Low-temperature rapid mixing preparation method of mRNA vaccine lipid nanoparticles
By employing an antifreeze and protective agent system at low temperatures, combined with the turbulent mixing technology of a microfluidic chip, the problems of thermal degradation and particle size inhomogeneity in the preparation of lipid nanoparticles for mRNA vaccines were solved, achieving efficient and stable nanoparticle preparation.
Patent Information
- Application Number
- CN202511446657.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-11
- Publication Date
- 2026-03-20
AI Technical Summary
Existing methods for preparing lipid nanoparticles for mRNA vaccines are prone to thermal degradation of mRNA at room temperature, while low-temperature mixing can lead to problems such as ethanol crystallization clogging microchannels and uneven particle size. There is a lack of effective low-temperature antifreeze mechanisms.
A dual system of antifreeze and protectant was adopted to rapidly prepare lipid nanoparticles by microfluidic chip under low-temperature turbulent mixing conditions. The process included pre-cooling, low-temperature microfluidic mixing, low-temperature ripening, and buffer replacement to ensure the stability and particle size uniformity of mRNA.
It significantly reduces the risk of thermal degradation of mRNA, avoids ethanol crystallization blockage at low temperatures, achieves instantaneous self-assembly and efficient encapsulation of nanoparticles, and improves the integrity of mRNA secondary structure and colloidal stability of LNP.
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Figure CN121695102A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of mRNA vaccine lipid nanoparticle preparation, in particular to a low-temperature rapid mixing preparation method of mRNA vaccine lipid nanoparticles. BACKGROUND
[0002] The breakthrough of lipid nanoparticle (LNP) delivery technology has promoted the development of RNA therapy, and mRNA-LNP preparation is a key step from mRNA stock solution to finished preparation of mRNA vaccine, involving lipid preparation or screening, mRNA encapsulation / loading, purification and other processes.
[0003] The existing microfluidic mixing method needs to be carried out at room temperature of 20-25°C, and high temperature environment is easy to cause thermal degradation of mRNA, and long time exposure accelerates the degradation of mRNA and reduces the integrity; if low-temperature mixing is tried, ethanol phase crystallization will block the microchannel, and water phase ice crystal will shear mRNA; lacking antifreeze mechanism, the lipid solvent phase separates at low temperature, and the laminar flow mixing method leads to high particle size dispersion. At present, there are explorations of LNP preparation under low temperature conditions, but there are defects such as ethanol crystallization, channel blockage and uneven particle size.
[0004] In order to solve the above problems, the applicant first proposes to realize large-scale controllable preparation under low-temperature turbulent mixing conditions through an antifreeze agent and a protective agent double system, and proposes a low-temperature rapid mixing preparation method of mRNA vaccine lipid nanoparticles. SUMMARY
[0005] In order to solve the above technical problems, the present application provides the following technical solutions:
[0006] The present application provides a low-temperature rapid mixing preparation method of mRNA vaccine lipid nanoparticles, comprising the following steps:
[0007] S1. Precooling treatment of lipid phase: dissolve ionized lipid, auxiliary lipid, cholesterol and PEG lipid in anhydrous ethanol containing antifreeze agent at a molar ratio of (45-55):(8-12):(36-40):(1-2), stir under inert gas protection until completely dissolved, then precool to 0-10°C and maintain constant temperature for more than 20 minutes, to ensure uniform dispersion of lipid molecules and no crystallization;
[0008] S2. Precooling treatment of mRNA phase: dissolve target mRNA sequence in pre-cooled acid buffer (pH 3.5-4.5), add low-temperature protective agent and RNase inhibitor, vortex mix at 0-10°C, then stand for equilibrium, to form a uniform aqueous system;
[0009] S3. Low-temperature microfluidic mixing: lipid phase and mRNA phase are input into the mixing chamber at a set flow rate ratio through the constant-temperature microfluidic chip, to realize millisecond-level mixing in a turbulent state at 0-10°C, to trigger the directional self-assembly of lipid molecules;
[0010] S4. Low-temperature maturation: the mixed solution flows into the constant-temperature retention pipe, to complete the rearrangement of the lipid bilayer and the encapsulation of mRNA in a static environment at 0-10°C;
[0011] S5. Low-temperature buffer replacement: the tangential flow filtration system is used to replace the buffer at 0-10°C, to gradually replace it with a physiological isotonic buffer, to remove organic solvents and free components;
[0012] S6. LNP suspension stabilization: the LNP suspension after purification is adjusted to a target concentration, is filled with nitrogen and sealed, and is stored at 0-10°C in the dark.
