Home Topics Bioprocessing Key Insights into Overcoming mRNA Process Challenges

Key Insights into Overcoming mRNA Process Challenges

March 1, 2022

Credit: Cytiva

Growing Interest In mRNA Capabilities

Although many people did not know about mRNA until it ushered us toward a return to normalcy during the COVID-19 pandemic, researchers have been studying the capabilities of RNA-based vaccines and therapeutics for almost 30 years. The potential of mRNA was first discovered by Hungarian scientist Katalin Kariko in the 1990s¹; yet, advancing it beyond an idea proved difficult, due to the body’s natural immune response to synthetic RNA. Kariko, along with immunologist Drew Weissman, eventually overcame this hurdle by incorporating modified nucleosides into mRNA, laying the foundation for its use in the vaccines later developed for COVID-19.¹

The expected growth of the mRNA market signals the success of the Moderna and Pfizer-BioNTech vaccines as only the beginning of a new era in the biopharmaceutical industry. In 2019—just before the COVID-19 outbreak—the mRNA vaccines and therapeutics market was valued at almost $600 million.² Now, recent reports show this number could be as high as $2,911.9 million by 2026,² with 155 therapies based on mRNA already in today’s clinical pipeline.³

There are three parameters driving interest in mRNA:

Safety. mRNA vaccines do not involve infectious elements like many conventional vaccines. mRNA is also degraded rapidly after injection by normal cellular processes.

2. Speed. It is produced more rapidly by cell-free processes than other biologics and is readily standardized and scaled up, improving responsiveness to large emerging outbreaks.

3. Efficacy. It induces expression of specific antigens that give rise to both humoral (antibodies) and cell-mediated immunity (T cells), which results in efficient and effective immune response.

Dr. Jing Zhu, associate director of mRNA development at GeneLeap Biotech, says the success of the Moderna and Pfizer-BioNTech vaccines drove significant changes in his company’s plans for the future. “Last year, GeneLeap Biotech had three areas of focus when it came to developing the vaccines and therapies in our pipeline: AAV [adeno-associated virus], oligonucleotides, and mRNA,” he explains. “But because of the success with the COVID-19 vaccines, we strategically shifted our main efforts to mRNA. Not only does it have the power to do things traditional modalities do not, but the development timelines and investment needed for mRNA are much less than other biologics.”

The impact of mRNA’s success in 2020 was also felt at the Centre for Process Innovation (CPI), a part of the UK government’s High Value Manufacturing network which has an objective of providing process development support and manufacturing expertise to the biopharma industry. Over the last year, CPI was a key part of the UK government response to support rapid vaccine development against COVID-19. As such it lead the work stream to develop scalable manufacturing solutions for Imperial College London’s saRNA (an mRNA type) vaccine candidate and was awarded £5 million early in 2021 to support the development of a COVID-19 variant mRNA vaccine library in the U.K.^4,5 “Prior to the pandemic, CPI was studying lipid nanoparticles and also working with other companies and organizations to develop cell-free expression platforms, which prepositioned us for what we’re doing now,” says Dr. John Liddell, chief technologist at CPI. “The attraction of mRNA as a vaccine is that it’s highly potent, and you can generate a lot of vaccine product from small volumes without requiring a large manufacturing footprint. It also offers a high transcription yield and reaction through enzymatic synthesis, allowing you to achieve four or five grams per liter within a few hours. These advantages mean that a company with access to the necessary capabilities and raw materials to produce a new mRNA vaccine could do so in a matter of weeks, while other approaches will take significantly longer.”

Overall, the characteristics of mRNA make it ideal for rapid response to infectious disease as well as for precision, i.e., personalized, medicine, a growing focus in a changing biopharma landscape—but only if the industry can overcome existing mRNA process development challenges.

Where Do We Go From Here?

The implications of what mRNA could achieve for modern medicine are far reaching; yet, advancing its capabilities means establishing a development toolbox that is fit for purpose, rather than relying on legacy methods designed for monoclonal antibodies (mAb) or other gene therapies. And while some aspects of mRNA seem simplified, the reality is that, technically, everything is new. “Compared to protein products, RNA is far more sensitive to degradation – by several orders of magnitude. It is very prone to RNase [ribonuclease] degradation,” explains Dr. Liddell. “RNase activity is ubiquitous and will destroy all of your products before you even know they’ve been destroyed. Therefore, we have to eliminate RNase activity during development and manufacture of mRNA-based products, which is very different from working with proteins. mRNA has the making of a platformable process, similar to what we have at the moment with mAbs. In that case, this has arisen from the advantage of having 20+ years of knowledge accumulated in that space, whereas with mRNA, there is still much to learn.”

