mRNA Manufacturing – The 5 process steps of mRNA production

daniel-tischler

Daniel Tischler

July 5, 2023

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mRNA manufacturing, or the production of messenger RNA (mRNA), has quickly become one of the most discussed topics in the biotechnology sphere. While the science of mRNA has been researched for quite some time, its application in drug manufacturing is a relatively recent development.

The novel use of mRNA technology to instruct our body cells to perform specific tasks - such as creating proteins to combat diseases - has marked an exciting shift in the landscape of medicine. Particularly in the realms of vaccine and therapeutic development, the potential of mRNA technology has been vividly demonstrated, instigating a wave of promising research and applications.

This article unveils the intricate world of mRNA manufacturing. We examine its processes, progress, and future outlook, including the transformative role of single-use technology in facilitating scalability and enhancing efficiency.

What is mRNA Manufacturing?

mRNA manufacturing involves the synthetic production of messenger RNA, a type of RNA that conveys genetic information from DNA to the machinery within cells responsible for protein synthesis. This process forms the basis of numerous biological functions, and harnessing it has opened up new avenues in biotechnology.

The ability to create mRNA molecules in a laboratory setting and use it to instruct host cells to produce specific proteins has the potential to revolutionize the way we treat diseases.

mRNA technology, the underpinning of mRNA manufacturing, employs this mechanism to fight diseases. By introducing laboratory-made nucleic acids into the body, scientists can essentially 'teach' cells how to fight off infections or diseases by targeted protein expression after cellular uptake. This has immense implications for the world of medicine and healthcare.

The Impact of mRNA Vaccines

The impact of mRNA vaccines, particularly Covid-19 vaccines designed to combat the COVID-19 virus, has been monumental. mRNA vaccines like those developed by Pfizer-BioNTech and Moderna have played a significant role in controlling the global pandemic.

Unlike traditional vaccine platforms, which introduce an inactivated or weakened virus into the body to stimulate an immune response, mRNA vaccines carry the genetic instructions that help human cells produce virus-like proteins. These proteins trigger the immune system, building a defensive response against the virus.

The rapid development and successful implementation of mRNA vaccines during the coronavirus pandemic showcased the potential of mRNA technology in tackling global health crises. It also highlighted the significance of maintaining robust mRNA production capabilities to respond swiftly to such outbreaks.

mRNA vaccines, through their quick production timelines and potency, have reshaped the landscape of vaccine development and deployment globally.

By successfully controlling the pandemic in many parts of the world, mRNA vaccines have demonstrated the power of biotechnology and mRNA manufacturing, ushering in a new era of disease control and prevention.

The Impact of mRNA Vaccines against COVID-19

Considerations in mRNA Manufacturing

Navigating the pathway of mRNA manufacturing involves a number of considerations beyond the primary biological procedures. These revolve around product quality, regulatory compliance, and adaptability to therapeutic needs.

Key Factors in Manufacturing Therapeutics

The manufacturing of mRNA-based therapeutics needs to be flexible and adaptable to the needs of different therapies. This includes considerations such as

  • dosage requirements
  • stability of the mRNA
  • delivery method (for instance, lipid nanoparticles or viral vectors)
  • targeted protein or antigen's nature

Regulatory and Quality Considerations

Biologics and vaccine manufacturing, including mRNA, requires strict adherence to regulatory guidelines and Good Manufacturing Practices (GMP). This ensures that the final product, be it an mRNA-based vaccine or therapeutic, is of high quality and safe for use. Regulatory considerations extend across all stages of mRNA production, including plasmid DNA manufacturing, mRNA synthesis, and purification. Maintaining rigorous quality control at every step is critical to achieving a product that meets all required specifications.

5 mRNA Production Steps

The simplicity, scalability, and speed of mRNA production make it a promising platform for drug development. This is particularly true for the production of vaccines, where speed is often of the essence. Unlike traditional vaccine development methods, which may take years, mRNA vaccines can be developed in a matter of months. 

