Process for large-scale purification of mRNA vaccines using gradient pore structure hollow fiber ultrafiltration membrane

Abstract

The present invention relates to a process for large-scale purification of mRNA vaccines using a gradient pore structure hollow fiber ultrafiltration membrane, which belongs to the technical field of vaccine production and purification, and comprises the following steps: (1) plasmid DNA purification: taking a cultured bacterial solution and performing cell lysis, and after lysis, using precipitation and / or salting-out treatment in combination with filtration using a gradient pore structure hollow fiber ultrafiltration membrane to obtain purified plasmid DNA; (2) mRNA purification: after the purified plasmid DNA is transcribed in vitro and modified with capping and tailing nucleotides to obtain target mRNA, DNA enzyme is added, and precipitation and / or salting-out treatment is used in combination with filtration using a gradient pore structure hollow fiber ultrafiltration membrane to obtain purified mRNA; (3) purification after mRNA encapsulation or loading: after the purified mRNA is encapsulated or loaded, precipitation and / or salting-out treatment is used in combination with filtration using a gradient pore structure hollow fiber ultrafiltration membrane to obtain an mRNA vaccine. The method of the present invention can reduce the use of organic solvents and is conducive to the large-scale, continuous and low-cost purification of mRNA vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine production and purification, and specifically relates to a process for large-scale purification of mRNA vaccines using a hollow fiber ultrafiltration membrane with a gradient pore structure. Background Art

[0002] mRNA vaccines involve introducing RNA encoding an antigen into the human body, where it is directly translated to produce the corresponding antigenic protein, thereby inducing a specific immune response and achieving preventive immunity. These vaccines have a relatively recent development history, with commercial products only beginning to be marketed in 2020. They utilize only viral genetic sequences and contain no viral components, posing no risk of infection. The human body uses mRNA as a template to translate the antigenic protein, stimulating an immune response and providing immune protection. The in vitro transcribed mRNA is ultimately degraded naturally by normal cellular metabolism.

[0003] The mRNA vaccine production process involves constructing a vector containing the antigen protein DNA, transferring it into bacterial culture to obtain plasmid DNA, in vitro transcription of the plasmid DNA, capping and tailing, and nucleotide modification to produce mRNA, which is then encapsulated using polymers or lipid nanoparticles to produce the vaccine. The production process is accompanied by impurities such as proteins, endotoxins, enzymes, free nucleotides, ribonucleoside triphosphates, and small DNA fragments, which are key factors limiting the purity of mRNA vaccines and causing significant side effects after clinical use.

[0004] Currently, the following methods are mainly used to remove impurities during the mRNA production process: for example, for the purification of plasmid DNA, the phenol-chloroform method can be used for extraction, but the use of excessive organic solvents is not conducive to improving the overall quality of the vaccine; centrifugation can also be used to separate plasmid DNA, but the centrifugation method cannot be operated continuously on a large scale, and cannot achieve large-scale purification of mRNA vaccines.

[0005] Therefore, reducing the use of organic solvents and achieving low-cost continuous operation are of great significance for the large-scale purification of mRNA vaccines. Summary of the Invention

[0006] The present invention discloses a process method for large-scale purification of mRNA vaccines using a hollow fiber ultrafiltration membrane with a gradient pore structure, which is conducive to the continuous, large-scale and stable purification of mRNA vaccines while reducing the use of organic reagents.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The process for large-scale purification of mRNA vaccines using a gradient pore structure hollow fiber ultrafiltration membrane comprises the following steps:

[0009] (1) Plasmid DNA purification:

[0010] The cultured bacterial solution is subjected to cell lysis, and after lysis, precipitation and / or salting-out treatment is performed in combination with filtration through a hollow fiber ultrafiltration membrane with a gradient pore structure to obtain purified plasmid DNA;

[0011] (2) mRNA purification:

[0012] Purified plasmid DNA is transcribed in vitro, capped, and tailed to obtain target mRNA, followed by the addition of DNase, and then subjected to precipitation and / or salting-out treatment combined with filtration through a gradient pore structure hollow fiber ultrafiltration membrane to obtain purified mRNA;

[0013] (3) Purification after mRNA encapsulation or loading:

[0014] After the purified mRNA is encapsulated or loaded, precipitation and / or salting-out treatment is used in combination with filtration through a gradient pore structure hollow fiber ultrafiltration membrane to obtain the mRNA vaccine.

[0015] The present invention adopts precipitation and / or salting-out treatment in combination with gradient pore structure hollow fiber ultrafiltration membrane filtration to purify mRNA vaccine at each stage of production. The precipitation and / or salting-out treatment reagents and parameters and gradient pore structure hollow fiber ultrafiltration membrane filtration parameters can be adjusted according to the impurities that need to be removed in each step. It has strong adaptability and can reduce the use of organic reagents compared with traditional purification methods. The final product has high purity and is suitable for continuous large-scale production.

[0016] Furthermore, in steps (1)-(3),

[0017] The material of the gradient pore structure hollow fiber ultrafiltration membrane is at least one of polyvinylidene fluoride PVDF, polyethersulfone PES, polysulfone PSF, sulfonated polyethersulfone SPES, sulfonated polysulfone SPSF, modified polyethersulfone mPES, modified polysulfone mPSF, and polyacrylonitrile PAN, and the cut-off molecular weight range of the gradient pore structure hollow fiber ultrafiltration membrane is 50KDa-750KDa.

[0018] Furthermore, in steps (1)-(3),

[0019] Internal pressure hollow fiber membrane modules are used for filtration. The diameter of the gradient pore structure hollow fiber ultrafiltration membrane in the module is 0.1-4 mm, and the ratio of the inner and outer diameters is 1.2-4.

[0020] The filtration method is tangential flow filtration or dead-end filtration;

[0021] Dead-end filtration pressure is 0.05-0.3MPa, tangential flow filtration flow rate is 15-150L / (h﹒m 2 );

[0022] After filtering in any of steps (1) to (3), backwashing is performed using reagent I;

[0023] The reagent I used for backwashing includes at least one of hydrochloric acid buffer Tris-HCl, phosphate buffer Tris-H3PO4, ethylenediaminetetraacetic acid buffer Tris-EDTA, and acetate buffer Tris-CH3COOH. The concentration range of the reagent I is 0.1 mol / L to 1 mol / L, and the pH range is 7-10.

[0024] Furthermore, in step (1), precipitation and / or salting-out treatment is combined with filtration through a gradient pore structure hollow fiber ultrafiltration membrane to remove cell debris, proteins, host genomic DNA, RNA, and endotoxins;

[0025] In step (2), precipitation and / or salting-out treatment is combined with filtration through a gradient pore structure hollow fiber ultrafiltration membrane to remove enzymes, free nucleotides, ribonucleoside triphosphates, immunogenic impurities in the reaction system, exogenous RNA fragments, dsRNA, and small DNA fragments;

[0026] In step (3), precipitation and / or salting-out treatment is combined with filtration through a gradient pore structure hollow fiber ultrafiltration membrane to remove unencapsulated or unloaded mRNA and other impurities.

[0027] Furthermore, in step (1),

[0028] After cell lysis, the protein is denatured and precipitated, and the supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane to remove cell debris and endotoxins to obtain concentrated solution I;

[0029] The host genomic DNA in the concentrated solution I is precipitated, and the supernatant is filtered through a gradient pore structure hollow fiber ultrafiltration membrane to obtain concentrated solution II;

[0030] The RNA in the concentrated solution II was precipitated, and the supernatant was filtered through a gradient pore structure hollow fiber ultrafiltration membrane to obtain concentrated solution III;

[0031] The plasmid DNA in the concentrated solution III is precipitated, the precipitated plasmid DNA is redissolved and filtered 2-3 times, and filtered using a gradient pore structure hollow fiber ultrafiltration membrane to obtain the final concentrated solution to obtain purified plasmid DNA.

[0032] Furthermore, in step (1),

[0033] The reagent used for cell lysis is reagent II, including at least one of potassium hydroxide KOH, sodium hydroxide NaOH, sodium citrate, sodium methoxide CH3ONa, and potassium ethoxide CH3COOK. The concentration of reagent II is 0.01-5 mol / L, and the treatment time is 5-200 min.