[0013] As a preferred technical solution of the present application, the antifreeze additive amount is 10-20% of the volume of ethanol, 15% of propylene glycol is selected, the freezing point depression effect makes the solution still maintain fluidity at -20°C, and microchannel blockage is avoided.
[0014] As a preferred technical solution of the present application, the cryoprotective agent is dextran, the addition amount is 3-8% of the mass of the water phase, the acidic buffer is a sodium citrate buffer, a sodium acetate buffer or a histidine buffer, and the ionic strength is less than or equal to 50 mM, so as to reduce the salting-out effect at low temperature; the cryoprotective agent forms a glassy matrix to encapsulate mRNA, to block the mechanical damage of ice crystals; the hydrogen bond network stabilizes the secondary structure of mRNA, and the melting temperature is increased by greater than or equal to 5°C.
[0015] As a preferred technical solution of the present application, the microfluidic mixing adopts a Y-shaped microfluidic chip, the flow rate ratio of the lipid phase to the water phase is strictly controlled to be (2.5-3.5):1; the mixing chamber is made of chemically inert polycarbonate or glass, the inner wall is modified by a hydrophilic coating, and the contact angle is less than or equal to 30°, so as to ensure that the low-temperature solution does not adhere to the wall.
[0016] As a preferred technical solution of the present application, the mixing fluid dynamics parameters need to meet the requirements that the Reynolds number Re is greater than or equal to 2000, the Weber number We is greater than 100, and a strong turbulent flow with a vortex scale of less than or equal to 50 μm is formed, so that the two phases are mixed at the molecular level within 10 ms; the temperature is controlled by an embedded semiconductor cooling module, the precision is ±0.5°C, and the thermal conduction interface is filled with thermal conductive silicone grease to eliminate local hot spots.
[0017] As a preferred technical solution of the present application, the low-temperature maturation process is carried out in a serpentine retention pipe with a diameter of 5-10 mm, and the residence time is 3-10 minutes; during the maturation stage, the lipid molecules undergo a triple transformation, the ionized lipid hydrophilic head electrostatically combines with the mRNA phosphate skeleton, cholesterol fills the gap of the lipid bilayer to enhance the stability of the membrane, and PEG lipid migrates outward to form a steric hindrance layer.
[0018] As a preferred technical solution of the present application, the low-temperature buffer replacement adopts a tangential flow filtration system, the membrane material is regenerated cellulose, the molecular weight cut-off is 30-100 kDa, the pressure of the replacement buffer is 0.2-0.5 bar, and the transmembrane flow rate to feed flow rate ratio (TMP / CFV) is less than or equal to 0.3, which prevents membrane pollution caused by low temperature and high viscosity.
[0019] As a preferred technical solution of the present application, the process of replacing the buffer is divided into two stages: the first stage uses a pH 5.0 buffer to replace ethanol, and the second stage uses a pH 7.4 phosphate buffer to adjust to physiological osmotic pressure; the replacement volume ratio is greater than or equal to 20:1, and the residual ethanol amount is within 0.01%.
[0020] As a preferred technical solution of the present application, the whole process operating temperature needs to be within 8°C, and the time from the beginning of mixing to the end of buffer replacement needs to be within 3 hours; the particle size of the obtained LNP is dynamically scattered and checked within 70-90 nm, the PDI is less than or equal to 0.15, the encapsulation rate is greater than or equal to 95% detected by fluorescence dye quenching method, and the endotoxin is less than 0.1 EU / mg detected by limulus reagent.
[0021] As a preferred technical solution of the present application, the mRNA-LNP prepared by the method has a particle size of 70-90 nm, a PDI ≤0.15, and an encapsulation rate ≥95%.
[0022] The mRNA vaccine lipid nanoparticle low-temperature rapid mixing preparation method has the advantages of low-temperature whole process operation, which greatly reduces the risk of thermal degradation of mRNA, protects the mRNA ice crystal damage through the synergistic effect of the antifreeze protective agent, inhibits ethanol phase crystallization, prevents low-temperature mixing channel blockage, realizes turbulent mixing through a Y-shaped microfluidic chip, realizes nanoscale instantaneous self-assembly, significantly improves the particle size uniformity, and maintains the integrity of the mRNA secondary structure and the colloidal stability of the LNP to the greatest extent. BRIEF DESCRIPTION OF DRAWINGS
[0023] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, which together with the embodiments of the present application, is used to explain the present application, and does not constitute a limitation on the present application. In the drawings:
[0024] Figure 1is a system flow chart of a low-temperature rapid mixing preparation method of an mRNA vaccine lipid nanoparticle of the present application;
[0025] Figure 2 is a system flow chart of a low-temperature rapid mixing preparation method of an mRNA vaccine lipid nanoparticle of the present application Figure 2 ;
[0026] Figure 3 is a low-temperature preparation core process parameter table of a low-temperature rapid mixing preparation method of an mRNA vaccine lipid nanoparticle of the present application;
[0027] Figure 4 is a LNP finished product quality index table of a low-temperature rapid mixing preparation method of an mRNA vaccine lipid nanoparticle of the present application; DETAILED DESCRIPTION
[0028] The preferred embodiments of the present application are described below in conjunction with the accompanying drawings, and it should be understood that the preferred embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.