Many of the challenges associated with the development and manufacture of mRNA are reminiscent of the early days of mAbs, when the industry struggled to overcome low titer and poor purification yields, leading to costly and inefficient commercial manufacturing. A focused effort to overcome those production challenges resulted in mAbs becoming the fastest growing class of biopharmaceutical products.⁶ A critical factor in driving mRNA forward is likely to be achieving consistency in process development of mRNA-based products. “The processes used for mRNA today have all been created by independent developers, each of which applied their own skills and knowledge,” says Dr. Liddell. “It should be that the same methods used to make a COVID vaccine are the same ones used for an influenza vaccine, which thus requires standardization across a range of areas.” One of the main challenges in mRNA process development and scale-up is the lack of dedicated equipment and consumables fit for the relatively small volumes and large size of the mRNA compared to traditional recombinant proteins. There is also a lack of experience and knowledge of scaling up mRNA processes as well as associated perceived regulatory uncertainties.

A Closer Look At mRNA Process Development Challenges

A more detailed examination of mRNA process development (Figure 1) uncovers several areas for improvement across multiple steps.

Plasmids

An integral raw material for mRNA-based therapies and vaccines is plasmid DNA (pDNA), which acts as a template for the mRNA. Yet, pDNA is also used for the production of viral vector-based therapies—another growing area of biopharma. This has led to a significant strain on GMP-quality pDNA supply. CPI has been fortunate to secure long-standing agreements with companies that make pDNA, but emerging companies like GeneLeap Biotech struggle to get the attention they need in an increasingly competitive environment. However, Dr. Liddell highlights a possible solution, “For DNA template production, there are a number of alternative cell-free technologies to generating pDNA, such as rolling circle DNA amplification approaches, which seem very promising for reducing process timelines and improving product quality,” he explains. “There has been a lot of pressure on plasmid manufacture, particularly to GMP, to support gene therapy and mRNA applications. Hence with these supply chain issues alternative approaches such as rolling circle amplification may see their use accelerated.”

In Vitro Transcription

After pDNA is manufactured in an E.coli-based fermentation process, it is harvested, linearized, purified, and used as templates for the enzymatic in vitro transcription (IVT) process, yielding the desired mRNA molecule. IVT is currently the cost-driving step in mRNA process development. “IVT is what generates the product, but it is a highly complex step. It requires the careful addition of a number of diverse components in addition to the DNA template, such as enzymes and nucleotides to synthesize the mRNA,” explains Dr. Liddell. “You also need a capping reagent that will be added to the five-prime end of the mRNA. Design of Experiments approaches are typically used for process optimization of these different components to optimize yield and quality. Currently, reactions are batch based, but alternate reactor designs could be devised to reduce inventory of expensive raw materials, eventually even moving to a continuous reaction scheme. This may be harder to develop, but it could make a big difference in overall productivity.”

Dr. Zhu says the need for additional optimization has led to issues and costs in GeneLeap Biotech’s work with mRNA. “We have found that each vendor’s T7 RNA polymerase has a different ‘flavor,’ which means we will optimize one IVT system using the T7 from one vendor but then have to optimize again if we switch to a different one. That is why sustainable supply from a reliable vendor is critical for us.” Equipment fit for the unique characteristics of mRNA is also essential. “For the upstream, we need equipment that is designed for cell-free expression platforms,” adds Dr. Zhu. “We can use some of the functionality on the bioreactors available now, but they are missing some important functions we need, like certain inline monitoring, which will help with scale-up by monitoring the different parameters that vary with mRNA models.”

Purification

Compounding raw material variability challenges is the impurity profile of mRNA molecules that can vary with each project, requiring different purification steps from case to case. “mRNA is a very large molecule—30 to 50 nanometers—which is way beyond the size of proteins and comparable to viral vectors. That means they do not interact well with conventional chromatography resins, where you will likely get only surface adsorption,” explains Dr. Liddell.

The varying impurity profiles of mRNA from the IVT step calls on options in purification technologies that would allow process development scientists to mix and match media based on the specific characteristics of the molecule. Dr. Zhu points to an internal case where GeneLeap Biotech used the Oligo dT purification platform for mRNA. “For some of our projects, the mRNA we worked on was very clean, with a purity profile higher than 90% after the IVT. Therefore, we applied TFF [tangential flow filtration] or SEC [size exclusion chromatography] to remove the small amount of remaining impurities,” he explains. “For other projects, the impurity profile was more complex and finding a common purification platform has been more difficult than we anticipated. Oligo dT mediated purification often works fine. For some mRNA variants, though, we found very strong double-stranded RNA product impurities, which required a specific polishing step to separate. That is why a mix-and-match approach based on the impurity profile would be ideal to form the final process. More adapted purification solutions for mRNA, such as Cytiva’s fiber-based Fibro chromatography, could also offer a potential alternative to help facilitate purification of mRNA in the future.”