[0034] The reagents used for protein precipitation include reagent I, which includes at least one of hydrochloric acid buffer Tris-HCl, sulfuric acid buffer Tris-H2SO4, phosphate buffer Tris-H3PO4, ethylenediaminetetraacetic acid buffer Tris-EDTA, and acetate buffer Tris-CH3COOH, the pH range of reagent I is 7-10, and the treatment time is 5-120 minutes;

[0035] The reagent used to precipitate the host genomic DNA includes reagent III, which includes at least one of KPVPK15, PVPK30, PVPK60, PVPK90, PVPK120, polyvinyl alcohol (PVA), and ethylene glycol. The concentration of reagent III is 0.001-1 mol / L, and the treatment time of reagent III is 10-60 min.

[0036] The reagent used for precipitating RNA includes reagent IV, which includes at least one of calcium chloride (CaCl2), sodium chloride (NaCl), sodium nitrate (NaNO3), barium chloride (BaCl2), potassium nitrate (KNO3), and calcium nitrate (Ca(NO3)2). The concentration of reagent IV is 0.01-5 mol / L, and the treatment time of reagent IV is 10-180 min.

[0037] The reagent used for precipitating plasmid DNA in the retentate I includes reagent V, which includes at least one of PEG200, PEG400, PEG800, PEG1000, PEG2000, PEG4000, PEG8000, PEG10000, and PEG20000, with a concentration of reagent V of 0.01-5 mol / L and a treatment time of reagent V of 10-60 min.

[0038] Furthermore, in step (2),

[0039] The target mRNA solution is directly filtered through a gradient pore structure hollow fiber ultrafiltration membrane to remove free nucleotides, ribonucleoside triphosphates, and immunogenic impurities in the reaction system to obtain concentrated solution IV;

[0040] The obtained concentrated solution IV is treated with DNA enzyme, and then the enzyme and protein are salted out. After salting out, the supernatant is filtered through a hollow fiber ultrafiltration membrane with a gradient pore structure to obtain concentrated solution V;

[0041] The exogenous RNA in the concentrated solution V is precipitated, and the supernatant is filtered through a gradient pore structure hollow fiber ultrafiltration membrane to obtain a concentrated solution VI;

[0042] The dsRNA in the concentrated solution VI is precipitated, and the supernatant is filtered through a gradient pore structure hollow fiber ultrafiltration membrane to obtain concentrated solution VII;

[0043] The mRNA in the concentrated solution VII is subjected to salt precipitation and directly filtered through a gradient pore structure hollow fiber ultrafiltration membrane to obtain a concentrated solution. The mRNA in the concentrated solution is redissolved and filtered 2-3 times to obtain the final concentrated solution to obtain purified mRNA.

[0044] Furthermore, in step (2),

[0045] The reagent used for the salting-out enzyme includes reagent VI, which includes at least one of sodium dodecylsulfonate, sodium dodecylbenzenesulfonate, sodium hexadecylsulfonate, sodium hexadecylbenzenesulfonate, and sodium ligninsulfonate. The concentration of reagent VI is 0.01-5 mol / L, and the treatment time of reagent VI is 10-60 min.

[0046] The reagent used to precipitate exogenous RNA includes reagent VII, which includes at least one of colchicine, 2-hydroxyethylamine, triisopropanolamine, tert-butylamine, and isobutylamine. The concentration of reagent VII is 0.01-2 mol / L, and the treatment time of reagent VII is 10-40 min.

[0047] The reagent used for precipitating dsRNA includes reagent VIII, which includes at least one of ethylene glycol, ethanol, n-butanol, glycerol, acetone, and propanol. The concentration of reagent VIII is 0.01-5 mol / L, and the treatment time of reagent VIII is 10-100 minutes.

[0048] The reagent used for salting out mRNA includes reagent IX, which includes at least one of ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, ammonium carbonate, and ammonium bicarbonate. The concentration of reagent IX is 0.01-5 mol / L, and the treatment time of reagent IX is 10-180 min.

[0049] Furthermore, in step (3),

[0050] The mRNA encapsulated or loaded solution is directly filtered through a gradient pore structure hollow fiber ultrafiltration membrane to remove other small molecular impurities to obtain concentrated solution VIII;

[0051] The unencapsulated or unloaded mRNA in the concentrate VIII is subjected to salting out. After salting out, the supernatant is filtered using a gradient pore structure hollow fiber ultrafiltration membrane to obtain a concentrate, which is the purified encapsulated / transferred mRNA.

[0052] Furthermore, in step (3),

[0053] The reagent used for salting out unencapsulated or unloaded mRNA includes reagent X, which includes at least one of tetramethylammonium chloride, dodecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, and spermidine. The concentration of reagent X is 0.01-5 mol / L, and the treatment time of reagent X is 10-100 min.

[0054] In summary, the method provided by the present invention can be applied to the continuous, large-scale, long-term and stable purification operation of mRNA vaccines from strain rupture to final encapsulation / transfer. Compared with the traditional mRNA vaccine purification processes at various stages, the method of the present invention is easy to implement industrially, has low overall purification costs, and high mRNA vaccine purity, providing an objective prospect for mRNA vaccine purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Shown is the SEM image of a hollow fiber ultrafiltration membrane with a gradient pore structure made of PVDF material and a cut-off molecular weight of 750KDa.

[0056] Figure 2 Shown is the SEM image of a hollow fiber ultrafiltration membrane with a gradient pore structure made of mPES material and a cut-off molecular weight of 300KDa. DETAILED DESCRIPTION

[0057] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0058] The gradient pore structure hollow fiber ultrafiltration membranes used in the examples are all manufactured by Tianjin Dingxin Membrane Technology Co., Ltd. This product is manufactured according to the company's patent CN115090134A. Figure 1 、 2 shown.

[0059] In the embodiment, the hydrochloric acid buffer is Tris-HCl, the sulfuric acid buffer is Tris-H2SO4, the phosphate buffer is Tris-H3PO4, and the acetate buffer is Tris-CH3COOH.

[0060] The E. coli stock solutions used in Examples 1-9 were all of the same strain, DH5a, and the concentrations were all 5-9*10 7 CFU / ml, and DNA enzyme model D806930, Shanghai MacLean Biochemical Technology Co., Ltd.

[0061] Example 1:

[0062] Step 1: Purification of plasmid DNA

[0063] Take 1000g of Escherichia coli stock solution, add 5L of 0.3mol / LKOH solution, mix thoroughly to fully lyse the bacterial cells, then add 1mol / L, pH=6.5 hydrochloric acid buffer, adjust the pH of the solution to 8.5-9.2, mix evenly for 30 minutes to allow the protein to denature and precipitate completely. The supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 1.8, model UC-PVDF750) with tangential flow. The membrane material is PVDF ( Figure 1 ), the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 750KDa, the pressure is 0.05MPa, and the flux of the tangential flow filtration is 25L / (h·m 2 ). Cell debris and endotoxins were removed to obtain a concentrated solution I. After each batch of liquid was processed, the ultrafiltration membrane was backwashed with hydrochloric acid buffer and ultrapure water, and then sterilized with alkali for later use.

[0064] To the obtained concentrated solution I, 0.8 mol / L PVPK302L reagent was added and mixed evenly. Then, 0.2 mol / L phosphate buffer with a pH of 7.5 was added and the pH of the solution was adjusted to 9.2-9.6. The solution was mixed evenly for 20 minutes to allow the host genomic DNA to precipitate completely. The supernatant was extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 1.6, model UC-PAN500) with tangential flow filtration. The membrane material was PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 500 KDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 35 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0065] 800 mL of 4 mol / L NaNO3 solution was added to the concentrated solution II and mixed evenly. Then, 1 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.3-9.7. The solution was mixed evenly for 25 minutes to allow the RNA to precipitate completely. The supernatant was extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1.5 mm, inner and outer diameter ratio of 2, model UC-PVDF300) by tangential flow filtration. The membrane material was PVDF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 300 KDa, the pressure was 0.05 MPa, and the flux of the tangential flow filtration was 20 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 1 mol / L, pH 7.8 hydrochloric acid buffer and ultrapure water.

[0066] At this point, the main substance in the concentrated solution III is plasmid DNA. For further purification, the following operation is performed. 0.9 mol / L PEG80002L is added to the concentrated solution III, mixed evenly, and then 0.2 mol / L phosphate buffer, pH = 7.5, is added to adjust the solution pH to 7.7-7.9. After mixing evenly for 25 minutes, the plasmid DNA is precipitated. The gradient pore structure mPES hollow fiber ultrafiltration membrane ( Figure 2 , diameter 1mm, inner and outer diameter ratio is 2, model UC-mPES300) dead-end filtration, hollow fiber ultrafiltration membrane cut-off molecular weight is 300KDa, pressure is 0.1MPa, dead-end filtration flux is 30L / (h·m 2 ) to obtain a concentrate, repeat the operation three times according to the above parameters, and re-dissolve the concentrate for the final time and continue to filter to obtain a high-purity plasmid DNA stock solution for in vitro transcription, capping, and tailing to produce the target mRNA.