[0029] As Figures 1-4 shown, a low-temperature rapid mixing preparation method of an mRNA vaccine lipid nanoparticle includes the following steps:
[0030] S1. Lipid phase precooling treatment: ionized lipids, auxiliary lipids, cholesterol and PEG lipids are dissolved in anhydrous ethanol containing an antifreeze agent at a molar ratio of (45-55):(8-12):(36-40):(1-2), stirred to complete dissolution under inert gas protection, then pre-cooled to 0-10°C and maintained at a constant temperature for more than 20 minutes, to ensure uniform dispersion of lipid molecules and no crystallization;
[0031] S2. mRNA phase precooling treatment: the target mRNA sequence is dissolved in a pre-cooled acidic buffer (pH 3.5-4.5), and a low-temperature protective agent and an RNase inhibitor are added, and after vortex mixing at 0-10°C, the system is balanced to form a uniform aqueous phase system;
[0032] S3. Low-temperature microfluidic mixing: the lipid phase and the mRNA phase are input into the mixing chamber at a set flow rate ratio through a constant-temperature microfluidic chip, and the millisecond-level mixing is realized under the condition of 0-10°C and turbulent flow, triggering the directional self-assembly of lipid molecules;
[0033] S4. Low-temperature maturation: the mixed solution flows into a constant-temperature retention tube to complete the reorganization of the lipid membrane and the encapsulation of mRNA in a static environment at 0-10°C;
[0034] S5. Low-temperature buffer replacement: a tangential flow filtration system is used to replace the buffer at 0-10°C, and the buffer is gradually replaced with a physiological isotonic buffer to remove organic solvents and free components;
[0035] S6. LNP suspension stabilization: Adjust the purified LNP suspension to the target concentration, seal with nitrogen and store at 0-10 °C in the dark.
[0036] Antifreeze agent is added in the amount of 10-20% of the volume of ethanol, and 15% propylene glycol is selected. The freezing point depression effect makes the solution remain fluid at -20 °C, avoiding microchannel blockage.
[0037] The cryoprotectant is dextran, and the addition amount is 3-8% of the mass of the aqueous phase. The acidic buffer solution is sodium citrate, sodium acetate or histidine buffer solution, and the ionic strength is less than or equal to 50 mM, in order to reduce the salting-out effect at low temperature; the cryoprotectant forms a glassy matrix to encapsulate mRNA, blocking the mechanical damage of ice crystals; the hydrogen bond network stabilizes the mRNA secondary structure, and the melting temperature is increased by greater than or equal to 5 °C.
[0038] Microfluidic mixing uses a Y-shaped microfluidic chip, and the flow rate ratio of the lipid phase to the aqueous phase is strictly controlled at (2.5-3.5):1; the mixing cavity material is chemically inert polycarbonate or glass, and the inner wall is modified by hydrophilic coating with a contact angle less than or equal to 30°, ensuring that the low-temperature solution does not adhere to the wall.
[0039] The mixing hydrodynamic parameters need to satisfy the Reynolds number Re greater than or equal to 2000 and the Weber number We greater than 100, forming a strong turbulent flow with a vortex scale less than or equal to 50 μm, so that the two phases are mixed at the molecular level within 10 ms; the temperature is controlled by an embedded semiconductor cooling module with an accuracy of ±0.5 °C, and the thermal conduction interface is filled with thermal conductive silicone grease to eliminate local hot spots.
[0040] The low-temperature maturation process is carried out in a serpentine retention pipe with a diameter of 5-10 mm, and the residence time is 3-10 minutes; during the maturation stage, the lipid molecules undergo a triple transition, the hydrophilic head of the ionized lipid is electrostatically combined with the mRNA phosphate backbone, the cholesterol fills the gap between the lipid bilayer to enhance the stability of the membrane, and the PEG lipid migrates outward to form a steric hindrance layer.
