The value of mRNA technology to human society was properly recognized in early October 2023 with the awarding of the Nobel Prize in Physiology or Medicine to Professors Katalin Kariko, Ph.D., and Drew Weissman, M.D., Ph.D., for the contributions each made to its development during their time as researchers at the University of Pennsylvania. Of course, that award was far from the only public recognition of the importance of their work, with millions worldwide having been protected by the mRNA-based vaccines developed in response to the COVID-19 pandemic.
Going forward, the market value of the mRNA vaccines and therapeutics is estimated to continue to grow rapidly for the foreseeable future. This market is estimated by several research firms
to be approximately $40–50 billion in 2022/2023 and to be expanding at a compound annual growth rate between 13% and 17%. Notably, the therapeutics sector is expected to be the major driver of further growth as demand for COVID-19 vaccines levels off.
Unlike traditional biologics, which are produced via microbial fermentation or mammalian cell culture, mRNA production does not require the use of living cells and is therefore simpler overall. However, that does not mean that consistent manufacture of high-quality mRNA is easy. Lab-scale kits cannot be practically implemented at commercial scale, process control can be challenging at large scale, and batch-based processes involve many steps for which there has yet to be standardization implemented across the industry. Furthermore, mRNA instability poses significant challenges. Raw material variability and the lack of fit-for-purpose, single-use manufacturing equipment exacerbate the growing pains in this young sector.
Wacker Biotech supports clients throughout the entire journey from pDNA to the LNP formulated mRNA product.
The sensitivity of mRNA molecules to temperature, shear, and degradation by ubiquitous enzymes creates the need for extensive process controls. Furthermore, processes for the generation of final products in the form of lipid nanoparticle (LNP) encapsulation of the mRNA active, which is necessary to improve stability and facilitate delivery into appropriate tissues, typically involve the use of proprietary lipids and large quantities of solvents.
Both biopharma companies and academic groups are working on solutions to these challenges to enable more robust, scalable, and cost-effective mRNA manufacturing processes. In the meantime, many developers of mRNA therapeutics and vaccines are turning to outsourcing partners with the expertise and specialized capabilities needed to produce mRNA drug substances and mRNA–LNP drug products.
The production of mRNA begins with transcribing the genetic code from linearized plasmid DNA (pDNA) into mRNA. While some manufacturers create pDNA through bacterial fermentation and linearization as initial steps, many acquire pDNA from external sources. The pDNA template carries a DNA-dependent RNA polymerase promoter and the mRNA construct’s corresponding sequence. In vitro transcription (IVT), a cell-free process employing RNA polymerases derived from bacteriophages and chemically modified ribonucleoside triphosphate molecules (rNTPs), then transforms pDNA into mRNA, enhancing stability with a 5’ cap and 3’ poly-(A) tail. Downstream purification includes tangential-flow filtration (TFF) and chromatography to remove immunogenic double-stranded RNA (dsRNA), residual DNA template, RNA polymerase, and elemental impurities, followed by mRNA encapsulation in lipid nanoparticles (LNPs) using various lipid types and ratios. LNPs are formed via rapid mixing of lipid solutions with mRNA, and the final product is prepared in the desired buffer solution and concentrated using TFF.
mRNA molecule
The specific steps in mRNA production vary from manufacturer to manufacturer and as a function of the genetic sequence, target application, dosage, and scale. In addition, the quantity and dosage level for mRNA products vary significantly depending on the application, from large-scale vaccines to fight global pandemics to very-small-scale personalized cancer therapies. Scalability — whether up, down, or out — is therefore essential, regardless of the manufacturing strategy. Processes must provide high yields of high-quality mRNA products cost-effectively over a range from micrograms to kilograms.
Although process parameters for the IVT step will need to be adjusted depending on the specific sequence, this step of the mRNA–LNP drug product production process is fairly platformizable. Downstream purification of the mRNA drug substance can also differ, depending on the nature of the mRNA. The optimum unit operation can also vary with scale, so in some cases scaling up and down is not simply a matter of changing equipment sizes but using different equipment entirely.
Also, the pDNA and LNP manufacturing need to be adjusted depending on the product; the size and other properties of the plasmid determine the optimum upstream process parameters for pDNA production, while the mRNA sequence dictates the choice of lipids, lipid ratios, and mixing parameters. Consequently, extensive process development and optimization is often needed for each of the three major steps of mRNA manufacturing and formulation.