[0067] Among them, linearized plasmid DNA is used as a template, T7 promoter or SP6 promoter is utilized, NTP is used as a substrate, and RNA polymerase is used to synthesize mRNA and then modify it; the mRNA capping process mainly involves adding 7-methylguanosine (m7G) to the first nucleotide at the 5′ end of the mRNA, which can protect the mRNA from degradation, increase its reproducibility and improve splicing efficiency. It is a key factor in promoting ribosome targeting to mRNA for translation. Capping is performed by capping enzyme, S-adenosylmethionine (SAM) and GTP. The enzymatic method uses the cowpox virus capping system and a natural cap. The system contains RNA triphosphatase, guanine methyltransferase and guanine methyltransferase, methyl donor SAM, GTP and reaction buffer. This reaction converts Cap 0 mRNA into Cap 1 mRNA. The reaction time is short, the cap structure is added in the correct direction, and the capping efficiency is close to 100%, which is highly efficient. The Poly A tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. The Poly A tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. By designing a sequence encoding a fixed length in the template, the target mRNA is directly transcribed in vitro.

[0068] After each batch of liquid is processed, the ultrafiltration membrane is backwashed with hydrochloric acid buffer and ultrapure water, and then sterilized with alkali and set aside.

[0069] The purity of the tested plasmid DNA was 96.8%.

[0070] Step 2: mRNA purification

[0071] The target mRNA solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner / outer diameter ratio of 1.2, model UC-mPES100). The membrane material was mPES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.05 MPa, and the flux of tangential flow filtration was 20 L / h / m 2 , remove free nucleotides, ribonucleoside triphosphates, immunogenic impurities and other small molecular impurities to obtain concentrated solution IV. After each batch of liquid treatment, the ultrafiltration membrane is backwashed with 0.2 mol / L, pH = 7.5 phosphate buffer and ultrapure water.

[0072] 5 g of DNA enzyme was added to the concentrate IV to fully degrade the residual DNA fragments, and then 2 L of 1 mol / L sodium dodecylbenzene sulfonate solution was added. After mixing for 20 minutes, 0.2 mol / L phosphate buffer with a pH of 7.5 was added to adjust the pH of the solution to 8.5-8.9. After mixing for 30 minutes, the enzyme and other proteins in the solution were completely precipitated by salting out. The supernatant was extracted and tangentially filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES / SPES150). The membrane material was PES / SPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 150 KDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 20 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH 7.5 phosphate buffer and ultrapure water.

[0073] 500 mL of 3 mol / L triisopropanolamine was added to the concentrated solution V and mixed evenly. Then, 0.2 mol / L phosphate buffer with a pH of 7.5 was added to adjust the pH of the solution to 8.5-8.8. After mixing evenly for 30 minutes, the exogenous RNA in the solution was completely precipitated. The supernatant was extracted and filtered by tangential flow through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-mPSF100). The membrane material was mPSF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.09 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0074] 1000 mL of 1 mol / L glycerol was added to the concentrated solution VI and mixed well. Then, 0.5 mol / L acetic acid buffer with a pH of 3.5 was added to adjust the pH of the solution to 8.2-8.4. After mixing for 20 minutes, the dsRNA in the solution was completely precipitated. The supernatant was extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner / outer diameter ratio of 2, model UC-PES100) with tangential flow filtration. The membrane material was PES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.05 MPa, and the flux of the tangential flow filtration was 20 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L acetic acid buffer solution with pH=3.5 and ultrapure water.

[0075] At this time, the main substance in the concentrated solution VII is mRNA. For further purification, the following operation is adopted. Add 400mL of 1mol / L ammonium chloride to the obtained concentrated solution VII, mix well, then add 0.5mol / L, pH=3.5 acetic acid buffer, adjust the solution pH to 7.8-8.1, mix well for 20 minutes, and then allow the mRNA to salt out. Use a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PES100) for dead-end filtration. The membrane material is PES, the hollow fiber ultrafiltration membrane cut-off molecular weight is 100KDa, the pressure is 0.15MPa, and the dead-end filtration flux is 30L / (h·m 2 ) to obtain a concentrate; the operation was repeated 4 times according to the above parameters, and the concentrate was re-dissolved and filtered to obtain a high-purity target mRNA stock solution for LNP encapsulation / loading.

[0076] Among them, the assembly and formation of LNPs are driven by hydrophobic and electrostatic forces. First, four lipids (dissociated lipids, saturated phospholipids, cholesterol lipids, and polyethylene glycol lipids) are dissolved in ethanol. Two volumes of lipid-containing ethanol solution are usually mixed with five volumes of mRNA in an acetate aqueous buffer at pH = 4. When the lipids come into contact with the aqueous buffer, they become insoluble in the 3:4 water / ethanol solvent, and the ionizable lipids become protonated and positively charged, which then drives it to electrostatically bind to the negatively charged mRNA phosphate backbone. At the same time, the lipids become insoluble, forming lipid particles that encapsulate the mRNA in the aqueous suspension. When the aqueous and lipid phases mix, the ionized lipids are protonated when the mixed phase pH approaches 5.8. When the mixed phase pH approaches 6.9, the mRNA is encapsulated. Subsequently, by dilution or dialysis to pH = 7.8, uncharged LNP nanolipid particles are formed.

[0077] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0078] The purity of the tested mRNA was 99.2%.

[0079] Step 3: mRNA encapsulation / loading and purification (fluid exchange and concentration)

[0080] The encapsulated solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.6, model UC-PES100). The membrane material was PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.2 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 ), removing small molecular impurities such as EDTA, sodium chloride, ethanol and sodium acetate to obtain concentrated solution VIII. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0081] 1000 mL of 1 mol / L dodecyltrimethylammonium bromide was added to the concentrate VIII and mixed evenly. Then, 1 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 8.2-8.5. After mixing evenly for 20 minutes, the unencapsulated or unloaded mRNA was completely salted out. The supernatant was extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 1.75, model UC-PAN150) with tangential flow filtration. The membrane material was PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.15 MPa, and the flux of the tangential flow filtration was 20 L / (h·m 2 After each batch of liquid is processed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH 3.5 acetic acid buffer and ultrapure water.

[0082] The purity of the encapsulated mRNA was tested to be 99.2%.

[0083] Example 2

[0084] Step 1: Purification of plasmid DNA

[0085] Take 1000g of Escherichia coli stock solution, add 3L of 1mol / L sodium citrate solution, mix thoroughly to fully lyse the bacterial cells, then add 0.2mol / L, pH=7.5 phosphate buffer, adjust the pH of the solution to 8.8-9.0, mix evenly for 30 minutes to completely denature and precipitate the protein. Extract the supernatant, and use a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PVDF750) for tangential flow filtration. The membrane material is PVDF, the hollow fiber ultrafiltration membrane has a cut-off molecular weight of 750KDa, the pressure is 0.05MPa, and the flux of tangential flow filtration is 35L / (h·m 2 ). Cell debris and endotoxins were removed to obtain a concentrated solution I. After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH = 7.5 phosphate buffer and ultrapure water.

[0086] To the obtained concentrated solution I, 200 g of reagent PVPK90 was added and mixed evenly. Then, 0.2 mol / L phosphate buffer with a pH of 7.5 was added and the pH of the solution was adjusted to 10.2-10.5. The solution was mixed evenly for 30 minutes to precipitate the host genomic DNA. The supernatant was extracted and tangentially filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 1.6, model UC-PAN500). The membrane material was PAN, the molecular weight cutoff of the hollow fiber ultrafiltration membrane was 500 KDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 15 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L acetic acid buffer solution with pH=3.5 and ultrapure water.

