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KR-20260067349-A - COMPOSITION FOR CRYOPRESERVATION OF MRNA-LNP AND METHOD FOR CRYOPRESERVATION OF MRNA-LNP

KR20260067349AKR 20260067349 AKR20260067349 AKR 20260067349AKR-20260067349-A

Abstract

The present invention relates to a composition for cryopreservation of mRNA-LNP and a method for cryopreservation of mRNA-LNP. The mRNA-LNP cryopreservation composition and the mRNA-LNP cryopreservation method of the present invention can exhibit an excellent cryoprotective effect even when the cryopreservation composition is added at a low concentration.

Inventors

  • 안동준
  • 원태경
  • 이고은
  • 박현지
  • 윤서빈

Assignees

  • 고려대학교 산학협력단

Dates

Publication Date
20260512
Application Date
20251104
Priority Date
20241105

Claims (14)

  1. A composition for cryopreservation of mRNA-LNP containing polyglycerol.
  2. In claim 1, A composition for mRNA-LNP cryopreservation in which the above polyglycerol has an average molecular weight of 9,000 to 10,000 Da.
  3. In claim 1, A composition for mRNA-LNP cryopreservation in which the above polyglycerol has a zeta potential value of -5 mV to -1 mV.
  4. In claim 1, A composition for cryopreservation of mRNA-LNP containing 8× 10⁻⁷ to 8 parts by weight of the above polyglycerol per 100 parts by weight of LNP that encapsulates and protects mRNA in the mRNA-LNP.
  5. In claim 1, A mRNA-LNP cryopreservation composition in which the mRNA encapsulated and protected by the LNP in the mRNA-LNP is Fluc mRNA or GFP mRNA.
  6. In claim 1, The LNP that encapsulates and protects the mRNA in the above mRNA-LNP is a mRNA-LNP cryopreservation composition having a diameter of 150 nm to 250 nm.
  7. In claim 1, A composition for cryopreservation of mRNA-LNP in which the LNP that encapsulates and protects the mRNA in the above mRNA-LNP has a zeta potential value of -30 mV to -5 mV.
  8. In claim 1, A mRNA-LNP cryopreservation composition in which the LNP that encapsulates and protects the mRNA in the mRNA-LNP is composed of a lipid composition including cationic lipids, phospholipids, cholesterol, and PEG-lipids.
  9. In claim 8, mRNA-LNP cryopreservation composition comprising a lipid composition including 40-59.5 wt% cationic lipid, 5-20 wt% phospholipid, 35-40 wt% cholesterol, and 0.5-5 wt% PEG lipid.
  10. In claim 8, A composition for cryopreservation of mRNA-LNP, wherein the cationic lipid comprises any one selected from the group consisting of ALC-0315, SM-102, and Dlin-MC3-DMA, and the PEG lipid comprises any one selected from the group consisting of ALC-0159 and DMG-PEG.
  11. In claim 1, A composition for cryopreservation of mRNA-LNP with a storage temperature of -80℃ to 4℃.
  12. In claim 1, A composition for cryopreservation of mRNA-LNP in which the concentration of the polyglycerol is 0.75 μM to 1.25 μM.
  13. A method for cryopreserving mRNA-LNP comprising the step of treating a pharmaceutical composition containing mRNA-LNP with polyglycerol.
  14. Polyglycerol; mRNA; and An mRNA vaccine comprising an LNP that encapsulates the mRNA to form an mRNA-LNP.

