Sudden demand for large-scale manufacturing of messenger RNA (mRNA) drug substances during the COVID-19 pandemic has few parallels in the history of pharmaceuticals. Some might posit penicillin production during World War II as an analog, or perhaps early insulin availability. Pandemics and epidemics such as smallpox, typhus, cholera, influenza, HIV/AIDS, or the bubonic plague, all relied more on infection control for resolution than on manufacturing capacity. New therapeutic uses for mRNA are now ramping up good manufacturing practice (GMP) demand, especially for clinical trial supply. Moderna collaborated with CARsgen Therapeutics to combine an mRNA therapeutic cancer vaccine with a solid tumor chimeric antigen receptor (CAR)-T cell therapy, aiming to treat multiple cancers (1). Pfizer partnered with Beam therapeutics to co-develop mRNA and base editing approaches (2), while BioNTech and DualityBio have a partnership to develop differentiated antibody-drug conjugates, for solid tumors (3). In terms of upstream manufacturing for novel mRNA drug products, this technical complexity forms a tidal wave of question marks.

A prominent paper (4) examined reducing complexity by evaluating what common process could be standardized through extrapolations of clinical trial complexity, based on four published papers Phase I-II dose ranging studies. While not the amounts of product required during the COVID-19 pandemic, clinical trial doses quickly mounted up in terms of both volume and also manufacturing challenges related to techniques pursued, but also formulation delivery requirements. The authors state, “from the four studies, the average mRNA usage was 5500 μg and for the 75 trials in 2021, 412 500 μg of RNA would need to be formulated, purified, sterile filtered, and fill-finished. At concentrations of 100 to 200 μg of mRNA per mL, this would amount to 2063 to 4125 mL of drug product” (4). As an interesting foray into possible future manufacturing, the authors went on to contrast traditional mRNA with a self-amplifying method. Often, this is cited as potentially a 100 to 1 decrease in required finished product due to higher potency performances, but this study chose a picturesque perspective, saying, “assuming the entire population consists of 7.7 billion people, and each person receives two doses, a total of 15.4 billion doses or 4.5 million L of the vaccine is required. In perspective, 4.5 million L would fill two Olympic-sized swimming pools (2500 m3), whereas saRNA [self-amplifying RNA] vaccines, which we assume are 100-fold more potent, would only require 0.02 Olympic-sized swimming pools. Assuming that production costs, which account for materials, RNA, and LNP [lipid nanoparticles] production, scale linearly, vaccinating the entire population with Pfizer/BioNTech’s mRNA vaccine would cost approximately 150 billion USD. In contrast, with saRNA, it would only cost approximately 1.5 billion USD” (4).

Commercial-scale mRNA is produced through chemical, recombinant, or enzyme-based methods. For chains under 100 nucleotides, the standard chemical technique “utilizes phosphoramidite chemistry and solid-phase support promoting chain elongation from the 3′ end to the 5′ end” (2). It is amenable to automation, as is recombinant production through host cells such as 

), which has some cost advantages as it can utilize legacy protein manufacturing lines, already in GMP compliance. Enzymatic processes are newer to arrive and have tradeoffs still being worked through. A study conducted by the Duke Human Vaccine Institute explored creating standardized robust manufacturing processing steps, saying they confronted the lack of established quality standards by, in part, surveying vendor operations “through questionnaire and/or audit relating to the vendors’ quality systems and operations as they pertained to manufacturing and/or testing of the materials” (5). The study adopted the Weismann–Karikó method of production, the upstream process requiring three enzymatic steps of plasmid linearization, mRNA transcription, and DNA template digestion. In the starting out phase, fundamentals the authors emphasized were that “for optimal 

 transcription yields, it is critical to start with high quality template DNA. Some key quality attributes that are required before use include the presence of only a single band of linearized DNA, a supercoiled ratio of at least 70%, and a sequencing-verified correct poly(A) tail length” (5). They also noted that “endotoxin control was the primary raw material risk due to its variability in raw material lots and the difficulty of its removal in subsequent downstream purification steps … reaction agitation, temperature, incubation time, and reactant concentrations were also carefully examined for each reaction step in order to yield a robust upstream platform process across multiple mRNA constructs” (5).

Chris Spivey is director, Industry Relations and Strategic Partnerships

Articles

Manufacturing

A tidal wave of questions floats the need for more upstream automation.

The Making of mRNA

Messenger RNA (mRNA) molecules are inherently unstable and readily degrade when exposed to ubiquitous enzymes such as RNase and undergo pH-dependent hydrolysis of phosphorus-oxygen bonds when exposed to water. That instability creates challenges for manufacture, formulation, storage, and transport of mRNA-based vaccines and therapeutics. While encapsulation of mRNA drug substances in lipid nanoparticles (LNPs) enhances their stability (and facilitates their delivery into cells), mRNA-LNP products still typically require low-temperature storage and present distribution challenges in areas where cold-chain management is limited.