Step 1: mRNA Template Design

The first step of the manufacturing process of mRNA therapeutics starts with the design of a plasmid DNA (pDNA) template, which consists of an RNA polymerase promoter and a specific mRNA sequence. After a plasmid DNA template has been established, it is amplified by using expression systems like e.Coli bacteria, for example. For enhanced integration, the plasmid DNA template is linearized.

To trigger the desired immune response and lead the cells to produce antibodies against a disease, this step has to be carried out with absolute accuracy. To ensure the stability of the sequence encoding the desired antigen, gene expression and translational qualities, it is important to protect it from any impurities.

Step 2: In vitro Transcription (IVT)

Following the design and preparation of the DNA template, the linear plasmid DNA has to be transcribed into mRNA. This is usually done by using in vitro transcription (IVT). In contrast to in vivo transcription, the mRNA is produced without using raw materials from animals. The synthesis of RNA is achieved through an enzymatic reaction of RNA polymerase and nucleotides.

After transcription, the mRNA has to be capped, to protect it from nuclease degradation.1 This takes place in the nucleus, where the mRNA capping enzyme equips the mRNA with a group of molecules that help keep the mRNA stable and enhance its translational and exporting qualities. There is also the option of co-transcription capping, which is less cost intensive than enzymatic capping, but also shows less effectivity in comparison.

These steps in mRNA modification are essential not only to enhance stability and translatability, but also to reduce immunogenicity.1 

Step 3: mRNA purification

As the DNA template is no longer needed after transcription, it has to be removed from the mRNA product. To isolate the mRNA, biotechnology has developed a number of strategies. mRNA can be purified through different chromatography steps and filtration techniques that use magnetic beads or affinity purification. For large scale purification chromatographic strategies are coupled with tangential flow filtration (TFF).2

The purification process is not only necessary to separate mRNA from the plasmid DNA template, but to further remove any impurities that could hinder the mRNA production process.2 

Step 4: mRNA Characterization

There are certain risk factors involved in the production of mRNA which call for efficient analytical tools to ensure that the mRNA product is not altered and safe for patients to use. During process development, the mRNA can degrade due to changes in temperature, exposure to light or ribonucleases. To minimize the threat of product alterations and impurities, different techniques for mRNA characterization have been developed to assess its quality. Among these methods are mass-spectrometry, RNase mapping and RNA-sequence mapping.3

With the emergence of mRNA in vaccine production during the recent coronavirus pandemic, mRNA technology has experienced a massive increase in interest. The development of two Covid-19-vaccines by Moderna and Biontech/Pfizer, shone the spotlight on mRNA-based vaccines. There are currently a number of mRNA vaccines for the treatment of infectious diseases in clinical trials or awaiting their FDA approval. Other components like viral vectors or lipid nanoparticles (LNPs)  that act as mRNA delivery systems have to be considered during mRNA vaccine development as well, as they have a strong influence on biological efficacy.3 4 

Step 5: Formulation and Stability Optimization

mRNA therapeutic and vaccine candidates have to face one of mRNA’s biggest challenges, which is its low stability. In order to prevent mRNA degradation, mRNA products require frozen storage, but also protective measures during mRNA manufacturing.

Many mRNA therapeutics and vaccines use LNPs and other lipids or carbohydrates which help to deliver the drug safely to the cells of the immune system. Further, LNPs can help to stabilize the mRNA formulation. After formulation, the drug product is sterilized, aseptically filled and packaged for further storage or transport.5 

mrna-manufacturing-process

mRNA Manufacturing Strategies

When it comes to mRNA manufacturing, various strategies have been developed and refined. They have to be adapted to the specific use of the subsequent mRNA product, such as vaccines or gene therapies, and leverage the unique capabilities of mRNA manufacturing. Nevertheless, common steps in the manufacturing of mRNA products include formulation, the creation of the drug product, and clinical trials.

mRNA for Vaccine Production

The production of mRNA vaccines involves designing lipid nanoparticles that effectively deliver the mRNA to cells. It is crucial that the encapsulated mRNA generates the right protein, eliciting the production of antibodies to fight the pathogen.