[0087] 1 L of 3 mol / L CaCl2 solution was added to the concentrated solution II and mixed well. Then, 1 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.2-9.8. After mixing well for 15 minutes, the RNA was precipitated. The supernatant was extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1.5 mm, inner and outer diameter ratio of 2, model UC-PVDF300) with tangential flow filtration. The membrane material was PVDF, the molecular weight cutoff of the hollow fiber ultrafiltration membrane was 300 KDa, the pressure was 0.08 MPa, and the flow rate of the tangential flow filtration was 25 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0088] At this point, the main substance in concentrated solution III was plasmid DNA. For further purification, the following procedure was employed: 500 g of PEG2000 was added to the concentrated solution III obtained and mixed uniformly. 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was then added to adjust the solution pH to 7.6-8.0. After mixing uniformly for 15 minutes, the plasmid DNA was salted out and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (1 mm in diameter, inner / outer diameter ratio of 2, model UC-mPES300). The mPES hollow fiber ultrafiltration membrane had a molecular weight cutoff of 300 kDa. The pressure was 0.1 MPa, and the flux of the tangential flow filtration was 50 L / (h·m 2 ), and this operation is repeated twice. The final concentrate is redissolved and filtered to obtain a high-purity plasmid DNA stock solution for in vitro transcription, capping, and tailing to produce the target mRNA.

[0089] Among them, linearized plasmid DNA is used as a template, T7 promoter or SP6 promoter is utilized, NTP is used as a substrate, and RNA polymerase is used to synthesize mRNA and then modify it; the mRNA capping process mainly involves adding 7-methylguanosine (m7G) to the first nucleotide at the 5′ end of the mRNA, which can protect the mRNA from degradation, increase its reproducibility and improve splicing efficiency. It is a key factor in promoting ribosome targeting to mRNA for translation. Capping is performed by capping enzyme, S-adenosylmethionine (SAM) and GTP. The enzymatic method uses the cowpox virus capping system and a natural cap. The system contains RNA triphosphatase, guanine methyltransferase and guanine methyltransferase, methyl donor SAM, GTP and reaction buffer. This reaction converts Cap 0 mRNA into Cap 1 mRNA. The reaction time is short and the cap structure is added in the correct direction. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. It is generated by direct transcription in vitro by designing a sequence encoding a fixed length in the template.

[0090] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=7.8 hydrochloric acid buffer and ultrapure water.

[0091] The purity of the tested plasmid DNA was 97%.

[0092] Step 2: mRNA purification

[0093] The target mRNA solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-mPES150). The membrane material was mPES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.05 MPa, and the flux of tangential flow filtration was 40 L / (h·m 2 ) to remove free nucleotides, ribonucleoside triphosphates, immunogenic impurities and other small molecular impurities to obtain concentrated solution IV. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH = 7.5 phosphate buffer and ultrapure water.

[0094] 5 g of DNA enzyme was added to the concentrated solution IV to fully dissolve the residual DNA fragments, and then 2 L of 2 mol / L sodium dodecyl sulfate solution was added. After mixing for 30 minutes, 0.2 mol / L phosphate buffer with a pH of 7.5 was added to adjust the pH of the solution to 7.5-7.9. The solution was mixed for 20 minutes to allow the enzyme and other proteins in the solution to salt out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES / SPES150). The membrane material was PES / SPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 150 KDa, the pressure was 0.05 MPa, and the flux of the tangential flow filtration was 40 L / (h·m 2 ) to obtain concentrated solution V. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0095] 500 mL of 3 mol / L 2-hydroxyethylamine was added to the concentrated solution V and mixed well. Then, 0.5 mol / L acetic acid buffer with a pH of 3.5 was added to adjust the pH of the solution to 8.2-8.8. The solution was mixed well for 20 minutes to allow the exogenous RNA in the solution to precipitate. The supernatant was extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-mPSF100) by tangential flow filtration. The membrane material was mPSF, the molecular weight cutoff of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.05 MPa, and the flux of the tangential flow filtration was 20 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0096] 500 mL of 3 mol / L ethylene glycol was added to the concentrate VI and mixed thoroughly. Then, 0.5 mol / L acetic acid buffer (pH 3.5) was added to adjust the pH of the solution to 9.2-9.5. Mix thoroughly for 10 minutes to allow the dsRNA in the solution to precipitate. The supernatant was extracted and tangentially filtered through a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES100). The membrane material was mPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 kDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0097] At this time, the main substance in the concentrated solution VII is mRNA. For further purification, the following operation is adopted. 500 mL of 1 mol / L ammonium sulfate is added to the obtained concentrated solution VII, mixed evenly, and then 0.5 mol / L, pH=3.5 acetic acid buffer is added, the pH of the solution is regulated to 8.8-8.9, mixed evenly for 30 minutes to allow the mRNA to salt out, and dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES100). The membrane material is PES, the hollow fiber ultrafiltration membrane has a cut-off molecular weight of 100 KDa, the pressure is 0.05 MPa, and the flux of dead-end filtration is 20 L / (h·m 2 ) to obtain a concentrated solution again; repeat the operation twice according to the above parameters, and the concentrated solution is re-dissolved and filtered to obtain a high-purity target mRNA stock solution for LNP encapsulation / loading.

[0098] Among them, the assembly and formation of LNPs are driven by hydrophobic and electrostatic forces. First, four lipids (dissociated lipids, saturated phospholipids, cholesterol lipids, and polyethylene glycol lipids) are dissolved in ethanol. Two volumes of lipid-containing ethanol solution are usually mixed with five volumes of mRNA in an acetate aqueous buffer at pH = 4. When the lipids come into contact with the aqueous buffer, they become insoluble in the 3:4 water / ethanol solvent, and the ionizable lipids become protonated and positively charged, which then drives it to electrostatically bind to the negatively charged mRNA phosphate backbone. At the same time, the lipids become insoluble, forming lipid particles that encapsulate the mRNA in the aqueous suspension. When the aqueous and lipid phases mix, the ionized lipids are protonated when the mixed phase pH approaches 5.8. When the mixed phase pH approaches 6.9, the mRNA is encapsulated. Subsequently, by dilution or dialysis to pH = 7.8, uncharged LNP nanolipid particles are formed.

[0099] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0100] The purity of the tested mRNA was 98%.

[0101] Step 3: mRNA encapsulation / loading and purification (fluid exchange and concentration)

[0102] The encapsulated solution was filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-PES100). The membrane material was PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.2 MPa, and the flux of the tangential flow filtration was 40 L / (h·m 2 ) to remove smaller molecular impurities such as EDTA, sodium chloride, ethanol, and sodium acetate to obtain concentrated solution VIII. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH 3.5 acetic acid buffer and ultrapure water.

[0103] Add 500 mL of 2 mol / L dodecyltrimethylammonium chloride to the concentrate VIII and mix well. Then add 0.5 mol / L acetic acid buffer with a pH of 3.5 and adjust the pH of the solution to 9.2-9.5. After mixing for 30 minutes, the unencapsulated or unloaded mRNA is salted out. The supernatant is extracted and filtered tangentially using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 2, model UC-PAN150). The membrane material is PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 150 KDa, the pressure is 0.2 MPa, and the flux of the tangential flow filtration is 30 L / (h·m 2 ) to obtain highly purified encapsulated mRNA vaccine. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0104] The purity of the encapsulated mRNA was tested to be 99%.

[0105] Example 3:

[0106] Step 1: Purification of plasmid DNA

[0107] Take 1000g of Escherichia coli stock solution, add 2L of 3mol / L sodium methoxide solution, mix thoroughly to fully lyse the bacterial cells, then add 0.5mol / L, pH=5.5 Tris-EDTA buffer, adjust the pH of the solution to 9.8-10.0, mix evenly for 20 minutes, and the protein denatures and precipitates. The supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PVDF750) with tangential flow filtration. The membrane material is PVDF ( Figure 1), the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 750KDa, the pressure is 0.1MPa, and the flux of the tangential flow filtration is 25L / (h·m 2 ), cell debris and endotoxin were removed to obtain concentrated solution I. After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0108] To the obtained concentrated solution I, 0.05 mol / L PVPK904L reagent was added and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 10.3-10.6. After mixing evenly for 20 minutes, the host genomic DNA was precipitated. The supernatant was extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 1.6, model UC-PAN500) with tangential flow filtration. The membrane material was PAN, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 500 KDa, the pressure was 0.05 MPa, and the flux of the tangential flow filtration was 26 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L acetic acid buffer solution with pH=3.5 and ultrapure water.

[0109] 500 mL of 3 mol / L BaCl2 solution was added to the concentrated solution II and mixed well. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.3-9.6. After mixing well for 25 minutes, the RNA was precipitated. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1.5 mm, inner and outer diameter ratio of 2, model UC-PVDF300). The membrane material was PVDF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 300 KDa, the pressure was 0.05 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=7.8 hydrochloric acid buffer and ultrapure water.

