Increasing mRNA Product Stability with Lyophilization

Challener,Cynthia;

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.

Using mild conditions

One approach for overcoming lyophilization challenges that is not appropriate, warns Pironti, is modification of the mRNA structure, as that would change the nature of the treatment and potentially undermine the efficacy of the finished therapy, she states.

The most important strategy is to ensure the chosen lyophilization process is as mild as possible, which requires accurate study of each freeze-drying phase to gain in-depth process knowledge and understanding. “While an accelerated method might save time, the risk of damage to the formulated mRNA product is much greater,” Pironti concludes.

New formulation approaches

In addition to mild conditions, formulation development is essential to establishing an effective lyophilization process for mRNA products. For instance, Pironti points out that the ionic strength of the chosen buffer can have a significant impact on the resulting cake post-lyophilization, potentially leading to better stability and easier reconstitution. She also notes that the concentration of the mRNA formulation pre-lyophilization can also affect the overall freeze-drying process, while use of an appropriate surfactant may address challenges with respect to LNP aggregation.

Increasing user friendliness

Lyophilization does more than increase the stability of mRNA-LNP products for simpler storage and distribution. It also, observes Dohmen, is the next step toward user-friendly drug products that physicians and patients can use without any concerns about stability and potential aggregates. “As we design future mRNA medicines, especially those for chronic conditions that will be administered on a regular basis, optimizing for stability in the hands of physicians and patients and reducing the potential for an immune reaction is of utmost importance,” he states.

CDMOs have a role to play

Lyophilization technology in general has advanced in recent years, with improvements in automation, such as automated loading and unloading, which according to Pironti has significantly reduced the time required for manufacture and filling of mRNA products. Automated unloading is important for these materials, because even post-lyophilization, mRNA formulations remain sensitive to temperature. “By automating and accelerating unloading, it is possible to minimize the risk of formulations being exposed to temperatures that are impactful on final stability,” Pironti says.

The advances in lyophilization technology are being leveraged by contract development and manufacturing organizations (CDMOs), particularly those invested in acquiring relevant experience in the production and handling of mRNA therapies and vaccines, Pironti adds. “CDMOs with this expertise have the knowledge and infrastructure to support pharmaceutical companies in accessing advances in lyophilization so they can harness it in a well-structured way to support the efficient commercialization of their mRNA products when they reach this stage,” Pironti contends.

The most successful CDMOs, according to Pironti, have deep knowledge of their mRNA manufacturing platforms, allowing the development of effective lyo cycles and identification of optimal formulations based on approaches that are well-known and already established.

Pironti also stresses the importance of working with an expert CDMO with experience in mRNA process and formulation development and manufacturing and the infrastructure for both lyophilization and subsequent sterile fill/finish activities. “Partnering with such CDMOs allows mRNA vaccine and therapy developers to benefit from existing knowledge about effective approaches, streamlining the development journey and eliminating the need to reinvent the wheel,” she observes.

Best tip for success: address lyophilization early in development

The development of optimal lyophilization processes takes time for even the simplest molecules. For complex mRNA-LNP products, it can be longer still. The lyophilization process itself also takes time, and as mentioned above, longer, and milder approaches are best for sensitive mRNA formulations.

Consequently, Pironti suggests that if a pharmaceutical company has a very short window in which to pass the mRNA candidate from early development into the clinic, it might be necessary to avoid lyophilization at the clinical manufacturing stage to accelerate the route to the clinic. However, Pironti believes that the earlier lyophilization is considered in the mRNA-LNP formulation development journey, the easier it will be to create an effective process that affords the maximum stability and simplified storage capabilities offer by lyophilization.

Learning curve at Arcturus Therapeutics

Arcturus Therapeutics began working on a lyophilization process for messenger RNA–lipid nanoparticles (mRNA-LNP) vaccines prior to emergence of the COVID-19 pandemic. The company, says President and CEO Joseph E. Payne, was looking at large-patient-population disease, for which the supply chain would be an issue. “We fortunately had strategic partners that enabled us to spend the necessary time, effort, and resources to develop a robust, repeatable lyophilization process to ensure we could provide a stable product for our late-stage trials taking place across the world,” he notes.

