Study examines freeze-dried mRNA and lipid nanoparticle vaccines with long-term stability

Vaccines have proven effective in controlling the spread of coronavirus disease 2019 (COVID-19). Among the different types of vaccines available, mRNA vaccines have emerged as the pioneers in mitigating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection due to its high efficacy and process. sequence-independent manufacturing, which could be quickly updated to a new variant, as well as low manufacturing cost.

Study: Lyophilized mRNA-lipid nanoparticle vaccines with long-term stability and high antigenicity against SARS-CoV-2. Image Credit: LookerStudio/Shutterstock

Currently approved mRNA vaccines depend on lipid or lipid-like delivery systems for their transfection efficiency. Due to the high requirements of lipid components and the sensitivity of mRNA to humidity, oxygen, pH and enzymes, cryogenic storage and transport (-20°C to -70°C) of mRNA vaccines are needed to maintain its stability. This hampers its accessibility in remote areas.

Freeze-drying is a gentle drying method that removes water by low-temperature sublimation under vacuum. This process could improve the stability of lipid nanoparticles as well as increase the storage temperature of mRNA at 4°C or room temperature for long periods of time. However, freeze-drying the mRNA-lipid complex is a sophisticated process as it would induce mechanical forces strong enough to deform the vehicle structure. This can cause vehicle aggregation, mRNA breakage, or mRNA leakage. Studies have revealed that this process can even reduce the transfection efficiency of the mRNA vaccine.

The study

A study has been published on bioRxiv* preprint server where an optimized freeze-drying technique has been proposed which is believed to be able to effectively maintain the bioactivity and physicochemical properties of mRNA-lipid nanoparticles (mRNA-LNP) whereby they could be stored at 2°C~8°C for long periods of time .

The wide applicability of this method was demonstrated by verifying the thermostability conferred by it with LNPs containing different mRNA molecules. The proposed freeze-drying technique was then used to prepare a thermostable mRNA-LNP vaccine – encoding the antigen for the wild-type, Delta and Omicron variants of SARS-CoV-2. The preventive capacity and the high level antibody response have been confirmed.

For this research, the SARS-CoV-2 N-terminal domain (NTD)-receptor-binding domain (RBD) region was used as the antigen. NLPs were prepared and characterized. Lyophilization was carried out by adding a solution of LNP with the cryoprotectant, filling the mixture into a penicillin bottle and performing the process with a freeze-dryer – using a designed procedure. This resulted in a powder which was characterized and stored at 4°C for later use.

The stability of the lyophilized LNPs was measured by incubating them at 4°C, 25°C or 40°C for a different time. mRNA integrity was measured using microfluidic capillary electrophoresis and gel retardation assay. 293T and HEK 293T/17 cells expressing ACE2 were cultured.

the live The transfection efficiency of LNP loaded with luciferase mRNA (mRNA-Luc LNP) and its lyophilized product Lyo-mRNA-Luc-LNP was examined. For the mouse immunization experiment, three types of LNPs were prepared with mRNA encoding the SARS-CoV-2 WA1, Delta or Omicron variant antigen. An ELISA test, a pseudotyped virus neutralization test, a SARS-CoV-2 Delta neutralization test and a SARS-CoV-2 Delta challenge in K18-hACE2 KI transgenic mice were then performed.


The four TNLs used in the study showed high encapsulation efficiency (EE) and narrow size distribution. After freeze-drying, the EE, size and polydispersity index (PDI) did not change, indicating that the process did not change the basic physical properties. mRNA integrity was also maintained, suggesting that the process did not damage mRNA structure.

When incubating Luc LNPs for different times to assess thermostability, no changes were found. EE was maintained at 4°C and 25°C after 18 days. However, this parameter increased at 40°C, although EE and mRNA integrity were maintained. From these results, it could be estimated that after lyophilization by the given method, LNPs could be stored in refrigerator for long durations at 2~8°C.

On the evaluation of the live transfection efficiency of lyophilized LNPs, well maintained bioactivity of mRNA-Luc LNPs was observed. The immunogenicity of lyophilized WT-mRNA LNPs was assessed following a priming regimen where mice received an intramuscular injection of WT-mRNA LNPs or wild-type Lyo-mRNA LNPs (WT ). Immunization was performed on day 0 and day 7. All vaccine-induced high IgG titers were found to be. The pseudotyped virus assay demonstrated that the freeze-drying process did not affect antibody bioactivity.

When evaluating a prepared mRNA-Delta vaccine for efficacy against Delta and other SARS-CoV-2 variants in mice, the vaccine was found to induce much higher neutralizing antibody titers against a wild-type antigen (WA1) than the delta variant.

In testing the antigenicity of the freeze-dried mRNA vaccine against Omicron (Lyo-mRNA-Omicron), robust neutralizing antibody titers against Omicron, as well as a lower degree of neutralization response against the Delta variant were been detected.

When evaluating the efficacy and immunogenicity of Lyo-mRNA-Delta in K18-hACE2 KI mice, the vaccine was found to fully protect mice against SARS-CoV-2 infection. Delta, proving its high immunogenicity.

The study proved the high immunogenicity and stability of freeze-dried mRNA-LNP vaccines. Therefore, it was inferred that this vaccine was a suitable choice for use as a control measure for the COVID-19 pandemic, and therefore its clinical trials could be quickly initiated.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.

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