[0110] At this point, the main substance in concentrated solution III is plasmid DNA. For further purification, the following procedure is employed: 1 L of 0.5 mol / L PEG20000 is added to the concentrated solution III obtained, mixed evenly, and then 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 is added to adjust the solution pH to 7.6-8.0. After mixing evenly for 25 minutes, the plasmid DNA is precipitated and dead-end filtered using a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES300). The mPES hollow fiber ultrafiltration membrane has a molecular weight cutoff of 300 kDa, the pressure is 0.05 MPa, and the dead-end filtration flux is 30 L / (h·m2 ), and the concentrate was obtained again. The operation was repeated three times according to the above parameters. The final retentate was redissolved and filtered again to obtain a high-purity plasmid DNA stock solution for in vitro transcription, capping, and tailing to produce the target mRNA.

[0111] Using linearized plasmid DNA as a template, T7 or SP6 promoters, and NTPs as substrates, RNA polymerase is used to synthesize mRNA and then modify it. The mRNA capping process mainly adds 7-methylguanosine (m7G) to the first nucleotide at the 5' end of the mRNA, which can protect the mRNA from degradation, increase its reproducibility, and improve splicing efficiency. It is a key factor in promoting ribosome targeting to mRNA for translation. Capping is performed using a capping enzyme, S-adenosylmethionine (SAM), and GTP. The enzymatic method uses the vaccinia virus capping system with a natural cap. The system contains RNA triphosphatase, guanine methyltransferase and guanine methyltransferase, methyl donor SAM, GTP, and reaction buffer. This reaction converts Cap 0 mRNA into Cap 1 mRNA. The reaction time is short and the cap structure is added in the correct direction. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. It is generated by direct transcription in vitro by designing a sequence encoding a fixed length in the template.

[0112] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=7.8 hydrochloric acid buffer and ultrapure water.

[0113] The purity of the tested plasmid DNA was 97.5%.

[0114] Step 2: mRNA purification

[0115] The target mRNA solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-mPES150). The membrane material was mPES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.08 MPa, and the flux of tangential flow filtration was 30 L / (h·m 2 ) to remove free nucleotides, ribonucleoside triphosphates, immunogenic impurities and other small molecular impurities to obtain concentrated solution IV. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH = 7.8 hydrochloric acid buffer and ultrapure water.

[0116] 5 g of DNA enzyme was added to the concentrated solution IV to fully degrade the residual DNA fragments, and then 2 L of 0.5 mol / L sodium lignin sulfonate solution was added. After mixing for 20 minutes, 0.2 mol / L phosphate buffer with a pH of 7.5 was added to adjust the pH of the solution to 8.5-8.9. After mixing for 30 minutes, the enzyme and other proteins in the solution were salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES / SPES100). The membrane material was PES / SPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.05 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 ) to obtain concentrated solution V. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0117] 500 mL of 2 mol / L tert-butylamine was added to the concentrated solution V and mixed well. Then, 0.5 mol / L acetic acid buffer with a pH of 3.5 was added to adjust the pH of the solution to 8.4-8.9. After mixing for 30 minutes, the exogenous RNA in the solution was precipitated. The supernatant was extracted and the supernatant was dead-end filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-mPES100). The membrane material was mPSF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.1 MPa, and the dead-end filtration flux was 15 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0118] 1000 mL of 1 mol / L propanol was added to the concentrated solution VI and mixed thoroughly. Then, 0.5 mol / L ethanol and a pH 3.5 acid buffer were added to adjust the pH of the solution to 9.3-9.8. After mixing for 20 minutes, the dsRNA in the solution was precipitated. The supernatant was extracted and tangentially filtered through a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES100). The membrane material was mPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 kDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0119] At this time, the main substance in the concentrated solution VII is mRNA. For further purification, the following operation is adopted. 1000 mL of 1 mol / L ammonium nitrate is added to the concentrated solution VII, mixed evenly, and then 0.5 mol / L acetic acid buffer with pH=3.5 is added to adjust the pH of the solution to 8.2-8.4. After mixing evenly for 20 minutes, the mRNA is precipitated and tangential flow filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES100). The membrane material is PES, the hollow fiber ultrafiltration membrane has a cut-off molecular weight of 100 KDa, the pressure is 0.04 MPa, and the flux of the tangential flow filtration is 30 L / (h·m 2 ) to obtain a concentrate again; repeat the operation three times according to the above parameters, and the concentrate was re-dissolved and filtered to obtain a high-purity target mRNA stock solution for LNP encapsulation / loading.

[0120] The assembly and formation of LNPs are driven by hydrophobic and electrostatic forces. Four lipids (dissociated lipids, saturated phospholipids, cholesterol lipids, and polyethylene glycol lipids) are first dissolved in ethanol. Two volumes of lipid-containing ethanol solution are typically mixed with five volumes of mRNA in an acetate aqueous buffer at pH 4. When the lipids come into contact with the aqueous buffer, they become insoluble in the 3:4 water / ethanol solvent, and the ionizable lipids become protonated and positively charged, which then drives them to electrostatically bind to the negatively charged mRNA phosphate backbone. At the same time, the lipids become insoluble, forming lipid particles that encapsulate the mRNA in the aqueous suspension. When the aqueous and lipid phases mix, the ionized lipids are protonated when the mixed phase pH approaches 5.8. When the mixed phase pH approaches 6.9, the mRNA is encapsulated. Subsequently, by dilution or dialysis to pH 7.8, uncharged LNP nanolipid particles are formed.

[0121] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0122] The purity of the tested mRNA was 98.6%.

[0123] Step 3: mRNA encapsulation / loading and purification (fluid exchange and concentration)

[0124] The encapsulated solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-mPES100). The membrane material was PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.08 MPa, and the flux of the tangential flow filtration was 20 L / (h·m 2), removing small molecular impurities such as EDTA, sodium chloride, ethanol and sodium acetate to obtain concentrated solution VIII. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0125] Add 500 mL of 1 mol / L tetramethylammonium chloride to the concentrate VIII and mix well. Then add 0.5 mol / L Tris-EDTA buffer with a pH of 5.5 and adjust the pH of the solution to 9.5-9.8. After mixing for 30 minutes, the unencapsulated or unloaded mRNA is salted out. The supernatant is extracted and filtered tangentially using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 2, model UC-PAN150). The membrane material is PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 150 KDa, the pressure is 0.1 MPa, and the flux of the tangential flow filtration is 40 L / (h·m 2 ) to obtain highly purified encapsulated mRNA vaccine. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0126] The purity of the encapsulated mRNA was tested to be 99%.

[0127] Example 4:

[0128] Step 1: Purification of plasmid DNA

[0129] Take 1000g of bacterial stock solution, add 2L of 1mol / L potassium ethanol solution, mix thoroughly to fully lyse the bacterial cells, then add 0.5mol / L, pH=5.5 Tris-EDTA buffer, adjust the pH of the solution to 9.8-10.0, mix evenly for 30 minutes, and the protein denatures and precipitates. The supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PVDF750) with tangential flow filtration. The membrane material is PVDF ( Figure 1 ), the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 750KDa, the pressure is 0.2MPa, and the flux of the tangential flow filtration is 35L / (h·m 2 ). Cell debris and endotoxins were removed to obtain a concentrated solution I. After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH = 7.5 phosphate buffer and ultrapure water.

[0130] Add 0.08 mol / L PVPK1202L reagent to the concentrate I and mix well. Then add 0.5 mol / L hydrochloric acid buffer with pH = 7.8 to adjust the pH of the solution to 9.3-9.6. After mixing for 20 minutes, the host genomic DNA is precipitated. The supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 1.6, model UC-PAN500) with tangential flow filtration. The membrane material is PAN, the hollow fiber ultrafiltration membrane has a molecular weight cutoff of 500 KDa, the pressure is 0.1 MPa, and the flux of tangential flow filtration is 30 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0131] 1 L of 1 mol / L KNO3 solution was added to the concentrated solution II and mixed well. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.4-9.7. After mixing for 15 minutes, the RNA was precipitated. The supernatant was extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1.5 mm, inner and outer diameter ratio of 2, model UC-PVDF300) by tangential flow filtration. The membrane material was PVDF, the molecular weight cutoff of the hollow fiber ultrafiltration membrane was 300 KDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=7.8 hydrochloric acid buffer and ultrapure water.