As a self-amplifying mRNA drug substance about three times larger than regular mRNA molecules, the Arcturus candidate had an extended learning curve, according to Payne. “Generally, the larger the mRNA, the more challenging is the development of manufacturing, purification, formulation, and lyophilization processes,” he observes.

The Arcturus process was developed after extensive empirical screening conducted in combination with the application of internal knowhow and innovative approaches. The product formulation includes a unique combination of excipients and buffers and a carefully established, sophisticated lyophilization process involving specifically designed cycles with optimum time, temperature, and vacuum settings.

The process is being implemented for the production of its Japanese-approved COVID-19 vaccine by its manufacturing partner in Japan (ARCALIS, joint venture between Axcelead and Arcturus), and Arcturus anticipates it will also transfer the technology to CSL, its partner for commercialization and distribution of the COVID-19 vaccine in Europe. In addition, Payne comments that the company is working to expand the lyophilization process to different types of vial presentation for different markets.

Significant advance for the mRNA field

Lyophilization is a great step toward further advancing the mRNA field, according to Dohmen, and he is excited to see lyophilized products reaching and advancing through clinical development to commercialization. “In the end, we need more user-friendly drugs, especially if mRNA technology is tofind use beyond vaccines,” he says.

For Pironti, the stability afforded by lyophilization has clear benefits for mRNA vaccines designed to tackle future pandemics, particularly when it comes to logistics. “A lyophilized product can simplify logistics in a scenario where vast numbers of doses of vaccine need to be manufactured and distributed across the globe, as there is no need for special cold-chain transport or storage,” she comments.

Scalability and cost of goods are two issues that still must be addressed, however, especially when thinking about vaccines where many doses are required, according to Dohmen. “The ability of lyophilization to improve access to mRNA vaccines in developing countries by removing the cold-storage requirements will only be realized if the vaccines are affordable as well,” he explains.

Finally, Payne notes that now that global mRNA vaccines have been commercialized, the stability of mRNA products is going to be highly scrutinized going forward. “As therapeutic candidates for personalized treatments and vaccines for widely spread viral infections advance through the clinic along with mRNA products formulated for many different delivery routes, such as nebulized or aerosolized powders, regulatory agencies will be looking closely at their safety and efficacy, which are both directly impacted by stability. Robust, repeatable lyophilization processes will become increasingly important as a result,” he concludes.

Formulating for delivery to the lungs

For its inhaled messenger RNA–lipid nanoparticles (mRNA-LNP) mRNA-LNP candidate, ETH47, for the treatment and prophylaxis of respiratory viral infections such as influenza and COVID-19, which is being investigated in a Phase I trial in the United Kingdom, Ethris faced additional challenges to optimizing the final formulation. “Delivery to the respiratory tract via an inhaled route of administration presents added challenges because it involves nebulization of the product, which induces additional stress to the formulation and can lead to LNP aggregation,” Christian Dohmen, executive director, technology development & CMC with Ethris, explains.

The company developed two strategies to reduce product deterioration and optimize the potency of lyophilized mRNA products, according to Dohmen. The first was to use a lipidoid rather than a traditional
cationic lipid as the basis for LNP generation to obtain more tightly packed particles and package twice the amount of mRNA with the same amount of lipids. Lipidoid LNPs are, in addition, more stable than traditional LNPs, such as those used in commercial COVID-19 vaccines, Dohmen says.

The second strategy was to further increase the stability of its final product formulation by adding a proprietary stabilizing excipient. The combination of these two solutions results in lyophilized mRNA products that exhibit “exceptional” stability of six months at room temperature, according to Dohmen. In addition, the drug product based on ETH47 can be nebulized for inhaled delivery to the respiratory tract without aggregation of the product.

Dohmen also emphasizes that the advances it has applied to enable robust lyophilization and increased mRNA product stability during storage can be used to improve mRNA beyond only those designed for inhaled delivery. “This technology has broad application for the optimization of many different types of mRNA vaccines and therapeutics,” he stresses.

Reference

About the author

Cynthia A. Challener, PhD, is a contributing editor to BioPharm International®.

Article details

When referring to this article, please cite it as Challener, C.A. Increasing mRNA Product Stability with Lyophilization. BioPharm International 2024 37 (2).