Additional considerations must also be made based on whether the mRNA is conventional or self-amplifying, with the latter having a much bigger structure that creates additional challenges in the purification step. “mRNA can be made using the four conventional nucleotides as well as modified mRNA, where you are using base analogs to increase the half-life, which typically means swapping out uridine for pseudo-uridine,” says Dr. Liddell. “For self-amplifying mRNA, you are using sequences derived from certain viruses to generate what is called a replicon, constructed from four non-structural proteins coded by the saRNA, which then makes copies of the transgene protein that the RNA is coding for.⁸ It’s quite cunning technology but is at an earlier stage of development.”

Encapsulation

Another critical step in mRNA processing is encapsulation using lipid nanoparticles. The specialized lipids used for mRNA provide a delivery system that protects the nucleic acid from degradation as the drug makes its way through the patient’s body. Prior to encapsulation, the lipids must all first be dissolved in an organic solvent, which is typically ethanol. Because ethanol is highly flammable, facilities must be properly equipped to ensure safe use of those types of materials, such as using flame-proof equipment.

If a company does not have these capabilities in-house, they may look to an outsourcing partner for encapsulation services; however, this could be challenging due to the limited number of CDMOs with experience in this area. There is also a significant number of patents in this area, leading to a complex intellectual property landscape. Dr. Liddell says the lipids used for mRNA today were originally developed to deliver small interfering RNA (siRNA) therapies. There may be better LNP formulations or alternative delivery technologies that might improve conditions required for storage stability, which is another challenge for mRNA products.

Storage

Due to the sensitivity of mRNA, an ultra-cold chain is necessary to deliver COVID-19 vaccines across the world. Paving a way toward a future with mRNA by addressing limitations during process development, though, could help minimize mRNA’s temperature requirements. “The low temperature needed to stabilize mRNA comes from the rough edges arising from rapid development and deployment,” explains Dr. Liddell. “We had to move very quickly for COVID, so people used what was available, but I really think there’s ample scope for improvement by selecting appropriate additives and excipients to achieve storage stability at higher temperatures. In addition to alternate lipid and excipient formulations, we may be able to utilize other formulation technologies, such as lyophilization to achieve better stability.”

The Future Of mRNA Beyond COVID

While the COVID-19 pandemic took so much from us—most importantly, over 3.8 million lives globally⁷—the white knight of mRNA that offers eligible vaccine recipients with invaluable protection will likely open the door to new opportunities to improve patient care; however, gaps in knowledge could slow progress in not only development but also regulatory approval. Both Drs. Liddell and Zhu agree that we will likely see continued focus on using mRNA for vaccines, such as replacing the egg-based production methods for vaccines, and even oncology applications, but we must address these process development issues and more to make the possibilities of mRNA a reality. Wherever the industry does go from here, though, is possible only through collaboration, communication, and a concerted effort to pave the way for the exciting future of mRNA.

Click here to learn more about equipment and solutions for efficient mRNA production.

References

Garde, Damian. (November 10, 2020). StatNews. The story of mRNA: How a once-dismissed idea became a leading technology in the COVID vaccine race.

2. PR Newswire. (April 5, 2021). mRNA Vaccines and Therapeutic Market Size is Projected To Reach USD 2911.9 Million 2026, Says Brandessence Market Research.

3. Roots Analysis. (January 2021). mRNA Therapeutics and Vaccines Market, 2020-2030.

4. Centre for Process Innovation. (April 2020). CPI joins national taskforce to develop COVID-19 vaccine.

5. Genetic Engineering & Biotechnology News. (June 2020). U.K. Government-Backed Centre for Process Innovation (CPI) Scaling Up Imperial’s Coronavirus Vaccine.

6. Ecker, Dawn M., et. al. (October 2020). The Therapeutic Monoclonal Antibody Product Market.

7. WHO Coronavirus Dashboard. (June 2021).

8. Blakney A et al. (Vaccines, Jan 2021). An Update on Self-Amplifying mRNA Vaccine Development DOI: 10.3390/vaccines9020097

To learn more about this topic, view this GEN eBook mRNA Vaccines: A New Era of Biotherapeutic Development