Finding Solutions to Manage mRNA Instability

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BP Elements, BioPharm International's BP Elements, January 2023, Volume 2, Issue 1

Messenger RNA is inherently unstable and thus requires unique solutions to protect its cohesion.

Oligonucleotide drug substances generally are unstable at room temperature, and some are also degraded in the presence of certain enzymes. That is why, in part, they are formulated using special delivery solutions such as lipid nanoparticles (LNPs). (These delivery technologies also facilitate entry of nucleotide APIs into target cells.) Degradation can occur via loss of terminal nucleotides or certain functional groups, oxidation, and other mechanisms. Oligonucleotide–oligonucleotide interactions that can occur at higher concentrations are an added concern. These instability issues must be managed throughout the entire manufacturing process.

The challenges are particularly great for messenger RNA (mRNA), which can be degraded by enzymes found commonly in the environment, temperature, and due to the overall shear sensitivity of the long-chained molecule.

Variety of oligonucleotide drug substances

The landscape of nucleotide-based therapeutics is based on more than half a century of research and development in these modalities, according to Julian Mochayedi, strategic marketing manager for mRNA solutions with MilliporeSigma, the Life Science business of Merck KGaA, Darmstadt, Germany. This knowledge translates to the clinic and beyond, with several approved therapeutic products on the market.

The different types of nucleotide-based therapeutics can be broadly classified by the type of nucleic acid, Mochayedi continues, such as those with DNA or RNA as a basis. Alternatively, they can be differentiated by their functional design, which determines the mode of action. “One can cite DNA-based adeno-associated virus (AAV) vectors for gene delivery, antisense oligonucleotides (ASOs), small-interfering RNAs (siRNAs) for gene expression disruption, and most recently, mRNA to express a protein of interest such as for immunization purposes, as the successful mRNA-based COVID-19 vaccines have demonstrated,” he explains.

mRNA instability particularly challenging

DNA-based oligonucleotide drug substances, such as AAV vectors and plasmid DNA, share the relatively stable characteristics of DNA, Mochayedi notes. The stability of shorter chemically synthesized RNAs such as ASOs and siRNA can be improved through chemical modifications during synthesis. Particularly long biologically synthesized mRNA molecules, however, are inherently unstable, and thus require additional management during manufacturing.

All nucleic acid-based platforms, however, must be formulated using delivery technologies that protect the therapeutic from degradation. Unique solutions such as LNPs help ensure stability in the patient setting, API stability and, ultimately, the efficacy of the therapeutic approach.

There are several reasons why mRNA is inherently unstable. The physiochemical properties of single-stranded mRNA molecules that lead to their instability include their very large size, high negative charge, sensitivity to rapid degradation by omnipresent enzymes such as endonucleases, shear sensitivity, and high viscosity through the formation of secondary structures in solution, according to Mochayedi.

Manufacturing strategies for managing mRNA instability

One of the main culprits causing mRNA degradation is the enzyme RNase. Avoiding it is difficult, however, because this enzyme is ubiquitous. “It is very difficult for mRNA to move freely in the environment,” Christian Cobaugh, CEO and founder of Vernal Biosciences, comments. “Extreme precautions must therefore be taken to avoid introducing this enzyme into manufacturing spaces or enclosures,” he stresses. This issue can often, he notes, be a barrier to entry into the mRNA market for laboratories not experienced in mRNA production.

Use of qualified processes, including sterile single-use technology, should be applied to mitigate contamination and carryover of endonucleases, Mochayedi says. Liberal use of RNase degrading chemicals for cleaning equipment surfaces, operator gloves, and so on is also essential, Cobaugh adds.


The instability of mRNA at room temperature creates further challenges, as the time available for holding generated mRNA sequences at room temperature is fairly restricted, according to Cobaugh. The drug substance must be moved seamlessly to downstream purification and formulation or quickly frozen.

“The choice of adopting a seamless manufacturing approach or having unit operations separated by controlled freezing and subsequent thawing steps must be considered early on, as the decision will impact the design and operation of the mRNA manufacturing facility, and once implemented, that design will essentially be locked in,” Cobaugh contends.

“Ideally, it is best to limit the number of freezing and thaw steps, as they can impact the quality of the mRNA drug substance and formulated mRNA-LNP products,” Cobaugh adds. Furthermore, gentle techniques must be employed. Continuous movement of the mRNA drug substance to purification and then LNP formulation avoids all but the final freezing step. “It is possible, though, with the right engineering solutions and validation procedures to surmount the challenges presented by either approach to mRNA manufacturing,” he concludes.

