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Freeze-drying protective agent for nucleic acid-lipid nanoparticles as well as preparation method and application of freeze-drying protective agent

A technology of lipid nanoparticles and drying protective agent, applied in the field of biomedicine, can solve the problems of energy consumption, increase the time and cost of product amplification and production, and achieve the effects of reducing energy consumption, inhibiting nucleic acid hydrolysis reaction, and high encapsulation rate

Active Publication Date: 2022-05-31
CANSINO BIOLOGICS INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The key parameters of the freeze-drying process in the prior art are often "empirical" applications. Generally, the freeze-drying process takes 30-100 hours from pre-freezing to the end of analytical drying. Energy consumption is one aspect, and on the other hand, it also greatly increases the production capacity. Amplify the time cost of production, such as the preferred freeze-drying program in the invention patent CN 110714015 B is "the pre-freezing temperature is -50 ℃, and the temperature is kept for 5 hours

Method used

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  • Freeze-drying protective agent for nucleic acid-lipid nanoparticles as well as preparation method and application of freeze-drying protective agent
  • Freeze-drying protective agent for nucleic acid-lipid nanoparticles as well as preparation method and application of freeze-drying protective agent
  • Freeze-drying protective agent for nucleic acid-lipid nanoparticles as well as preparation method and application of freeze-drying protective agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Preparation of Lyoprotectant

[0067] Prepare a lyoprotectant in a solvent according to the formula in Table 1, wherein the solvent is nuclease-free water, and filter and sterilize with a 0.22 μm sterile filter membrane. With the added amount of the lyoprotectant as 10% (w / w), the samples of Examples 1-1~1-12 and Comparative Examples 1-1~1-28 were used as the protective agent and the model sample lipid nanoparticles (ie mRNA- LNP) were mixed evenly, and then packed into vials; Comparative Example 1-29 was to mix the protective agent of Example 1-1 with the liposome sample (ie, nucleic acid-Lip), that is, to replace the model sample with the liposome sample Packed into vials.

[0068] Table 1 Prescription of lyoprotectant

[0069]

[0070]

[0071] Take 500 μl of samples from Examples 1-1~1-12 and Comparative Examples 1-1~1-29 into 3ml vials for freeze-drying. Freeze-drying procedure: pre-freezing stage: -60°C for 4 hours; sublimation drying stage: -45...

Embodiment 2

[0077] Example 2 Scanning Electron Microscope (SEM) Morphological Observation

[0078] Scanning electron microscope (SEM) operation steps: use a needle to take a small amount of freeze-dried powder samples of Example 1-1 and Comparative Example 1-1 and place them on the conductive gel, and then use SEM to scan and image. For the results, see the attached Figure 2a , attached Figure 2b. It can be seen that Example 1-1 has a "ginger-shaped" wrapped scaffold microstructure, while in Comparative Example 1-1 no scaffold structure was formed between LNP and the protective agent, and the protective agent can only play the role of dispersive filling and reduce stress damage.

Embodiment 3

[0079] Example 3 Morphological observation by transmission electron microscope (TEM)

[0080] Dilute the model sample and Example 1-1, Example 1-4, Example 1-8, Example 1-12, and Comparative Example 1-10 by adding an appropriate amount of nuclease-free water, and then drip the diluted solution on the copper grid. For the sample, absorb the excess sample from the edge of the copper grid with filter paper, then add 2% phosphotungstic acid to stain for 5 seconds, absorb the excess phosphotungstic acid from the edge of the copper grid with filter paper, and use a transmission electron microscope to scan and image. The results are shown in the appendix Figure 3a , Figure 3b , Figure 3c , Figure 3d , Figure 3e , Figure 3f , visible embodiment 1-1 (see Figure 3b ), embodiment 1-4 (see Figure 3c ), Examples 1-8 (see Figure 3d ), Examples 1-12 (see Figure 3e ), Comparative Examples 1-10 (see Figure 3f ) have similar particle size distribution and microscopic morphol...

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Abstract

Based on physical and chemical principles of a freeze drying process, freeze drying conditions, namely freeze drying procedures and freeze drying protective agents, of the nucleic acid-lipid nanoparticles are systematically studied, and an efficient freeze drying method suitable for the nucleic acid-lipid nanoparticles is preferably designed. According to the method, the total time of a nucleic acid-lipid nanoparticle freeze-drying process can be shortened to 8-18 hours, the energy consumption and the time cost of product amplified production are remarkably reduced, the freeze-dried nucleic acid-lipid nanoparticles are rapid in rehydration (within 10 seconds) and high in nucleic acid total amount, entrapment efficiency and nucleic acid integrity, and in addition, the freeze-dried nucleic acid-lipid nanoparticles have good application prospects. The cell transfection efficiency of the freeze-dried and rehydrated preparation is not significantly different from that of an unfreeze-dried nucleic acid-lipid nanoparticle stock solution, and the in-vivo immune response is high and even exceeds that of the unfreeze-dried nucleic acid-lipid nanoparticle stock solution.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a nucleic acid-lipid nanoparticle freeze-drying protective agent, a preparation method and application thereof. Background technique [0002] In the novel coronavirus pneumonia (COVID-19) epidemic, the mRNA vaccine has the advantages of safety, high immune response and low production cost, and has been approved for marketing in the United States and the European Union. In recent years, studies have found that transient protein expression induced by mRNA has great application value in many fields such as other infectious disease vaccines, cancer vaccines, cardiovascular diseases, protein replacement therapy, and genetic diseases, and can even achieve autonomous production of CAR through in vivo injection. -T effect. However, unlike the storage conditions of most vaccines (2-8°C), the marketed mRNA vaccines have poor long-term stability and require ultra-low temperature storage ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/19A61K9/51A61K39/00A61K39/12A61K47/26A61P31/14A61P31/16A61P31/20A61P31/22A61J3/02
CPCA61K9/19A61K9/5146A61K9/5123A61K47/26A61K39/00A61K39/12A61P31/14A61P31/20A61P31/16A61P31/22A61J3/02A61K2039/53A61K2039/54Y02A50/30
Inventor 王浩猛李明媛严志红贾琳刘健马文林邱东旭谢焱博宇学峰郁彭朱涛
Owner CANSINO BIOLOGICS INC
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