Lyophilization of mRNA vaccines: Stability innovation

The last article takes you to understand whether mRNA vaccines can be freeze-dried? In this article, we take a look at the advantages of freeze-dried mRNA vaccines. How big is the advantage?

Zhao et al. studied lyophilized mrNA-LNP encoding firefly fluorescein, and confirmed that the redissolved product maintained mRNA expression efficiency through bioluminescence imaging studies in mice. Hong et al. developed a freeze-dried SARS-CoV-2 mRNA-LNP vaccine formulation and showed that the vaccine can induce a strong immune response in mice.

Importantly, none of these published studies provide information on the stability of freeze-dried mRNA-LNP formulations over time.

This study will introduce a very efficient lyophilization process that can be used to produce lyophilized preparations of mRNA-LNPs. Lyophilized mRNA-LNPs were produced at -80 ° C, -20 ° C, 4 ° C, 25 ° C (room temperature) and 42 ° C for 4, 12, or 24 weeks, respectively. The results showed that the physicochemical properties of mRNA-LNPs did not change significantly after being stored at room temperature for 12 weeks or at 4℃ for at least 24 weeks and then redissolved with water. Under the same storage conditions, through bioluminescent imaging studies in mice, we found that the mRNA-LNPs encoded by firefly fluorescein did not lose their high translational properties. Importantly, we demonstrated in a mouse comparative immunity study that, as a freeze-dried preparation, the mRNA-LNP vaccine does not lose potency after storage at room temperature for 12 weeks or at 4 ° C for at least 24 weeks.

Bonus: Advantages of trehalose over sucrose

Suitable lyophilized protective agents need to have the following four characteristics: high glass transition temperature, poor water absorption, low crystallization rate and no reduction group.

In the freeze-drying process of many biological products, disaccharides (sucrose, trehalose) are often used as protective agents, because disaccharides can not only act as low temperature protective agents in the freezing process, but also play the role of dehydration protective agents in the drying and dehydration process, and do not contain reductive groups, which will not cause the protein Browning reaction of biological products to deteriorate and deactivate. Therefore, sucrose and trehalose are two commonly used protective agents in the freeze-drying formulation of biological drugs.

Trehalose has a higher glass transition temperature than sucrose, so trehalose solutions are less likely to form ice crystals.

Trehalose also has a magical hydration ability: according to each glucose unit, the number of non-frozen water molecules around trehalose is more than that of sugar, and its solution can form a more rigid trehalose/water structure, which is more resistant to freezing dehydration.

In addition, trehalose has low hygroscopic properties, and trehalose is almost non-hygroscopic when placed in a place with relative humidity above 90% for more than 1 month. Therefore, when trehalose is selected as a lyophilized protective agent, there is no need to worry about the decrease of the glass transition temperature due to its hygroscopic properties.

For biological products, trehalose, as a stable non-reducing disaccharide, does not contain reducing groups, does not react with active substances such as proteins, and has non-specific protective effects on biological macromolecules and organisms, is an ideal choice of lyophilized protective agents or protein protective agents.

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