In addition, stringent optimization of reaction conditions and the process parameters for key unit operations, such as the synthesis step (in vitro transcription) and filtration (via tangential-flow filtration), can overcome viscosity challenges and ensure low shear during processing to maintain the stability of the mRNA at each stage of the manufacturing process, Mochayedi remarks. “Bespoke process development and optimization are necessary for each unique mRNA sequence to address and overcome the highlighted challenges,” he concludes.

Minimizing formulation time

While the concept of leveraging mRNA for the prevention or treatment of various diseases is straightforward, the safe and effective delivery of these fragile nucleic acids into cells where their message can be translated into proteins has proved to be a huge challenge, Mochayedi observes. “For the COVID-19 mRNA vaccines, the solution to this problem came through the development of LNPs. These particles, typically composed of four different lipids (ionizable lipids whose positive charges bind to the negatively charged backbone of mRNA, pegylated lipids that help stabilize the particle, and phospholipids and cholesterol molecules that contribute to the particle’s structure), provide a protective bubble for the delicate mRNA molecules, enabling their safe and efficient delivery into cells,” he explains.

The structure of the nucleotide API has a strong impact on the selection of the excipients for the formulation. The manufacturing process must, in addition, be highly controlled. “The buffer solution containing the mRNA drug substance used to produce the LNPs is of low pH (typically aqueous sodium citrate) that places additional stress on the nucleotide’s stability, leading to even more rapid breakdown than occurs at neutral or slightly basic pH,” Cobaugh says.

A low pH of approximately 4.5 is needed to ionize the mRNA so that it forms nanoprecipitates with the lipids. The use of stabilizing process aids is, Cobaugh adds, generally avoided because they would interfere with the ionization of the mRNA and could also potentially end up in the product. Most molecules that would stabilize mRNA against pH-mediated degradation would, furthermore, likely be hazardous chemicals.

The key to successful LNP production, therefore, is minimization of the time required for the LNP manufacturing process. That, Cobaugh emphasizes, requires extensive understanding of the process from a quality-by-design basis combined with thorough process validation. “It is absolutely essential to have a deep awareness of what levers can be pulled from both process and engineering perspectives to minimize the time for all aspects of the formulation process,” he states.

Rapid fill/finish important too

With respect to fill/finish of mRNA therapeutics and vaccines, the strategy is generally no different than that of any other biological API, according to Mochayedi. The goal, agrees Cobaugh, is to fill the product—whether drug substance in sterile single-use plastic bags and containers or formulated drug product into sterile glass vials—and then freeze them as rapidly as possible but in a controlled manner. In this case, the challenges are largely engineering and equipment-related.

Maintaining the cold chain

For storage and shipment of mRNA, the requirement to maintain the cold chain to ensure its stability and integrity all the way to the patient is of utmost importance, Mochayedi says. “Central storage is usually performed at very low temperatures (-80 °C). Similarly, formulations containing nucleotides require shipment at low temperatures, which is typically achieved using dry-ice packaging,” he notes. In some cases, mRNA drug products may require the addition of a cryoprotectant to protect the particles. The adjustment of excipients to protect the nanoparticles may also enable some formulations to be shipped at approximately 4 °C.

Looking to lyophilization

The production of mRNA-LNP formulations that are stable at warmer temperatures is a key focus of developers of candidate therapeutics and vaccines. One approach noted by Cobaugh involves the development of higher-quality DNA templates that are free of breaks (commonly referred to as nicks) that result in the shorter, more unstable mRNA sequences. Mochayedi points to new types of modified nucleotides and new capping technologies to physically altered forms of mRNA, such as circular RNA. “These new approaches may overcome some of the stability issues and enable a broader application of mRNA as a technology platform,” he believes.

Lyophilization of mRNA products as an alternative to freezing is the other main area of investigation. “Theoretically, there is no reason why freeze-dried mRNA products cannot be stored at room temperature as long as water is kept out of the storage containers,” Cobaugh says. He notes that there has been preliminary success in this area, and he is hopeful that lyophilization will be applicable for mRNA products in the not-too-distant future.

Mochayedi agrees, noting that significant investigations into formulation optimizations to overcome the ultra-low temperature requirements of mRNA drug products during shipment and storage are underway across the industry, with lyophilization of formulated mRNA-LNPs as an extremely promising alternative for liquid-based products. “The complete drying of formulations afforded by freeze-drying would unlock the possibility of much more easily providing mRNA therapeutics in countries with higher temperatures,” he remarks.

About the author

Cynthia A. Challener, PhD has been a freelance technical writer for over 20 years, leveraging her education from Stanford University (BS) and University of Chicago (PhD) and 10+ years of industry experience. She currently focuses on pharma/biopharma topics, writing technical articles, white papers, blogs, and other content for a variety of clients in addition to contributing regularly to BioPharm International and Pharmaceutical Technology.