October 24, 2022 PAO-10-022-CL-04
To date, mRNA manufacturing at large scale has taken place in bioreactors with capacities of less than a hundred liters at most. That scale can provide millions of doses of an mRNA vaccine. Many mRNA candidates in development are intended to treat much smaller patient populations compared with a global vaccine — even as small as one dose per patient for personalized cancer vaccines. Accordingly, production capability will be needed at scales from milliliters to several hundreds of liters. The question for mRNA manufacturing, therefore, is not just one of achieving cost-effective scale-up but also of realizing cost-effective product at small scale and, potentially, cost-effective scale-out, which requires a unique approach.
While one of the benefits of mRNA technology is that its manufacturing processes can be readily standardized, to date most mRNA processes have been developed relatively independently, without significant cross-industry collaboration. There is a need to reach a state — similar to what exists for monoclonal antibodies today — in which mRNA production platforms are at least relatively consistent across companies. This will require not only industry-wide development efforts but also the introduction of fit-for-purpose production equipment and consumables specifically designed for small-volume mRNA production.
Some specific aspects of the mRNA production process must also be considered for production at large or small scales. These include the replacement of solvent extraction with tangential-flow filtration and the elimination of RNases from all raw materials, equipment, reagents, and the general manufacturing environment. Other important considerations are the choice of delivery system and the stability of the final product and methods to assure sterility if sterile filtration is not possible.
Today, mRNA is produced via fermentation using plasmid DNA (pDNA) templates. While significant additional pDNA capacity has recently come online or is being constructed, the supply chain remains constrained, and wait times for production of custom GMP-quality pDNA can be more than one year.
Because pDNA templates determine the mRNA structure, it is essential to have access to pDNA constructs that have been carefully designed and manufactured with high quality and purity. Plasmid production involves amplification in bacterial cells, typically Escherichia coli, to yield circular pDNA, which is then harvested and purified. For mRNA production, the pDNA may also be linearized to facilitate in vitro transcription. Linearization involves incubating the circular pDNA in the presence of a restriction enzyme, which can introduce additional impurities (e.g., the enzyme, bovine serum albumin DNA fragments, endotoxin) that must then be removed. At the lab scale, purification is often performed using solvent extraction, but for GMP production, TFF and chromatography are more appropriate.
Issues with the process include the time required for the fermentation step, the need to perform cell lysis carefully to avoid denaturing of the pDNA, and the high viscosity and sensitivity of pDNA to shearing, which must be taken into consideration for all manufacturing steps. Some companies are exploring the development of non-fermentation-based processes for pDNA manufacturing to alleviate some of the current capacity and processing concerns.
In vitro transcription (IVT) is a crucial process for synthesizing desired mRNA, as well as to control process- and product-related impurities. It requires product-specific process optimization development, based on understanding the dynamic of the transcription reaction (transcription rate with concentration of raw material components, such as pDNA, NTPs, MgSO4 concentration). For example, during the reaction, depletion of some reaction components (e.g., NTPs) may lead to misincorporations by RNA polymerase that impact the integrity of the RNA integrity and the fidelity to the pDNA template.
Linearized pDNA is used as a template for mRNA production via an enzymatic IVT process. While IVT is well-established process, it is a complex step that involves the use of not only the pDNA template but also enzymes and nucleotides. The pDNA may then be degraded using DNases. If enzymatic capping is used, that step is performed after the mRNA is purified.
Reaction temperature is also an important control parameter that can affect process performance and quality (accumulation of NTPs at lower temperatures may lead to inefficient 5’ capping and generate higher immunogenic double-stranded RNA (dsRNA)). Temperature-stable enzymes (e.g., RNA polymerase) are recommended to maintain high fidelity during the reaction.
Different reactivities of T7 RNA polymerases from different vendors are a challenge to establishing a single, optimum platform process for mRNA manufacture, even within a single company. IVT is also a cell-free process, and single-use technologies on the market today have been designed for cell culture applications. As a result, they generally lack many of the sensors needed for real-time monitoring of IVT reactions.
After IVT, the mRNA must be purified, which involves removal of excess reagents (e.g., residual DNA template, RNA polymerase), as well as endotoxins, immunogenic dsRNA, elemental impurities, and so on. TFF is useful for separating the mRNA from smaller impurities and also enables concentration and diafiltration into the appropriate buffer in the same step.
However, if the DNA template has been degraded, it is important to realize that it is possible during TFF for small DNA fragments to hybridize to the mRNA, generating additional new impurities. As an alternative, chromatography (reverse-phase ion pair, anion exchange (AEX), and affinity using poly(dT) capture) can be effective for removing impurities, including the DNA template. However, it is considerably more expensive, and a TFF step may still be required for concentration and diafiltration. If enzymatic capping is performed, a chromatography step is needed afterward.
For GMP production, affinity chromatography poly(dT) uses a fit-for-purpose resin that removes pDNA, other nucleotides, and buffer impurities but cannot distinguish between single-stranded and double-stranded RNA. Therefore, affinity chromatography is typically followed by an AEX polishing step. TFF is then performed again to concentrate the mRNA and transfer it into the final buffer solution.
Because the raw materials used in the IVT process can vary significantly with respect to quality and impurity profiles, the output of the IVT process can also vary dramatically, leading to the need for the development of different purification processes for each mRNA molecule. This issue also limits the ability of companies to establish platform processes for downstream operations. A range of purification technologies is needed to address the potential variations so that mRNA developers and manufacturers could select from a known portfolio rather than have to conduct extensive studies for each project to identify the best solutions.