One solution for overcoming mRNA instability is lyophilization, or freeze-drying, which is used in the manufacture of many biologic and small-molecule drugs that exhibit instability in aqueous solutions. Lyophilization involves the removal of frozen water (ice crystals) via sublimation under vacuum at low temperature to ideally produce a solid cake that typically can be stored at room temperature for extended periods. The challenge with mRNA-LNP products is to find the right lyophilization conditions that do not harm the product particles (change their size and polydispersity, among other attributes) and produce a solid with desirable properties.

While there was insufficient time during development of the mRNA-LNP COVID-19 vaccines to identify an effective lyophilization process, several companies have since then developed effective lyophilization solutions. Pfizer/BioNTech and Moderna are two well-known firms with candidates in the clinic. But it was the small biopharma company Arcturus Therapeutics, in collaboration with CSL, that received the first marketing approval of a lyophilized mRNA-LNP product. Its self-amplifying mRNA COVID-19 vaccine ARCT-154 was approved by Japan’s Ministry of Health, Labor, and Welfare (MHLW) in November 2023 (1).

The main challenge: maintaining integrity

While lyophilization is an important means of addressing stability issues associated with mRNA therapeutics, allowing the transport and storage of these products without the need for ultracold chain infrastructure, there have been challenges to effectively harnessing the benefits of lyophilization for these products, according to Vincenza Pironti, strategic marketing director at Recipharm.

The sensitivity of large, highly charged mRNA molecules, even when encapsulated in LNPs, to oxygen, light, heat, and temperature must be taken into consideration, contends Joseph E. Payne, president and CEO of Arcturus Therapeutics. The temperature transfer during lyophilization and the use of a vacuum places a strain on the LNPs. “It is necessary to develop a subtle and sophisticated method that does not perturb or discombobulate the nanoparticles during both freeze-drying and reconstitution,” he says.

Doing so, Payne adds, requires a considerable amount of resources and years of innovation, and typically involves the use of the right combination of buffers and excipients to stabilize the mRNA-LNPs during dehydration and rehydration.

These challenges, Pironti says, include ensuring the lyophilization process is effective at optimizing product stability and protecting the integrity of the LNPs. “In practice this means there needs to be a good final cake within the vial that can be readily reconstituted when it is time to administer without detrimental effects to the formulation,” she explains.

That can be particularly difficult to achieve for low-dosage formulations such as mRNA-based therapies due to the ratio of ingredients to dose volume, observes Pironti. “What is needed in these cases is the use of appropriate cryoprotectants or bulking agents within the formulation, which must be compatible with the formulation and the vial and ensure the stability of final drug product,” she observes. She also notes that comprehensive analytical testing of both the formulation and the LNPs well ahead of commercialization can help identify the right lyophilization methods to support product stability and shelf-life once the therapy is in commercial use.

Equally important, according to Christian Dohmen, executive director, technology development & CMC with Ethris, is to ensure that the LNPs are fully intact after reconstitution. There are many factors that contribute to product deterioration of lyophilized mRNA/LNPs, including particle size, mRNA integrity, and encapsulation efficiency. “If left unaddressed, the combined effect of these factors leads to deterioration of the product and decreased biological activity as well as undesired immune recognition (e.g., due to formation of aggregates), ultimately leading to reduced therapeutic potency,” he explains. In addition, he comments that aggregation of LNPs during the reconstitution process after lyophilization is another challenge for lyophilized mRNA products.

Pironti recommends using long freeze-drying cycles to prevent any damage to the LNPs within the formulation that could undermine performance and stability. Ethris has, Dohmen says, optimized its product design and lyophilization process to account for these factors and improve the potency of the resulting product as well as a reconstitution process that prevents aggregation from occurring.

Cynthia A. Challener, PhD, is a contributing editor to 

Developing freeze-drying processes requires patience and deep product and process understanding.

Increasing mRNA Product Stability with Lyophilization

The development of effective vaccines to combat COVID-19 has showcased the potential of messenger RNA (mRNA) technology, which is now under exploration for various clinical areas, including oncology, HIV, rare diseases, and personalized medicine. Given that the application of mRNA technology is relatively new, regulatory guidelines and industry standards that address specific aspects of mRNA quality during process development are still evolving, with existing resources primarily focused on mRNA vaccines.

How this guidance will translate for developers of mRNA products for other therapeutic modalities remains to be seen. Developers will need to remain nimble during this period of evolution and stay aware of emerging regulatory guidelines applicable for their unique therapies. Collaborating with a strategic contract development and manufacturing organization (CDMO) partner that has the appropriate industry knowledge and experience can help developers navigate these challenges and bring novel mRNA products to market faster.

Current and developing regulatory guidance for mRNA products

mRNA-based products are currently regulated within existing frameworks of both FDA and the European Medicines Agency (EMA). FDA oversees mRNA-based products under its Center for Biologics Evaluation and Research (CBER), and the responsible review division is assigned based on indication (e.g., vaccine, oncology). In Europe, mRNA-based vaccines can fall under different regulatory statuses depending on their target (infectious disease or not).