[0132] At this point, the main substance in concentrated solution III is plasmid DNA. For further purification, the following procedure is employed: 1L of 0.35 mol / L PEG800 is added to concentrated solution III and mixed thoroughly. Then, 0.5 mol / L hydrochloric acid buffer (pH 7.8) is added to adjust the solution pH to 7.7-8.0. After mixing thoroughly for 25 minutes, the plasmid DNA is precipitated. Dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES300). The mPES hollow fiber ultrafiltration membrane has a molecular weight cutoff of 300 kDa. The pressure is 0.2 MPa, and the dead-end filtration flux is 36 L / (h·m 2 ), and the concentrate was obtained again. This operation was repeated three times according to the above parameters. The concentrate was redissolved and filtered again to obtain a high-purity plasmid DNA stock solution for in vitro transcription, capping, and tailing to produce the target mRNA.

[0133] Using linearized plasmid DNA as a template, T7 or SP6 promoters, and NTPs as substrates, RNA polymerase is used to synthesize mRNA and then modify it. The mRNA capping process mainly adds 7-methylguanosine (m7G) to the first nucleotide at the 5' end of the mRNA, which can protect the mRNA from degradation, increase its reproducibility, and improve splicing efficiency. It is a key factor in promoting ribosome targeting to mRNA for translation. Capping is performed using a capping enzyme, S-adenosylmethionine (SAM), and GTP. The enzymatic method uses the vaccinia virus capping system with a natural cap. The system contains RNA triphosphatase, guanine methyltransferase and guanine methyltransferase, methyl donor SAM, GTP, and reaction buffer. This reaction converts Cap 0 mRNA into Cap 1 mRNA. The reaction time is short and the cap structure is added in the correct direction. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. It is generated by direct transcription in vitro by designing a sequence encoding a fixed length in the template.

[0134] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=7.8 hydrochloric acid buffer and ultrapure water.

[0135] The purity of the tested plasmid DNA was 98.8%.

[0136] Step 2: mRNA purification

[0137] The target mRNA solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-mPES150). The membrane material was mPES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.06 MPa, and the flux of tangential flow filtration was 35 L / (h·m 2 ) to remove free nucleotides, ribonucleoside triphosphates, immunogenic impurities and other small molecular impurities to obtain concentrated solution IV. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH = 7.8 hydrochloric acid buffer and ultrapure water.

[0138] 5 g of DNA enzyme was added to the concentrate IV to fully degrade the residual DNA fragments, and then 2 L of 0.5 mol / L sodium hexadecylsulfonate solution was added. After mixing for 20 minutes, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.5-9.8. After mixing for 20 minutes, the enzyme and other proteins in the solution were completely salted out and precipitated. The supernatant was extracted and tangentially filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES / SPES100). The membrane material was PES / SPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 25 L / (h·m 2 ) to obtain concentrated solution V. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0139] 1000 mL of 2 mol / L isobutylamine was added to the concentrated solution V and mixed well. Then, 0.5 mol / L acetic acid buffer with a pH of 3.5 was added to adjust the pH of the solution to 8.7-8.9. After mixing for 30 minutes, the exogenous RNA in the solution was precipitated. The supernatant was extracted and the supernatant was dead-end filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-mPSF100). The membrane material was mPSF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.08 MPa, and the dead-end filtration flux was 35 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0140] 1000 mL of 2 mol / L ethanol was added to the concentrated solution VI and mixed well. Then, 0.5 mol / L acetic acid buffer with a pH of 3.5 was added to adjust the pH of the solution to 9.3-9.8. After mixing for 20 minutes, the dsRNA in the solution was precipitated. The supernatant was extracted and tangentially filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner / outer diameter ratio of 2, model UC-PES100). The membrane material was PES, the molecular weight cutoff of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 ) The precipitated dsRNA was removed to obtain concentrated solution VII. After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0141] At this time, the main substance in the concentrated solution VII is mRNA. For further purification, the following operation is adopted. 1000mL of 2mol / L ammonium acetate is added to the concentrated solution VII and mixed uniformly. Then, 0.5mol / L acetic acid buffer with pH=3.5 is added to regulate the solution pH to 9.2-9.6. After mixing uniformly for 20min, the mRNA is salted out and precipitated. Dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-mPES100). The membrane material is mPES, the molecular weight cutoff of the hollow fiber ultrafiltration membrane is 100KDa, the pressure is 0.05MPa, and the flux of the dead-end filtration is 30L / (h·m 2 ) to obtain a concentrated solution again; repeat the operation 4 times according to the above parameters, and finally the concentrated solution is re-dissolved and filtered to obtain a high-purity target mRNA stock solution for LNP encapsulation / loading.

[0142] The assembly and formation of LNPs are driven by hydrophobic and electrostatic forces. Four lipids (dissociated lipids, saturated phospholipids, cholesterol lipids, and polyethylene glycol lipids) are first dissolved in ethanol. Two volumes of lipid-containing ethanol solution are typically mixed with five volumes of mRNA in an acetate aqueous buffer at pH 4. When the lipids come into contact with the aqueous buffer, they become insoluble in the 3:4 water / ethanol solvent, and the ionizable lipids become protonated and positively charged, which then drives them to electrostatically bind to the negatively charged mRNA phosphate backbone. At the same time, the lipids become insoluble, forming lipid particles that encapsulate the mRNA in the aqueous suspension. When the aqueous and lipid phases mix, the ionized lipids are protonated when the mixed phase pH approaches 5.8. When the mixed phase pH approaches 6.9, the mRNA is encapsulated. Subsequently, by dilution or dialysis to pH 7.8, uncharged LNP nanolipid particles are formed.

[0143] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0144] The purity of the tested mRNA was 97.6%.

[0145] Step 3: mRNA encapsulation / loading and purification (fluid exchange and concentration)

[0146] The encapsulated solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-PES100). The membrane material was PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.03 MPa, and the flux of the tangential flow filtration was 10 L / (h·m 2), removing small molecular impurities such as EDTA, sodium chloride, ethanol and sodium acetate to obtain concentrated solution VIII. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0147] 800 mL of 1 mol / L spermidine was added to the concentrate VIII and mixed evenly. Then, 0.5 mol / L acetic acid buffer with a pH of 3.5 was added to adjust the pH of the solution to 9.5-9.8. After mixing evenly for 20 minutes, the unencapsulated or unloaded mRNA was salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 2, model UC-PAN150). The membrane material was PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 ) to obtain highly purified encapsulated mRNA vaccine. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0148] The purity of the encapsulated mRNA was tested to be 99.3%.

[0149] Example 5:

[0150] Step 1: Purification of plasmid DNA

[0151] Take 1000g of Escherichia coli stock solution, add 3L of 1.5mol / L potassium hydroxide solution, mix thoroughly to fully lyse the bacterial cells, then add 0.5mol / L, pH=5.5 Tris-EDTA buffer, adjust the pH of the solution to 9.8-10.0, mix evenly for 40 minutes, and the protein denatures and precipitates completely. The supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PVDF750) with tangential flow filtration. The membrane material is PVDF ( Figure 1 ), the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 750KDa, the pressure is 0.3MPa, and the flux of the tangential flow filtration is 25L / (h·m 2 ). Cell debris and endotoxins were removed to obtain a concentrated solution I. After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH = 7.5 phosphate buffer and ultrapure water.

[0152] Add 0.05 mol / L PVPK602L reagent to the concentrate I and mix well. Then add 0.5 mol / L hydrochloric acid buffer with pH = 7.8 to adjust the pH of the solution to 9.3-9.5. After mixing for 20 minutes, the host genomic DNA is precipitated. The supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 1.6, model UC-PAN500) with tangential flow filtration. The membrane material is PAN, the hollow fiber ultrafiltration membrane has a cut-off molecular weight of 500 KDa, the pressure is 0.1 MPa, and the flux of tangential flow filtration is 30 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L acetic acid buffer solution with pH=3.5 and ultrapure water.

[0153] 800 mL of 1 mol / L KNO3 solution was added to the concentrated solution II and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.3-9.7. After mixing evenly for 15 minutes, the RNA was precipitated. The supernatant was extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1.5 mm, inner and outer diameter ratio of 2, model UC-PVDF300) by tangential flow filtration. The membrane material was PVDF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 300 KDa, the pressure was 0.05 MPa, and the flux of the tangential flow filtration was 35 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=7.8 hydrochloric acid buffer and ultrapure water.