mRNA vaccines against SARS-CoV-2, developed by companies like Pfizer and Moderna, offer examples of this strategy. These vaccines introduced the genetic code of the virus' spike protein to the body, stimulating an immune response. This approach of vaccine technology has proven successful, with the vaccines demonstrating high efficacy in clinical trials.

mRNA for Gene Therapy and Immune Response

mRNA's role isn't limited to vaccine production. It's also a promising tool in gene therapy, offering a potentially safer modality to DNA-based methods. By delivering genetic information directly to the body's cells, mRNA can stimulate the production of therapeutic proteins, potentially treating a range of genetic diseases.

mRNA also plays a pivotal role in evoking immune responses, thereby contributing to the development of therapies for infectious diseases. In this context, mRNA acts as a platform technology, where the same manufacturing process can be used to produce therapies for different diseases.

Challenges and Bottlenecks in mRNA Drug Manufacturing

Despite the manifold possibilities offered by mRNA, manufacturing poses several challenges to stakeholders. Scaling up mRNA drug manufacturing to meet global demand presents one of them, along with process optimization and quality control issues.

Scaling-Up mRNA Manufacturing

Increasing production volumes while ensuring product quality is a complex task. Challenges encompass securing consistent supplies of raw materials, maintaining the quality of drug substance, and refining various steps in the process for efficiency and cost-effectiveness. The ability to adapt rapidly to changing demands and emerging disease threats adds an additional layer of complexity, requiring high process flexibility.

Quality Control and Optimization

Despite the urgency of scale-up, strict quality control measures must not be compromised. From controlling the absence of contaminants to ensuring the final product meets all specifications, the quality control process needs to be rigorous and precise from small scale to large scale. Accurate fluid management is essential for ensuring quality and effectiveness of the final mRNA product.2

Aseptic filling, a crucial step in the production chain, can pose significant contamination risks. Further, maintaining a consistent cold chain, particularly for mRNA vaccines that require ultra-low storage temperatures, is another significant challenge. This necessitates the deployment of innovative technologies capable of ensuring temperature stability throughout the product's lifecycle.

These challenges, while substantial, also provide opportunities for innovation and advancement in mRNA drug manufacturing. This is exactly where Single Use Support comes in with its innovative single-use systems.6 

Single Use Support's mRNA Manufacturing Solutions

Stepping up to the challenges related to mRNA manufacturing, Single Use Support offers automated closed systems based on single-use technology designed for mRNA manufacturing. These solutions address critical aspects of the process, including filling & aliquoting and freeze & thaw processes. 

Single Use Support's RoSS.FILL platform allows for fully automated and aseptic filling & aliquoting of mRNA, adhering to stringent cGMP regulations. Its modularity allows for unlimited scalability and process versatility. Additionally, their advanced freeze & thaw platforms ensure that mRNA or Lipid Nanoparticles (LNPs) are handled in a controlled manner, mitigating risks associated with temperature fluctuations.

RoSS.FILL_Sealer-Stepper Valve Rack_2

Their solutions extend to the cold chain storage and shipping of mRNA substances too. Frozen single-use bags are housed in a protective single-use shell and can be securely stored in a cold storage freezer. For shipping, RoSS.SHIP offers stable temperature control for at least six days, ensuring that the integrity and cold chain of the mRNA substances is maintained.

Single Use Support's end-to-end solutions pave the way for a streamlined and robust mRNA manufacturing process, contributing significantly to the field's rapid advancement.

App Note: The Chilled Future of RNA Therapeutics Filling & Freezing Applications

Traditional and rigid manufacturing processes are not for Advanced Therapies. Since novel therapies push limits in biotechnology, life science needs to keep up with adequate process solutions around commercialization of these specific therapies. Yes, SARS-CoV-2 has largely boosted the acceptance of mRNA in vaccines. And RNA vaccines and therapeutics won’t stop to fulfil unmet medical needs. But biotechs and biopharma companies continuously advancing novel treatments while solution provider drive process optimization to increase efficiency. With different product characteristics come different challenges and risks for fluid management. Therefore, modular, and flexible technologies facilitate ATMPs (Advanced Therapy Medicinal Products) manufacturing. Adjustability of process solutions to different requirements and scenarios will satisfy the needs of biomanufacturing and biotechnology companies as well as CMOs and CDMOs along the journey from drug development to commercialization.