[0041] Low-temperature buffer replacement uses a tangential flow filtration system, the membrane material is regenerated cellulose, the molecular weight cutoff is 30-100 kDa, the pressure of the replacement buffer is 0.2-0.5 bar, and the transmembrane flow rate to the feed flow rate ratio (TMP / CFV) is less than or equal to 0.3, preventing membrane contamination caused by high viscosity at low temperature.
[0042] The process is divided into two stages: the first stage uses a pH 5.0 buffer to replace ethanol, and the second stage uses a pH 7.4 phosphate buffer to adjust to physiological osmotic pressure; the replacement volume ratio is greater than or equal to 20:1 throughout the process, and the residual ethanol amount needs to be within 0.01%.
[0043] The whole process operating temperature needs to be within 8°C, from the beginning of mixing to the end of buffer replacement, the time consumption needs to be within 3 hours; the particle size of the obtained LNP needs to be within 70-90 nm by dynamic light scattering, the PDI is less than or equal to 0.15, the encapsulation rate is greater than or equal to 95% measured by fluorescence dye quenching method, and the endotoxin is less than 0.1 EU / mg detected by limulus reagent.
[0044] Example 1: Low-temperature preparation
[0045] The mRNA is dissolved in Tris buffer containing 5% sucrose (4°C), and the lipids are dissolved in ethanol (4°C), wherein the molar ratio of the lipids is: ionizable cationic lipid: DSPC: cholesterol: DMG-PEG2000 = 50:10:38:2.
[0046] The preliminary nanoparticles are obtained by rapid mixing at a volume ratio of 1:3 through a Y-shaped microfluidic chip at 4°C.
[0047] Table 1: Low-temperature preparation process condition table
[0048]
[0049] Example 2: Comparative experiment
[0050] Room temperature mixing: the average particle size of the obtained LNP is 145 nm, the PDI is 0.28, and the encapsulation rate is 82%;
[0051] Low-temperature mixing (the present application): the average particle size of the LNP is 82 nm, the PDI is 0.12, and the encapsulation rate is 95%.
[0052] Table 2: Comparative experiment data table
[0053]
[0054] From the above experiment, it can be seen that the LNP prepared by the method has uniform particle size and high encapsulation rate, which is significantly better than the room temperature process.
[0055] Example 3: Stability test
[0056] The mRNA-LNP obtained in Example 1 is stored at 4°C for one week, one month, and stored at -20°C for 1 month, 3 months, and 6 months, and the particle size change is less than 10%, and the encapsulation rate is maintained above 90%.
[0057] Table 3: Stability test experiment data table
[0059] From the above experiment, it can be seen that the LNP product prepared by the method has good stability at 4°C and -20°C.
[0060] Example 4: Batch repeatability test
[0061] Five batches of mRNA-LNP were prepared successively according to the procedure of Example 1, and the particle size, PDI, and encapsulation efficiency were measured respectively. The results of the five times are shown in Table 4, which shows that the method has good repeatability.
[0062] Table 4 Repeatability results of 5 batches
[0063]
[0064] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.
Claims
1. A method for the low-temperature rapid mixing preparation of mRNA vaccine lipid nanoparticles, characterized in that, Includes the following steps: S1. Pre-cooling treatment of lipid phase: Ionized lipids, auxiliary lipids, cholesterol and PEG lipids are dissolved in anhydrous ethanol containing antifreeze at a molar ratio of (45-55):(8-12):(36-40):(1-2). The mixture is stirred under inert gas protection until completely dissolved, and then pre-cooled to 0-10°C to ensure that the lipid molecules are uniformly dispersed and no crystals are precipitated. S2. Pre-cooling treatment of mRNA phase: Dissolve the target mRNA in pre-cooled acidic buffer (pH 3.5-4.5), add cryoprotectant and RNase inhibitor, vortex mix at 0-10°C and let stand to equilibrate to form a homogeneous aqueous phase system; S3. Low-temperature microfluidic mixing: The lipid phase and mRNA phase are input into the mixing chamber at a set flow rate ratio through a constant-temperature microfluidic chip, achieving millisecond-level mixing in turbulent conditions at 0-10°C, triggering the directional self-assembly of lipid molecules; S4. Low-temperature curing: The mixture flows into a constant-temperature retention tube and completes lipid membrane recombination and mRNA encapsulation in a static environment of 0-10°C. S5. Low-temperature buffer replacement: A tangential flow filtration system is used to replace the buffer at 0-10°C, gradually replacing it with physiological isotonic buffer, removing organic solvents and free components; S6. Stabilization of LNP suspension: Adjust the purified LNP suspension to the target concentration, seal it with nitrogen, and store it at 0-10°C in the dark.