Description

Composition for Cryopreservation of mRNA-LNP and Method for Cryopreservation of mRNA-LNP The present invention relates to a composition for cryopreservation of mRNA-LNP and a method for cryopreservation of mRNA-LNP. With the recent commercialization of mRNA vaccines due to the COVID-19 pandemic, interest in novel therapeutics such as DNA, mRNA, and siRNA is gradually increasing. In particular, lipid-based nanoparticles (LNPs) are showing excellent efficacy in the storage and in vivo delivery of these novel therapeutics. However, mRNA vaccines and similar products still require mandatory ultra-low temperature storage, which complicates logistics and limits accessibility, making distribution particularly difficult in low-income countries. Consequently, recent research has focused on raising the storage temperature of mRNA vaccines and improving the stability of LNPs during freezing periods and freeze-thaw cycles. To this end, cryopreservation compositions containing cryoprotectants are used, and high-concentration sugar-based sucrose is particularly widely used. Sucrose forms a glassy matrix around LNP particles during the freezing process, which prevents the LNP particles from being damaged, prevents aggregation of LNP particles, and improves long-term storage stability. However, if sucrose is included in mRNA-LNP vaccines, it can cause serious side effects in patients with fructose intolerance, and there are several problems such as increased viscosity of the vaccine composition because sucrose is required in high concentrations to function as a cryoprotectant. Figure 1 is a diagram showing mRNA-LNP, a cryoprotectant, and their characteristics according to the present invention. Figure 1a is a schematic diagram showing polyglycerol used as a cryoprotectant and its preservation mechanism, Figure 1b shows the z-mean diameter of mRNA-LNP frozen using sucrose, PG, PVA, and P188, respectively, as cryoprotectants after 3 months, and Figure 1c is an SEM image of mRNA-LNP frozen using sucrose, PG, PVA, and P188, respectively, as cryoprotectants after 3 months. Figure 2 is a nanoparticle tracking analysis image of the number and size distribution of LNP particles frozen with the cryopreservation composition of Example 1 and Comparative Example. Figure 2a shows a scatter plot of the size of each mRNA-LNP after 3 months, and Figure 2b shows the results of analyzing the concentration of each mRNA-LNP particle after 1 week, 1 month, and 3 months. FIG. 3 shows additional data obtained from mRNA-LNPs after cryopreservation using the cryopreservation compositions of Example 1 and Comparative Example. FIG. 3a and FIG. 3b are SEM and cryo-TEM images of mRNA-LNPs before freezing, respectively, and FIG. 3c and FIG. 3d show the z-mean diameter and SEM images of each mRNA-LNP after 1 week and 1 month, respectively. The inset shows the cryo-TEM images. Figure 4 shows the results of size and particle concentration of mRNA-LNP after sub-zero storage at various PG concentrations. Figures 4a and 4b show the z-average diameter and particle concentration of mRNA-LNP frozen with the addition of PG at 1 mM, 1 μM, 1 nm, and 1 pm after 1 week and 1 month. Figure 5 is a diagram showing the in vitro cytotoxic effect of a cryoprotective substance. Figure 6 is a figure showing the evaluation of mRNA-LNP transfection efficiency. Figure 6a is a fluorescence optical microscope image of a cell treated with mRNA-LNP that was frozen for 3 months after treatment with Example 1 and Comparative Examples 1-3, respectively, and Figure 6b is a figure showing the analysis of mRNA transfection efficiency using Fluc mRNA that was frozen by adding the cryopreservation compositions of Example 1 and Comparative Examples 1-3 to HSC-3 cells for 1 week, 1 month, and 3 months. Figure 7 is a diagram analyzing the distribution and interaction of mRNA-LNP and PG within ice crystals during thawing. Figure 7a is an image showing the locations of cy3-labeled mRNA-LNP (yellow-green) and cy5-labeled PG (red) observed using a fluorescence microscope with the splat method, and Figure 7b is a diagram showing the observation of cy3-labeled mRNA-LNP and cy5-labeled PG in-situ fluorescence microscopy during the thawing process (λ ex : 450–480 nm, λ em is filtered to observe wavelengths greater than 515 nm). Figure 7c is the emission spectrum of cy3-labeled mRNA-LNP and cy5-labeled PG in PBS solution during thawing (λ ex : 460 nm), Figure 7d is the 680 nm relative PL intensity of cy3 and cy5 during the thawing process, and Figure 7e is a diagram showing the zeta potentials of mRNA-LNP, PG, PVA, and P188 in PBS solution. Figure 8 is a diagram showing the ice recrystallization inhibitory activity of a cryoprotective agent. Figure 8a is an optical microscope image taken after 30 minutes using the splat method at -6℃, and Figure 8b is a diagram showing the results of the experiment showing the inhibition of ice recrystallization of the cryoprotective polymer in PBS buffer. Figure 9 i