[0154] At this point, the main substance in concentrated solution III is plasmid DNA. For further purification, the following procedure is employed: 2L of 0.25 mol / L PEG1000 is added to concentrated solution III and mixed thoroughly. Then, 0.5 mol / L hydrochloric acid buffer (pH 7.8) is added to adjust the solution pH to 7.8-8.5. After mixing thoroughly for 45 minutes, the plasmid DNA is precipitated. Dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES300). The mPES hollow fiber ultrafiltration membrane has a molecular weight cutoff of 300 kDa. The pressure is 0.25 MPa, and the dead-end filtration flux is 60 L / (h·m 2 ) to obtain the concentrate again, repeat the operation three times according to the above parameters, and the final concentrate is redissolved and filtered to obtain a high-purity plasmid DNA stock solution for in vitro transcription, capping, and tailing to produce the target mRNA.

[0155] Using linearized plasmid DNA as a template, T7 or SP6 promoters, and NTPs as substrates, RNA polymerase is used to synthesize mRNA and then modify it. The mRNA capping process mainly adds 7-methylguanosine (m7G) to the first nucleotide at the 5' end of the mRNA, which can protect the mRNA from degradation, increase its reproducibility, and improve splicing efficiency. It is a key factor in promoting ribosome targeting to mRNA for translation. Capping is performed using a capping enzyme, S-adenosylmethionine (SAM), and GTP. The enzymatic method uses the vaccinia virus capping system with a natural cap. The system contains RNA triphosphatase, guanine methyltransferase and guanine methyltransferase, methyl donor SAM, GTP, and reaction buffer. This reaction converts Cap 0 mRNA into Cap 1 mRNA. The reaction time is short and the cap structure is added in the correct direction. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. It is generated by direct transcription in vitro by designing a sequence encoding a fixed length in the template.

[0156] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=7.8 hydrochloric acid buffer and ultrapure water.

[0157] The purity of the tested plasmid DNA was 97.6%.

[0158] Step 2: mRNA purification

[0159] The target mRNA solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-mPES150). The membrane material was mPES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.05 MPa, and the flux of tangential flow filtration was 40 L / (h·m 2 ) to remove free nucleotides, ribonucleoside triphosphates, immunogenic impurities and other small molecular impurities to obtain concentrated solution IV. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH = 7.8 hydrochloric acid buffer and ultrapure water.

[0160] 5 g of DNA enzyme was added to the concentrated solution IV to fully dissolve the residual DNA fragments, and then 4 L of 0.4 mol / L sodium hexadecylbenzenesulfonate solution was added. After mixing for 30 minutes, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.3-9.6. After mixing for 30 minutes, the enzyme and other proteins in the solution were salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES / SPES100). The membrane material was PES / SPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.2 MPa, and the flux of the tangential flow filtration was 15 L / (h·m 2 ) to obtain concentrated solution V. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=7.8 hydrochloric acid buffer and ultrapure water.

[0161] 2000 mL of 1 mol / L colchicine was added to the concentrated solution V and mixed evenly. Then, 0.2 mol / L phosphate buffer with a pH of 7.5 was added to adjust the pH of the solution to 8.0-8.3. After mixing evenly for 30 minutes, the exogenous RNA in the solution was completely precipitated. The supernatant was extracted and then dead-end filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-mPSF100). The membrane material was mPSF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.06 MPa, and the dead-end filtration flux was 25 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0162] 800 mL of 1.5 mol / L n-butanol was added to the concentrated solution VI and mixed well. Then, 0.5 mol / L acetic acid buffer with a pH of 3.5 was added to adjust the pH of the solution to 8.3-8.8. After mixing for 20 minutes, the dsRNA in the solution was completely precipitated. The supernatant was extracted and tangentially filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner / outer diameter ratio of 2, model UC-mPES100). The membrane material was mPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0163] At this time, the main substance in the concentrated solution VII is mRNA. For further purification, the following operation is adopted. 2000mL of 1.5mol / L ammonium carbonate is added to the concentrated solution VII, mixed evenly, and then 0.5mol / L acetic acid buffer with pH=3.5 is added to adjust the pH of the solution to 9.3-9.6. After mixing evenly for 20 minutes, the mRNA is salted out and dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PES100). The membrane material is PES, the hollow fiber ultrafiltration membrane has a cut-off molecular weight of 100KDa, the pressure is 0.08MPa, and the flux of dead-end filtration is 20L / (h·m 2 ) to obtain a concentrate again; the operation was repeated 4 times according to the above parameters, and the concentrate was re-dissolved and filtered to obtain a high-purity target mRNA stock solution for LNP encapsulation / loading.

[0164] The assembly and formation of LNPs are driven by hydrophobic and electrostatic forces. Four lipids (dissociated lipids, saturated phospholipids, cholesterol lipids, and polyethylene glycol lipids) are first dissolved in ethanol. Two volumes of lipid-containing ethanol solution are typically mixed with five volumes of mRNA in an acetate aqueous buffer at pH 4. When the lipids come into contact with the aqueous buffer, they become insoluble in the 3:4 water / ethanol solvent, and the ionizable lipids become protonated and positively charged, which then drives them to electrostatically bind to the negatively charged mRNA phosphate backbone. At the same time, the lipids become insoluble, forming lipid particles that encapsulate the mRNA in the aqueous suspension. When the aqueous and lipid phases mix, the ionized lipids are protonated when the mixed phase pH approaches 5.8. When the mixed phase pH approaches 6.9, the mRNA is encapsulated. Subsequently, by dilution or dialysis to pH 7.8, uncharged LNP nanolipid particles are formed.

[0165] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0166] The purity of the tested mRNA was 98.8%.

[0167] Step 3: mRNA encapsulation / loading and purification (fluid exchange and concentration)

[0168] The encapsulated solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-PES100). The membrane material was PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.05 MPa, and the flux of the tangential flow filtration was 10 L / (h·m 2), removing small molecular impurities such as EDTA, sodium chloride, ethanol and sodium acetate to obtain concentrated solution VIII. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0169] 800 mL of 1.5 mol / L spermidine was added to the concentrate VIII and mixed evenly. Then, 0.5 mol / L acetic acid buffer with a pH of 3.5 was added to adjust the pH of the solution to 9.6-9.8. After mixing evenly for 40 minutes, the unencapsulated or unloaded mRNA was salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 2, model UC-PAN150). The membrane material was PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 35 L / (h·m 2 ) to obtain highly purified encapsulated mRNA vaccine. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0170] The purity of the encapsulated mRNA was tested to be 99.2%.

[0171] Example 6:

[0172] Step 1: Purification of plasmid DNA

[0173] Take 1000g of the bacterial stock solution, add 2L of 1.2mol / L sodium hydroxide solution, mix thoroughly to fully lyse the bacterial cells, then add 0.5mol / L Tris-EDTA buffer with pH=5.5, adjust the pH of the solution to 9.1-9.5, mix evenly for 30 minutes, and the protein denatures and precipitates. The supernatant is extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PVDF750). The membrane material is PVDF, the hollow fiber ultrafiltration membrane has a cut-off molecular weight of 750KDa, the pressure is 0.25MPa, and the flux of the tangential flow filtration is 28L / (h·m 2 ). Cell debris and endotoxins were removed to obtain a concentrated solution I. After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH = 7.5 phosphate buffer and ultrapure water.

[0174] To the obtained concentrated solution I, 0.08 mol / L PVPK601L reagent was added and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.4-9.8. After mixing evenly for 40 minutes, the host genomic DNA was completely precipitated. The supernatant was extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 1.6, model UC-PAN500) with tangential flow filtration. The membrane material was PAN, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 500 KDa, the pressure was 0.2 MPa, and the flux of the tangential flow filtration was 35 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L acetic acid buffer solution with pH=3.5 and ultrapure water.

[0175] 1 L of 1.5 mol / L calcium nitrate solution was added to the concentrated solution II and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.1-9.4. After mixing evenly for 25 minutes, the RNA was precipitated. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1.5 mm, inner / outer diameter ratio of 2, model UC-PVDF300). The membrane material was PVDF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 300 KDa, the pressure was 0.03 MPa, and the flux of the tangential flow filtration was 36 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=7.8 hydrochloric acid buffer and ultrapure water.