2. The low-temperature rapid mixing preparation method for mRNA vaccine lipid nanoparticles according to claim 1, characterized in that, The amount of antifreeze added is 10-20% of the volume of ethanol, and 15% propylene glycol is selected. The freezing point inhibition effect allows the solution to remain fluid at -20°C, avoiding microchannel blockage.
3. The low-temperature rapid mixing preparation method for mRNA vaccine lipid nanoparticles according to claim 1, characterized in that, The cryoprotectant is dextran, added at 3-8% of the aqueous phase mass. The acidic buffer is sodium citrate, sodium acetate, or histidine buffer with an ionic strength of less than or equal to 50 mM to reduce the salting-out effect at low temperatures. The cryoprotectant forms a glassy matrix to encapsulate mRNA and prevent mechanical damage from ice crystals. The hydrogen bond network stabilizes the secondary structure of mRNA, increasing the melting temperature by more than or equal to 5°C.
4. The method for low-temperature rapid mixing preparation of mRNA vaccine lipid nanoparticles according to claim 1, characterized in that, The microfluidic mixing uses a Y-type microfluidic chip, and the flow rate ratio of the lipid phase to the aqueous phase is strictly controlled at (2.5-3.5):
1. The mixing chamber is made of chemically inert polycarbonate or glass, and the inner wall is modified with a hydrophilic coating with a contact angle of less than or equal to 30° to ensure that the low-temperature solution does not adhere to the wall surface.
5. The method for low-temperature rapid mixing preparation of mRNA vaccine lipid nanoparticles according to claim 4, characterized in that, The mixing fluid dynamics parameters must meet the following requirements: Reynolds number Re greater than or equal to 2000, Weber number We greater than 100, forming strong turbulence with vortex scale less than or equal to 50 μm, enabling molecular-level mixing of the two phases within 10 ms; the temperature is controlled by an embedded semiconductor cooling module with an accuracy of ±0.5°C, and the thermal interface is filled with thermally conductive silicone grease to eliminate local hot spots.
6. The method for low-temperature rapid mixing preparation of mRNA vaccine lipid nanoparticles according to claim 1, characterized in that, The low-temperature curing process is carried out in a serpentine retention tube with a diameter of 5-10 mm and a residence time of 3-10 minutes. During the curing stage, lipid molecules undergo a triple transformation: the hydrophilic head of ionized lipids electrostatically binds to the phosphate backbone of mRNA, cholesterol fills the gaps in the lipid bilayer to enhance membrane stability, and PEG lipids migrate outward to form a steric hindrance layer.
7. The low-temperature rapid mixing preparation method for mRNA vaccine lipid nanoparticles according to claim 1, characterized in that, The low-temperature buffer replacement adopts a tangential flow filtration system with a membrane material of regenerated cellulose, a molecular weight cutoff of 30-100 kDa, a pressure of 0.2-0.5 bar, and a transmembrane flow rate to feed flow rate ratio (TMP / CFV) of less than or equal to 0.3 to prevent membrane fouling caused by low temperature and high viscosity.
8. The method for low-temperature rapid mixing preparation of mRNA vaccine lipid nanoparticles according to claim 7, characterized in that, The buffer replacement process consists of two stages: the first stage uses pH 5.0 buffer to replace ethanol, and the second stage uses pH 7.4 phosphate buffer to adjust to physiological osmotic pressure; the total replacement volume ratio is greater than or equal to 20:1, and the residual ethanol content must be less than 0.01%.
9. The method for low-temperature rapid mixing preparation of mRNA vaccine lipid nanoparticles according to claim 1, characterized in that, The entire process must be carried out at a temperature below 8°C, and the time from mixing to buffer replacement must be within 3 hours. The particle size of the obtained LNPs is 70-90 nm as determined by dynamic light scattering, the PDI is less than or equal to 0.15, the encapsulation efficiency is greater than or equal to 95% as determined by fluorescent dye quenching, and the endotoxin content is less than 0.1 EU / mg as determined by Limulus amebocyte lysate (LAL) reagent.
10. The low-temperature rapid mixing preparation method for mRNA vaccine lipid nanoparticles according to claim 1, wherein the mRNA-LNP prepared by the method has a particle size of 70–90 nm, a PDI ≤0.15, and an encapsulation efficiency ≥95%.