The regulatory framework for advanced therapy medicinal products (ATMPs) includes both gene therapy medicinal products (GTMPs) and somatic cell therapy medicinal products (sCTMPs). mRNA-based products may be classified under either category, depending on their intended use.

To help navigate the regulatory complexity in Europe, the Committee for Advanced Therapies, an advisory committee that reports into EMA, provides non-binding opinions including classifications of ATMPs. This legal status is critical to understand as it can have implications for different controls and other specificities that are intended to limit risks for different product types.

Navigating a region’s regulations can be challenging and sometimes cause for confusion. Guidance documents serve an important role in providing industry with the health authority’s interpretation of policy on a regulatory issue while not being legally enforceable. Naturally, guidance documents become an important part of the region’s regulatory framework. The process for the development and issuance of guidelines is based on policy itself, involves public consultation, and can be quite lengthy.

specific guidelines related to chemistry, manufacturing, and control (CMC) considerations for mRNA-based products. Past guidance documents issued by FDA, such as the 2018 FDA final guidance document on liposome drug products and the 2022 final guidance document on drug biological products containing nanomaterials, offer some regulatory insight to mRNA developers that employ liposome delivery strategies, but these guidelines are written broadly and are not specific to mRNA (1,2).

The integration of mRNA-based vaccines into clinical and market settings during the COVID-19 pandemic highlighted the need for a collaborative approach to standardize quality expectations, facilitating their successful development. Early efforts are focused on vaccines for infectious disease, but undoubtably there will be principles and considerations that will be relevant to other mRNA therapies.

Michele Duggan is senior manager, Regulatory Affairs, at Thermo Fisher Scientific.

When referring to this article, please cite it as Duggan, M. Navigating an Uncertain Regulatory Environment for mRNA-based Products. 

Pharmaceutical Technology Innovations in mRNA 

Quality

Regulations for mRNA products are evolving as the market for mRNA expands. 

Navigating an Uncertain Regulatory Environment for mRNA-based Products

Much of the current development of messenger RNA (mRNA) in the biopharma industry is focused on using mRNA-based therapeutics in the targeted treatment of cancers. Obstacles still persist, however, in the delivery method of the therapeutic. One solution lies in the formulation stage, where, for instance, lipid nanoparticle (LNP) technology has become a common mode of delivery, owing to its success in the mRNA-based COVID-19 vaccines. In an interview with Stefaan De Koker, vice-president, Technology and Innovation, etherna, 

® explores the criteria that make mRNA a good choice as a therapeutic and looks at how LNP technology is becoming a well-established vehicle for delivering mRNA. 

According to De Koker, mRNA combines three significant advantages compared to other drug modalities. First, mRNA is an extremely flexible platform. For instance, mRNA sequences can be easily modulated, and, hence, they can encode virtually any therapeutic protein, “whether it’s in the cytosol of the cell, on the membrane, or even a secreted protein,” said De Koker. The ability to easily modulate mRNA sequences was on reason why mRNA was well suited to a pandemic case, such as COVID-19. It also makes mRNA well suited for developing personalized immunity, such as, a cancer vaccine. In this instance, the vaccine can be adapted to the antigen profile of a tumor.

A second major advantage of mRNA is the ease of manufacturing, says De Koker. It can be produced quickly, which, again, was exemplified by the COVID crisis, where, within a few months, mRNA vaccines were in clinical studies, he emphasizes. “They were also clearly much faster to reach approval compared to more conventional therapeutic modalities, [such as] protein-based vaccines or viral vector-based vaccines,” De Koker adds.

The reason behind mRNA’s quick synthesis is that production can be done entirely 

, and thus there is no need for cells to produce it, De Koker emphasizes. mRNA manufacture is based on an 

 transcription reaction, which uses a viral polymerase to amplify the mRNA. Moreover, De Koker explains, mRNA contains only four building blocks, which means it has constant physical and chemical properties. “Thus, regardless of the protein [one] wants to encode, [they] would have the same physical/chemical properties. This, [therefore], allows [one] to develop generic manufacturing processes,” De Koker says.

A third beneficial aspect of mRNA is the fact that the immunogenicity of the platform can be modulated. “This means that the mRNA and the formulation can either be made less (for therapeutics) or more immunogenic (for vaccines) based on the application. It really makes the immune system believe that the viral infection is ongoing, and it needs to respond to that infection by mounting a strong immune response,” De Koker explains.

“These three things together—the flexibility, the speed of manufacturing, and ability to modulate the immunogenicity of the platform—makes mRNA a versatile platform for vaccines and other uses,” De Koker states.

Pharmaceutical Technology’s Innovations in mRNA eBook

When referring to this article, please cite it as Mirasol, F. Evaluating the Partnership between mRNA and LNPs. 

Formulation

The LNP success in mRNA vaccines leads to their establishment as a standard in mRNA delivery.

Innovations in mRNA, April 2024 eBook

The Making of mRNA

Increasing mRNA Product Stability with Lyophilization

Navigating an Uncertain Regulatory Environment for mRNA-based Products

Evaluating the Partnership between mRNA and LNPs