity greater than 95% and free from process-related impurities, meeting GMP standards.
Synthesis and Purification of mRNA
The steps of mRNA synthesis and purification mainly include in vitro transcription, capping, tailing, and purification. In vitro transcription and capping/tailing reactions of mRNA are safer and faster compared to most other vaccine production methods, but they rely on relatively expensive raw materials.
- In vitro transcription: In vitro transcription of RNA generally uses DNA fragments as templates and incorporates triphosphate nucleotides (adenine, guanine, cytosine, uracil) providing nucleotide bases, along with RNA polymerase, ribonuclease inhibitor, pyrophosphatase (to degrade pyrophosphates to prevent transcription inhibition), magnesium ions (as polymerase cofactors), and buffer containing antioxidants and polyamines. Currently, the in vitro transcription process is batch-produced under GMP conditions.
- Capping: In eukaryotes, the 5' Cap of mRNA regulates multiple aspects such as post-transcriptional splicing, nuclear export, translation initiation, and protection against degradation by nucleases. Additionally, the 5' Cap can mark RNA from endogenous sources for the innate immune system, thereby protecting these RNAs from attack. Currently, there are three main methods for generating capped RNA in vitro: the first method involves using vaccinia virus capping enzymes after transcription to cap RNA, resulting in RNA with Cap 0 or Cap 1. Although this method is highly efficient in capping reactions, it adds an additional 2'-O-Methyltransferase during scale-up production to increase the capping rate of Cap 1, making the entire system more expensive and complex. Moreover, enzymatic reactions cannot produce Cap 2 or m6Am cap. The second method involves adding excess Cap analogs during transcription to cap RNA. This method produces highly capped mRNA, but with lower transcription efficiency and faster reaction speed. The third method is the Cleancap method, which produces highly capped RNA with higher yield (approximately 4 mg/mL).
- Tailing: The poly(A) tail is a segment of adenosine residues at the 3' end of most mature mammalian mRNA. The poly(A) tail of mature mRNA in mammals consists of tens to hundreds of adenosine residues, and it can bind to poly(A)-binding proteins (PABPs) to form cytoplasmic ribonucleoprotein (RNP) complexes. This interaction is necessary for efficient translation and controlling mRNA stability. For in vitro transcription of mRNA, the poly(A) tail can be encoded into DNA templates (which can be PCR products or plasmids) or added to mRNA through enzymatic reactions after in vitro transcription. Poly(A) segments are prone to recombination during the amplification of plasmid DNA in bacteria, leading to sequence deletions. Although the template encoding poly(A) tail method has the aforementioned drawbacks, it is more advantageous in industry than the enzymatic method. For example, template encoding can produce determined and reproducible poly(A) lengths, ensuring product consistency. Conversely, enzymatic reaction-generated poly(A) lengths vary, making it difficult to control the composition of the final product and may not meet regulatory requirements.
- mRNA purification: The mRNA product obtained through the above enzymatic reactions will contain impurities such as proteins, incomplete mRNA, double-stranded RNA (dsRNA), and other reaction impurities. These impurities need to be removed in subsequent mRNA purification steps. Currently, mRNA purification techniques mainly include oligo(dT) affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), and ion pair reverse chromatography. Different purification methods have different purification principles, may remove different types of impurities, and have different requirements for product purity. The specific selection of which purification method or combination of purification
Development of mRNA Delivery System
mRNA delivery systems are mainly divided into viral vectors and non-viral vectors. Viral vectors are more commonly used in gene therapy, but their immunogenicity, tumorigenicity, and limited loading capacity have limited their application in nucleic acid therapeutics. Non-viral vectors, on the other hand, are used more extensively, such as polymer-based vectors, lipid-based vectors (liposomes or LNPs), and can be conjugated with specific ligands to target specific cells, such as GalNAc, peptides, antibodies, etc. Lipid nanoparticles (LNPs) are a relatively mature technology platform currently used for delivering RNA drugs, vaccines, or gene editing tools. Compared to other types of nucleic acid delivery systems, LNPs have many advantages. For instance, they have high encapsulation efficiency of nucleic acids and can effectively transfect cells, exhibit strong tissue penetration, low cytotoxicity, and immunogenicity, making them advantageous for drug delivery. These advantages make LNPs an excellent nucleic acid delivery system.
* Only for research. Not suitable for any diagnostic or therapeutic use.
