Increasing mRNA Product Stability with Lyophilization

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.

Click here to read the article in the Innovations in mRNA eBook.

About the author

Cynthia A. Challener, PhD, is a contributing editor to Pharmaceutical Technology®.

Article details

Pharmaceutical Technology
Innovations in mRNA eBook
April 2024
Pages: 8-12