The instability of mRNA, including its sensitivity to temperature, shear, and ubiquitous RNase enzymes, must be managed not only during production but also during analysis and storage/distribution. Minimizing processing and handling times and freeze-thaw steps is crucial, as are monitoring and control of all process steps; all become more challenging when large process volumes are involved. Rapid analytical methods suitable for product characterization, quality control release, and stability testing, are also necessary. One way to help minimize overall production times and handling of mRNA is to establish end-to-end manufacturing capabilities from pDNA through mRNA–LNP fill/finish.
Sourcing difficulties, meanwhile, create additional challenges for scaling mRNA–LNP production. Regulatory authorities today expect that not only critical raw materials but also important ancillary materials meet GMP requirements. Access to a reliable supply of high-quality and GMP-compliant raw materials for mRNA production, including pDNA and the specialized enzymes and lipids required for IVT and LNP formation, are of particular note. The limited number of suppliers that can reliably provide high-quality and GMP-grade specialty enzymes and lipids in large quantities presents cost and availability issues.
Different mixing technologies can be leveraged to produce LNPs: microfluidics, such as the continuous technology developed by Precision NanoSystems, and more traditional mixing in classical mixers. Both are widely used depending on the needs and preferences of each drug developer. Development of effective processes using both approaches involves trial and error to some extent, however, because full and deep knowledge of the LNP formation process remains lacking.
Management of large quantities of flammable solvents is a separate challenge associated with current LNP processes. Such materials are not found in typical biopharmaceutical manufacturing facilities and require the implementation of special safety measures. Containment infrastructure, solvent waste disposal, and operator training are just three additional items that add cost and complexity to LNP manufacturing. Some companies elect to run multiple smaller batches rather than install the costly infrastructure required to perform larger-scale runs. In addition, because single-use equipment is not compatible with most organic solvents used in LNP processes, stainless-steel equipment, with necessary cleaning and sterilization systems, must be employed.
Given the large number of manufacturing steps involved in the production of mRNA products through fill/finish and final packaging, it is not surprising that manufacturers lacking appropriate in-house capabilities frequently end up working with multiple service providers. That adds time, cost, and risk to the overall process, as well as greater supplier management requirements.
These challenges can be greatly reduced by working with a CDMO that provides an end-to-end, integrated offering starting with plasmid design, cell banking, and plasmid production and continuing through fill/finish of mRNA–LNP formulated products. Planning and oversight can be greatly simplified, with each step in the process well aligned. Even when transfer from one site to another is still required, those transfers occur within the same company with much reduced delays, cost, and risk, particularly if all of the sites operate under the same management, quality, and regulatory systems, have the documentation styles and review expectations, and approach raw material procurement in the same manner. Development, analytical, and production teams can also meet regularly to ensure that all activities are completely and continuously aligned across the entire value chain.
Wacker Biotech’s mRNA Competence Center in Halle, Germany (expected to be operational by April 2024)
Finding such a CDMO can be a challenge, however, because very few have experience in large-scale manufacture of mRNA drug substances and mRNA–LNP drug products. Those that do have typically focused on one of the steps — pDNA manufacture, mRNA production, mRNA–LNP formation, or fill/finish operations. Drug developers must therefore coordinate the efforts of a multitude of suppliers and manage longer timelines, greater risk, and higher costs. Effective process improvements are inhibited, and the instability of mRNA magnifies the challenges.
Flexibility is in fact crucial for mRNA drug developers and hence for CDMOs offering support for the development and manufacture of mRNA products. The variability in mRNA processes is further complicated by a complex intellectual property (IP) landscape, which drives each developer of mRNA vaccines and therapeutics to seek different methods to skirt existing patents, often in downstream purification techniques (e.g., preparative high-performance chromatography, ethanol precipitation, clarification chromatography) or in the LNP production step. If the CDMO offers a (platform) production process, the CDMO becomes responsible of making sure it has the freedom to operate within this landscape.
It is also worth noting that regulatory requirements for mRNA drug substances and drug products have not yet been fully established. Both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) are still learning as candidates move through the development and approval process, and further guidance is expected. More changes are anticipated with regard to analytics compared with processing.
Originally published on PharmasAlmanac.com on April 8, 2024.