Outlook for mRNA drug manufacturing

The achievements in mRNA therapeutics have solidified mRNA's position as a revolutionary force in the pharmaceutical industry. Looking forward, mRNA drug manufacturing is poised to catalyze advancements in diverse therapeutic areas, beyond infectious diseases.

As techniques continue to improve and our understanding of the underlying biology deepens, we can anticipate the development of mRNA-based therapies for genetic disorders, cancers, autoimmune diseases, and more.

In addition, continual innovations in single-use technologies and automated systems will further streamline mRNA manufacturing processes, improving scalability, efficiency, and quality control.

It is essential to remember that success in mRNA drug manufacturing will require overcoming the complex challenges and bottlenecks we've explored in this article. The role of pioneering process solution providers, such as Single Use Support, will only grow in importance as we step into this exciting future.

More about mRNA manufacturing

mRNA technology simply explained

mRNA technology simply explained

New advances in mRNA technology are happening regularly and rapidly. The prevention and treatment of viruses and immunogenic diseases has become possible through new therapeutics that use messenger RNA to help the body produce specific proteins. Nevertheless, the heightened demand for mRNA drugs leads to challenges for its production.

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mRNA vaccines: How to safely ship and store them

mRNA vaccines require high diligency along their supply chain. This is why every step in the mRNA vaccine supply chain must be carefully planned, carried out in a safe and controlled manner and requires appropriate, innovative technologies.

Freezing mRNA: best practice

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Considering the manifold applications of mRNA, it is essential to establish optimal freezing processes that maintain viability and effectiveness. In this article, we will compare different freezing methods for mRNA.

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Filling, Filtering & Dispensing mRNA

As the demand for messenger RNA-based products continues to rise, efficient fluid management plays a crucial role in ensuring the high-quality production of mRNA drug products. This article is about challenges and the latest advancements in fluid management techniques, focusing on filling, filtering, and aliquoting mRNA during the manufacturing process.

How to produce lipid nanoparticles (LNPs)

Lipid nanoparticle (LNP) manufacturing: Challenges & Solutions

Being promising tools in life sciences and medicine, lipid nanoparticles are drawing attention on their manifold advantages. In this article, however, we take a step back and discover how LNPs are produced. Solutions based on single-use systems for increasing quality and efficiency in production are also covered. 

Single-use technology in mRNA manufacturing

Single-use technology in mRNA manufacturing

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Sources

  1. mRNA capping: biological functions and applications, http://dx.doi.org/10.1093/nar/gkw551, Published 2016-06-18
  2. mRNA vaccines manufacturing: Challenges and bottlenecks, http://dx.doi.org/10.1016/j.vaccine.2021.03.038, Published 2021-03-24
  3. Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry, http://dx.doi.org/10.1021/acs.analchem.2c00765, Published 2022-05-12
  4. The Storage and In-Use Stability of mRNA Vaccines and Therapeutics: Not A Cold Case, http://dx.doi.org/10.1016/j.xphs.2022.11.001, Published 2022-11-06
  5. The Storage and In-Use Stability of mRNA Vaccines and Therapeutics: Not A Cold Case, http://dx.doi.org/10.1016/j.xphs.2022.11.001, Published 2022-11-06
  6. mRNA vaccines manufacturing: Challenges and bottlenecks, http://dx.doi.org/10.1016/j.vaccine.2021.03.038, Published 2021-03-24
daniel-tischler

Daniel Tischler

Chief Commercial Officer (CCO)

Daniel Tischler is Director Commercial Operations and Deputy of Chief Commercial Operations with experience in product line management, application engineer at Single Use Support.

Proximity to mechatronic systems and to customers were two constants for Daniel Tischler. At Single Use Support he started as a project manager. Later he provided customers technical insights as sales application engineer. Daniel was Head of Product Line Management working on new innovations for customers in the biopharmaceutical industry, before he has started as Director of Commercial Operations.

Daniel has gained 10+ years of expertise in design engineering and project management before he joined Single Use Support.

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