mRNA drugs are a novel class of therapeutics that use messenger RNA molecules to deliver genetic instructions to cells, enabling them to produce proteins of interest. Compared to conventional drugs, mRNA drugs have several advantages, such as cost-effectiveness, modularity, and targeting previously undruggable pathways. For example, mRNA drugs can be synthesized rapidly and cheaply by in vitro transcription and can be easily modified by changing the nucleotide sequence. Moreover, mRNA drugs can induce the expression of various types of proteins, such as enzymes, antibodies, and antigens, and modulate the immune system and cellular functions. The field of mRNA therapeutics witnessed a breakthrough in 2020, when two mRNA vaccines for COVID-19, developed by Pfizer/BioNTech and Moderna, were granted emergency use authorization by the US Food and Drug Administration. These vaccines have demonstrated high efficacy and safety in preventing COVID-19 infection and transmission and have been widely distributed and administered around the world. The success of mRNA vaccines has not only provided a solution to the global pandemic, but also revolutionized the field of RNA therapeutics, attracting more attention and investment from researchers, industry, and regulators.
Fig.1 Critical stages of mRNA drug manufacturing. (Webb C, 2022)
mRNA drug substance manufacturing refers to the process of producing mRNA molecules that encode the desired protein sequence. The most common method of mRNA synthesis is in vitro transcription (IVT), which uses a DNA template, a bacteriophage RNA polymerase, and four nucleoside triphosphates (NTPs) to generate a single-stranded RNA transcript. IVT is a simple, fast, and scalable method that can produce large amounts of mRNA in a single reaction. However, IVT also faces several challenges, such as the incorporation of unwanted impurities, the generation of truncated and misfolded transcripts, and the degradation of mRNA by nucleases.
One of the key steps in IVT is mRNA capping, which involves the addition of a 7-methylguanosine (m7G) cap at the 5' end of the mRNA molecule. The cap protects the mRNA from exonucleases, enhances its stability and translation efficiency, and facilitates its recognition by the cellular machinery. There are different methods of mRNA capping, such as co-transcriptional capping, post-transcriptional capping, and enzymatic capping. Co-transcriptional capping uses a cap analog, such as anti-reverse cap analog (ARCA), to replace a fraction of the GTP in the IVT reaction, resulting in a mixture of capped and uncapped transcripts. Post-transcriptional capping uses chemical or enzymatic reactions to modify the 5' end of the mRNA after the IVT reaction, resulting in a fully capped transcript. Enzymatic capping uses a combination of enzymes, such as vaccinia virus capping enzyme (VCE) and guanylyltransferase (GTase), to mimic the natural capping process in cells, resulting in a more authentic and functional cap. Each method has its own advantages and disadvantages, such as the yield, purity, cost, and complexity of the capping process.
Fig.2 Process of IVT and DS purification and upstream processes to produce the DNA template. (Webb C, 2022)
mRNA drug product manufacturing refers to the process of formulating mRNA molecules into a delivery system that can transport them to the target cells. The most widely used delivery system for mRNA drugs is lipid nanoparticles (LNPs), which are spherical particles composed of a lipid bilayer enclosing an aqueous core that contains the mRNA. LNPs can protect the mRNA from degradation, enhance its cellular uptake, and facilitate its endosomal escape. LNPs can also be modified by adding different lipids or ligands to improve their stability, biocompatibility, and targeting specificity.
There are different methods of LNP formulation, such as microfluidic mixing, thin-film hydration, and ethanol dilution. Microfluidic mixing uses a microfluidic device to mix the mRNA solution and the lipid solution in a controlled manner, resulting in uniform and reproducible LNPs. Thin-film hydration uses a rotary evaporator to form a thin film of lipids on a glass flask, which is then hydrated with the mRNA solution, resulting in heterogeneous and large LNPs. Ethanol dilution uses ethanol to dissolve the lipids, which are then injected into the mRNA solution, resulting in small and polydisperse LNPs. Each method has its own advantages and disadvantages, such as the scalability, efficiency, and cost of the LNP formulation process.
One of the challenges in LNP characterization is to measure the physical and chemical properties of LNPs, such as size, polydispersity, encapsulation efficiency, and zeta potential. These properties can affect the stability, biodistribution, and efficacy of LNPs. There are different techniques for LNP characterization, such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), high-performance liquid chromatography (HPLC), and electrophoretic light scattering (ELS). DLS measures the size and polydispersity of LNPs by analyzing the fluctuations of scattered light. NTA measures the size and concentration of LNPs by tracking their Brownian motion. HPLC measures the encapsulation efficiency of LNPs by separating the free and encapsulated mRNA. ELS measures the zeta potential of LNPs by applying an electric field. Each technique has its own limitations and challenges, such as the accuracy, sensitivity, and reproducibility of the LNP characterization process.
mRNA drug manufacturing scale-up and distribution refers to the process of producing and delivering mRNA drugs to the target population. The global demand and capacity for mRNA drug manufacturing have increased significantly in the past year, due to the urgent need for COVID-19 vaccines and the growing interest in mRNA therapeutics for other diseases. According to a recent report, there are more than 200 clinical trials, 3000 publications, and 10 approved products related to mRNA drugs as of November 2023. However, the current mRNA drug manufacturing supply chain faces several bottlenecks and gaps, such as the availability and quality of raw materials, the scalability and robustness of equipment and facilities, and the training and safety of personnel.
One of the solutions to address these challenges is to adopt emerging technologies and trends that can improve mRNA drug manufacturing efficiency, quality, and accessibility. For example, self-amplifying mRNA (saRNA) is a type of mRNA that can replicate itself in the cytoplasm, resulting in a higher protein expression and lower dose requirements. Continuous flow reactors are devices that can perform IVT and LNP formulation in a continuous and automated manner, resulting in higher yield and quality of mRNA drugs. Distributed manufacturing is a concept that involves decentralizing the production and distribution of mRNA drugs to local and regional sites, reducing transportation and storage cost, and increasing the accessibility and equity of mRNA drugs.
Taken together, mRNA drug manufacturing scale-up and distribution is a critical and challenging process that requires the collaboration and coordination of multiple stakeholders, such as researchers, industry, regulators, and governments. By adopting emerging technologies and trends, mRNA drug manufacturing can become more efficient, quality, and accessible.
mRNA drugs have shown remarkable results in the prevention and treatment of various diseases, such as COVID-19, cancer, and genetic disorders. However, mRNA drug manufacturing still faces many challenges and limitations, such as the complexity, variability, and scalability of the manufacturing processes, the stability, biocompatibility, and targeting of the delivery systems, and the availability, quality, and cost of the raw materials and equipment. Therefore, more research and development are needed to optimize the manufacturing processes, enhance product performance, and expand the application scope of mRNA drugs. By overcoming these obstacles and capitalizing on these opportunities, mRNA drug manufacturing can become more efficient, high-quality, and accessible, meeting worldwide demand and expectations for mRNA therapeutics.
Creative Biolabs is a CRO solutions company founded and managed by a team of scientists and experts with extensive knowledge and experience in cell therapy research and development. We provide high-quality mRNA synthesis services for clients worldwide. Whether it's chemical synthesis or IVT synthesis, we can customize the most unique strategies for you, completing the synthesis in the shortest time possible to accelerate your research.
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