Lyophilization Of Lipid Nanoparticle-formulated mRNA

Lipid nanoparticle (LNP)-formulated nucleoside-modified mRNA vaccines have validated to be very a success in the combat towards the coronavirus disease 2019 (COVID-19) pandemic. They are effective, safe, and may be produced in huge quantities. However, the long-term storage of mRNA-LNP vaccines without freezing remains a challenge. Here, Seattle Genova offers that nucleoside-modified mRNA-LNPs with lyophilization option. The physicochemical properties of the lyophilized material do not substantially change for 12 weeks after storage at room temperature and for at least 24 weeks after storage at 4°C. Lyophilization (freeze-drying) is commonly used in the pharmaceutical industry to increase the stability and shelf life of various products by removing the water from drug formulations.

Mechanism

Among potential mRNA degradation pathways, hydrolysis plays a major role in mRNA stability. We first screened for buffer solutions that could protect LNPs from potential damage during the freeze-drying process, where sucrose was selected as a cryoprotectant. The concentration of sucrose in the solution varied (0%, 4%, 8%, 12%, and 16% w/v) with 20 mM Tris at pH 7.5. LNP formulations were lyophilized using a lyophilizer. 

Conditions applied during lyophilization

The lyophilization cycle consisted of:

◆ A freezing step at −40°C for 4 h under vacuum.

◆ A primary drying step at −40°C for 45 h under vacuum (∼3 Pa).

◆ A secondary drying step at +10°C for 66 h under vacuum (∼3 Pa).

The pressure within the shelf was returned to atmospheric pressure using dry nitrogen to obtain lyophilized samples. A solid cake was formed after freeze-drying LNP- mRNA with at least 4% sucrose, and no aggregation after reconstitution with water.