As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began to encircle the world with devastating effects during the coronavirus disease 2019 (COVID-19) pandemic, scientists began searching for the right way to stop the spread of the virus by inducing host immunity before or soon after infection.
A new paper reports on the current and future perspectives on messenger ribonucleic acid (mRNA) technology, the platform on which the first successful COVID-19 vaccine, from Pfizer/BioNTech, was based.
The researchers looked at the global demand for this technology in the fields of drug substance (DS) and drug products (DP), the estimated quantity that will be required and the current aspects in the manufacturing processes that offer scope for improvement.
From being investigated for a few key applications, the mRNA platform became known worldwide when it was used to distribute billions of doses of the COVID-19 vaccines from Pfizer and Moderna. These companies were already looking into this area to make cancer immunotherapies, protein replacement therapies, and other vaccines.
These plans were rapidly diverted to perfecting and commercializing the COVID-19 vaccines, a feature made possible by the versatility of this platform. The greatest appeal of the mRNA platform is that it is a modular technology. That is, mRNA can be easily tweaked to encode most proteins without a significant alteration in its chemical nature.
Using mRNA, modified nucleosides and lipid nanoparticle (LNP)-based formulations can be produced within the host cell without a whole new delivery system.
Preclinical trials of the current mRNA vaccines were done in mouse models to study dosage ranges. These studies used low amounts of raw materials, thus reducing the supply chain demand and mitigating the limited production of these reagents. In this way, the companies were able to identify the dosages at which high levels of neutralizing antibodies and T cell-mediated immunity was elicited while minimizing the adverse drug reactions.
Mouse models also helped compare the immunogenicity of different candidate vaccines in preclinical trials, but required twice as much RNA-LNPs for the trials, at a dosage of 0.2−1 μg with each candidate. Moderna used four different strains of mouse, varying the spike antigen dosage from 0.0025−20 μg.
Overall, other preclinical studies in nonhuman primates and ferrets used an average RNA dose of over 5,000 μg each, which puts a significant strain on RNA production, especially with the growth in such studies.
It is interesting that since 1990, RNA vaccine studies are being ever more frequently published. In fact, the rise from 2020 to 2021 was >400%, vs >300% from 2019 to 2020. If just one in ten of the 2,000 published studies use a second species, the demand would be >1.1 million μg of RNA formulation each year.
With gene replacement studies, the RNA doses are 100-fold higher at 10−1 vs 10−3 mg/kg for vaccine studies. The dosage goes up to 1 mg/kg with microRNA (miRNA) and small interfering RNA (siRNA). The estimated demand for RNA could thus be much higher in the foreseeable future.
RNA DS and formulated DP demand really took off with the large-scale demand for COVID-19 vaccines. Moderna and Pfizer planned to produce 1 and 2 billion doses of their vaccines, respectively, in 2021 – over 100 kg and 60 kg of mRNA-DP formulation, respectively, excluding manufacturing process losses and the consumption of reagents in analytical tests.
Clinical trials also use up significant amounts of RNA. This was estimated from early phase Moderna and BioNTech trials, adjusted for the expected proliferation of clinical trials of other RNA DPs, microRNA and siRNA, gene replacement therapies, CRISPR-Cas9 gene editing and RNA transfected gene therapies. With four studies describing phase 1-2 dosage studies for Moderna and Pfizer mRNA vaccines, they found a mean mRNA usage of 5,500 μg.
Extrapolating this to the 75 trials carried out in 2021, this would mean over 400,000 μg of RNA was required. At 100-200 μg mRNA per mL, this comes to ~2,000-4,000 mL of DPs of many different kinds, at higher expense vs vaccine mRNA because of the lack of economies of scale.
Three types of processes are used at present for large-scale mRNA production, namely, chemical, recombinant and enzymatic, depending on the required mRNA.
Chemical vs recombinant techniques
Chemical synthesis is a standard automated process of short RNA production, but is costly, and limited to the production of <100 nucleotides, with DNA splint ligation being required for longer molecules.
Recombinant RNA production uses the same principle as recombinant protein production: the DNA of interest is delivered by a vector into the host cell, where it is transcribed to the corresponding mRNA. The transcribed mRNA is vulnerable to breakdown by host nucleases, and cannot incorporate modified ribonucleotides. Recent studies describe shielding the transcribed mRNA and using circular mRNA that has superior stability.
Recombinant technology may be the most cost-effective method. Recombinant protein manufacturing units could be repurposed, since they already comply with Good Manufacturing Practice (GMP) codes.
Enzyme synthesis and IVT
Enzymatic synthesis of RNA is carried out via either the polymerase chain reaction (PCR) or the polymerase chain transcription (PCT). The former, used for decades, produces amplified oligonucleotides, but relies on thermolabile RNA polymerase. Instead, the modified double-mutant DNA polymerase TGK polymerase allows initiation with ribonucleotides.