One of the reasons that current commercial mRNA production batches are performed at no larger than a few hundred liters can be attributed to limitations with the lipid-based encapsulation process leveraged to generate lipid nanoparticles (LNPs). The high sensitivity of mRNA means that the time for carrying out the encapsulation process must be limited, which in turn restricts the batch size. The process is also complex, requiring the use of multiple different lipids at specified ratios, some of which are highly specialized, as well as organic solvents. This step is often outsourced, but like with pDNA, there are only a few contract development and manufacturing organizations (CDMOs) with expertise in this area, and the intellectual property landscape is complex.
Given that the composition of LNPs was originally developed to deliver siRNA, there is potential to screen the optimized LNPs depending on the RNA payload. Self-amplifying RNA (saRNA) is rising as an alternative candidate in the field of RNA-based therapeutics to overcome concerns related to repetitive injections and relatively large doses of mRNA–LNP vaccines. In the case of saRNA, the injection dose is significantly lower, since it can replicate the gene of interest (GOI) inside of the body. This implies that smaller batch size is required — and for this reason, CDMOs should consider scalability to accommodate various types of RNA payloads in the long term. To overcome the drawbacks of commercially available LNPs, mostly with regard to immune response and target specificity, studies are actively ongoing to develop non-lipid cargos, such as nanoemulsions and polymeric nanoparticles, and these will be prospective candidates for the future mRNA CDMO market.
The instability of mRNA when formulated as LNPs remains an additional issue, as in most cases these products must be stored and shipped at very cold temperatures, creating ultra-cold-chain concerns. Advances are needed that will allow the storage and transport of mRNA products at refrigerated or ideally room temperature. Here again, the rapid development of the COVID-19 mRNA vaccines did not allow time for process and product optimization. It is anticipated that the cold-chain challenge will be resolved through novel formulation approaches using different additives and excipients.
Production of mRNA therapies and vaccines involves a complex process that starts with pDNA manufacture and concludes with the fill/finish of formulated products using some type of delivery technology. With commercial-scale manufacture of mRNA vaccines having been underway for just a little more than one year, there are unsurprisingly few CDMOs with extensive expertise in this field.
In addition, those CDMOs that have been involved in the commercial production of COVID-19 mRNA vaccines and the clinical production of mRNA candidates in late-stage trials generally only support one aspect of the process, such as pDNA manufacture, mRNA production, LNP formation, or fill/finish operations. As of yet, there are no CDMOs that offer end-to-end services starting with pDNA for mRNA manufacturing and including both drug substance and drug product manufacturing.
Having the right partner with the ability to take charge of all of these activities and eliminate the need to manage multiple service providers would be a huge advantage for developers of mRNA therapies and vaccines. Such a partner would have the capability to support the different manufacturing steps as well as the regulatory aspects of mRNA product development, including validation and application filing.
The benefits of mRNA technology — its potential for platform processing and reduced development timelines — make it highly attractive for the manufacture of global vaccines, personalized cancer vaccines, and therapies for the treatment of a host of different indications. In addition, unlike traditional vaccines, mRNA products can be produced in the same facility as other biopharmaceuticals. However, one challenge for many of the companies developing mRNA products will be to find CDMO partners with the knowledge and capability to support this emerging field.
Samsung Biologics has a well-earned reputation for delivering high-quality biologic products in short timelines with a high standard of service. This same spirit and energy are now being applied to support companies developing mRNA products. We have tech-transferred 139 biologics products, which translates to 13 products per year. While these projects involved antibodies, all of the knowledge gained during downstream purification and analytical testing can be leveraged for mRNA programs.
In addition, Samsung Biologics is one of the few CDMOs with actual commercial mRNA production experience, as we have been providing fill/finish services for Moderna’s COVID-19 mRNA vaccine product, including all of the accompanying analytical development and testing for product release. New investments are being made to enable end-to-end support of mRNA projects, with RNA vaccine drug substance manufacturing capacity added in April 2022. All of the capabilities will be located at our single campus in South Korea, with R&D and manufacturing experts in close communication with one another.
Frequent handling of mRNA in multiple locations increases contamination and degradation risks, but when the entire work stream from pDNA to vial is coordinated by one partner from a single location, transitions across development and production tasks run smoothly, maximizing efficiency and eliminating these potential risks.
Samsung Biologics offers integrated manufacturing services from clinical to commercial, including aseptic fill/finish, labeling, packaging, and cold-chain storage — all at a single site — to successfully manufacture mRNA therapeutics. As a one-stop-shop mRNA manufacturing partner, we are fully equipped with a dedicated mRNA MSAT lab where we offer an integrated solution to facilitate our clients’ molecules to commercial production through tech transfer, and process development and characterization.
This move into mRNA with end-to-end support is part of Samsung Biologics’ sustainable growth plan, which also includes venturing into multimodal products, such as next-gen vaccines and cell and gene therapies. We are proactively expanding our capabilities and expertise in mRNA manufacturing through partnerships with our valued clients, and our teams of experts are working around the clock to solidify Samsung Biologics as a manufacturing hub for scientific innovations to help save the lives of people worldwide.
Pierre Catignol is the Executive Vice President and Head of Manufacturing at Samsung Biologics. He is a recognized industry expert with a demonstrated history of 26+ years in the biopharmaceutical and pharmaceutical industries. Prior to joining Samsung Biologics, Mr. Catignol held a leadership role at Lonza serving as the Head of the Portsmouth, NH (USA) and Porriño, Spain sites. He also served as Site Head at Virbac & Sanofi Pasteur, as well as Senior Vice President of Technical Operations & Supply Chain at Stallergenes Greer earlier in his career. Mr. Catignol holds a master’s degree in general engineering from ECAM LaSalle.
.svg)