PCT has been reported to successfully synthesize RNA from unmodified and 2′-F-modified rNTP using DNA polymerase. This budding technology may one day allow large-scale RNA production.
In vitro transcription (IVT) of mRNA is the gold standard for mRNA synthesis, using bacteriophage-derived modified RNA polymerases to synthesize the mRNA from chemically modified ribonucleoside triphosphate (rNTPs). It needs sequence-specific promoters, and the terminal regions show variability because of the polymerase’s non-specific run-off.
It is also costly, and the enzyme is vulnerable to oxidation, fragmenting the mRNA. Nonetheless, the versatile template DNA permits the synthesis of any mRNA. This could speed up regulatory approval for novel DS manufacture.
Altogether, with imperative improvements, IVT is the current state-of-the-art technology that offers cell-free, cost-effective, straightforward, and coherent large-scale production of clinical use mRNA with the help of modified RNA polymerases.”
Purification of mRNA is another essential step to get usable mRNA. Laboratory-grade mRNA is purified by DNases, but for clinical use, chromatography is among the standard pharmaceutical methods, being adaptable, scalable and cost-effective. Various chromatographic methods are suitable for large-scale efficient purification. Any of these can be used depending on the product-specific process developed by the manufacturer.
LNP-based RNA formulations carry charged nucleic acids across the lipid membrane into the cytoplasm, to exert their bioactivity. A popular technique is continuous self-assembly, in acidic conditions that allow the LNPs to enter the cytoplasm via receptor-mediated endocytosis pathways, from which the RNA is released.
Lipid film hydration is a more established method, but the above method works better for the in vivo production of siRNAs with intact activity compared to the preformed vesicle method. The former is limited by the lack of control, poor reproducibility and the need for further processing.
Microfluidic mixing is another method, used to create RNA-LNPs for use in cancer, protein replacement therapies, gene editing and vaccine production, with good scalability from prototype level to in-line analytic methods. Different techniques are used, including the staggered herringbone mixer (SHM), with the next-generation toroidal mixers (TrM) enhancing the SHM flow rate by an order of magnitude.
The future of mRNA
Scientists look forward to self-amplifying RNA (saRNA), mRNA that can amplify itself on entering the cell. Early research suggests a 6-100-fold increase in potency with saRNA compared to mRNA, while saRNA production reduces the need for RNA and lipids by 6-100-fold.
Thus, in the context of a pandemic, it would theoretically be possible to make 6- to 100-times more doses of a vaccine with the same batch volume and subsequently have a lower cost of and time required for production.”
A direct comparison shows the price of each 0.3 mL dose of the Pfizer vaccine to be $19.50, which would mean 4.5 million liters of vaccine fluid – enough to fill two Olympic-size swimming pools! In contrast, saRNA vaccines would require only 0.02 of one pool. The corresponding costs of vaccination for the entire population of the earth would be 150 billion USD vs 1.5 billion USD for the Pfizer vs the theoretical saRNA vaccine, respectively.
Once developed, saRNA could boost plant capacity many-fold for operating plants. Microfluidics also offers large commercial opportunities for scalable high-throughput drug development.
Decentralized RNA production
The ongoing pandemic has shown the limitations of large centralized drug manufacture in times of increased demand. The two mRNA vaccines were distributed from relatively few centers in Europe and the USA, leading to a supply chain crunch. The high demand for COVID-19 vaccines led to a shortage of DS and DP in the world at large, excluding the most developed countries in Europe and the USA.
In response, many countries plan to set up their own mRNA drug manufacturing sites, which will help achieve greater flexibility to adapt to local requirements, including personalized drugs. Precision medicine will likely benefit greatly from this approach. However, a lot of changes will be necessary, including in the regulatory oversight, the need to train highly skilled workers and set up a knowledge database locally, and the higher initial investment for each local site.
This will likely be offset by the ability to produce multiple DPs that use the same technology and manufacturing processes. Again, mRNA DPs are ideal for this model as they retain their chemical properties even with major changes in the nucleotide sequence.
Many companies are investing in this field, while China is likely to be the world leader, once their new plant in Zhenjiang, Jiangsu Province, is set up. It is expected that this single plant will double the company’s manufacturing capacity.
Future bottlenecks include a stable scalable lipid supply; mRNA production; and integration of multiple activities into a single suite as smaller plants are set up in different locations.
Envisioning manufacturing facilities that can churn out RNA medicines according to need, the authors compare them to the chip manufacturing revolution that ushered in today’s information technology age.
The introduction of semiconductor foundries that employed standardized manufacturing processes, suitable for producing any number of circuit designs, decoupled chip design from manufacturing capabilities and infrastructure. The concept of an RNA medicine foundry, enabled by modular manufacturing technologies such as single-use bioreactors, microfluidics, and in-line analytical capabilities, represents a potential for democratizing the design of future RNA medicines.”