[0176] At this point, the main substance in concentrated solution III is plasmid DNA. For further purification, the following procedure is used: 2L of 0.35 mol / L PEG1000 is added to concentrated solution III and mixed thoroughly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 is added to adjust the solution pH to 7.9-8.4. After mixing thoroughly for 35 minutes, the plasmid DNA is precipitated. Dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES300). The mPES hollow fiber ultrafiltration membrane has a molecular weight cutoff of 300 kDa. The pressure is 0.15 MPa, and the dead-end filtration flux is 32 L / (h·m 2 ), and the concentrate was obtained again. The operation was repeated three times according to the above parameters. The final concentrate was redissolved and filtered to obtain a high-purity plasmid DNA stock solution for in vitro transcription, capping, and tailing to produce the target mRNA.

[0177] Using linearized plasmid DNA as a template, T7 or SP6 promoters, and NTPs as substrates, RNA polymerase is used to synthesize mRNA and then modify it. The mRNA capping process mainly adds 7-methylguanosine (m7G) to the first nucleotide at the 5' end of the mRNA, which can protect the mRNA from degradation, increase its reproducibility, and improve splicing efficiency. It is a key factor in promoting ribosome targeting to mRNA for translation. Capping is performed using a capping enzyme, S-adenosylmethionine (SAM), and GTP. The enzymatic method uses the vaccinia virus capping system with a natural cap. The system contains RNA triphosphatase, guanine methyltransferase and guanine methyltransferase, methyl donor SAM, GTP, and reaction buffer. This reaction converts Cap 0 mRNA into Cap 1 mRNA. The reaction time is short and the cap structure is added in the correct direction. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. It is generated by direct transcription in vitro by designing a sequence encoding a fixed length in the template.

[0178] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=7.8 hydrochloric acid buffer and ultrapure water.

[0179] The purity of the tested plasmid DNA was 98.4%.

[0180] Step 2: mRNA purification

[0181] The target mRNA solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-mPES150). The membrane material was mPES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.04 MPa, and the flux of tangential flow filtration was 42 L / (h·m 2 ) to remove free nucleotides, ribonucleoside triphosphates, immunogenic impurities and other small molecular impurities to obtain concentrated solution IV. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH = 7.8 hydrochloric acid buffer and ultrapure water.

[0182] 5 g of DNA enzyme was added to the concentrated solution IV to fully dissolve the residual DNA fragments, and then 1 L of 0.3 mol / L sodium hexadecylbenzenesulfonate solution was added. After mixing for 44 minutes, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.2-9.4. After mixing for 20 minutes, the enzyme and other proteins in the solution were salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES / SPES100). The membrane material was PES / SPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.2 MPa, and the flux of the tangential flow filtration was 28 L / (h·m 2 ) to obtain concentrated solution V. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=7.8 hydrochloric acid buffer and ultrapure water.

[0183] 1000 mL of 1 mol / L colchicine was added to the concentrated solution V and mixed well. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 8.1-8.3. After mixing well for 33 minutes, the exogenous RNA in the solution was completely precipitated. The supernatant was extracted and the supernatant was dead-end filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PSF100). The membrane material was mPSF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.07 MPa, and the dead-end filtration flux was 35 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0184] 600 mL of 1.7 mol / L n-butanol was added to the concentrated solution VI and mixed well. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 8.5-8.7. After mixing for 20 minutes, the dsRNA in the solution was completely precipitated. The supernatant was extracted and tangentially filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner / outer diameter ratio of 2, model UC-mPES100). The membrane material was mPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.1 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0185] At this time, the main substance in the concentrated solution VII is mRNA. For further purification, the following operation is adopted. 2000 mL of 2.5 mol / L ammonium bicarbonate is added to the obtained concentrated solution VII, mixed evenly, and then 0.5 mol / L hydrochloric acid buffer with pH = 7.8 is added to adjust the pH of the solution to 9.3-9.6. After mixing evenly for 20 minutes, the mRNA is precipitated and dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES100). The membrane material is PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 100 KDa, the pressure is 0.07 MPa, and the flux of the dead-end filtration is 24 L / (h·m 2 ) to obtain a concentrated solution again; the operation was repeated three times according to the above parameters, and the concentrated solution was re-dissolved and filtered to obtain a high-purity target mRNA stock solution for LNP encapsulation / loading.

[0186] The assembly and formation of LNPs are driven by hydrophobic and electrostatic forces. Four lipids (dissociated lipids, saturated phospholipids, cholesterol lipids, and polyethylene glycol lipids) are first dissolved in ethanol. Two volumes of lipid-containing ethanol solution are typically mixed with five volumes of mRNA in an acetate aqueous buffer at pH 4. When the lipids come into contact with the aqueous buffer, they become insoluble in the 3:4 water / ethanol solvent, and the ionizable lipids become protonated and positively charged, which then drives them to electrostatically bind to the negatively charged mRNA phosphate backbone. At the same time, the lipids become insoluble, forming lipid particles that encapsulate the mRNA in the aqueous suspension. When the aqueous and lipid phases mix, the ionized lipids are protonated when the mixed phase pH approaches 5.8. When the mixed phase pH approaches 6.9, the mRNA is encapsulated. Subsequently, by dilution or dialysis to pH 7.8, uncharged LNP nanolipid particles are formed.

[0187] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0188] The purity of the tested mRNA was 98.5%.

[0189] Step 3: mRNA encapsulation / loading and purification (fluid exchange and concentration)

[0190] The encapsulated solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-PES100). The membrane material was PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.05 MPa, and the flux of the tangential flow filtration was 16 L / (h·m 2), removing small molecular impurities such as EDTA, sodium chloride, ethanol and sodium acetate to obtain concentrated solution VIII. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0191] 900 mL of 1.5 mol / L dodecyltrimethylammonium bromide was added to the concentrate VIII and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.4-9.8. After mixing evenly for 40 minutes, the unencapsulated or unloaded mRNA was salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 2, model UC-PAN150). The membrane material was PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.08 MPa, and the flux of the tangential flow filtration was 38 L / (h·m 2 ) to obtain highly purified encapsulated mRNA vaccine. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0192] The purity of the encapsulated mRNA was tested to be 99.3%.

[0193] Example 7:

[0194] Step 1: Purification of plasmid DNA

[0195] Take 1000g of bacterial stock solution, add 1.4mol / L sodium citrate solution 1.5L, mix thoroughly to fully lyse the bacterial cells, then add 0.5mol / L, pH=5.5 Tris-EDTA buffer, adjust the pH of the solution to 8.2-8.4, mix evenly for 30 minutes, and the protein denatures and precipitates completely. The supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PVDF750) with tangential flow filtration. The membrane material is PVDF ( Figure 1 ), the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 750KDa, the pressure is 0.35MPa, and the flux of tangential flow filtration is 38L / (h·m 2 ). Cell debris and endotoxins were removed to obtain a concentrated solution I. After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0196] To the obtained concentrated solution I, 1.5 L of 0.09 mol / L PVP K90 was added and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.3-9.6. After mixing evenly for 30 minutes, the host genomic DNA was completely precipitated. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 1.6, model UC-PES / SPES500). The membrane material was PES / SPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 500 KDa, the pressure was 0.3 MPa, and the flux of the tangential flow filtration was 24 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L acetic acid buffer solution with pH=3.5 and ultrapure water.

[0197] 1 L of 2.5 mol / L sodium chloride solution was added to the concentrated solution II and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.2-9.5. After mixing evenly for 35 minutes, the RNA was completely precipitated. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1.5 mm, inner and outer diameter ratio of 2, model UC-PVDF300). The membrane material was PVDF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 300 KDa, the pressure was 0.02 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0198] At this point, the main substance in concentrated solution III is plasmid DNA. For further purification, the following procedure is used: 0.36 mol / L PEG20003L is added to concentrated solution III and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 is added to adjust the solution pH to 7.8-8.4. After mixing evenly for 33 minutes, the plasmid DNA is precipitated. Dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner / outer diameter ratio of 2, model UC-mPES300). The mPES hollow fiber ultrafiltration membrane has a molecular weight cutoff of 300 kDa. The pressure is 0.15 MPa, and the dead-end filtration flux is 36 L / (h·m 2 ) to obtain the concentrate again, repeat the operation three times according to the above parameters, and the final concentrate is redissolved and filtered to obtain a high-purity plasmid DNA stock solution for in vitro transcription, capping, and tailing to produce the target mRNA.

[0199] Using linearized plasmid DNA as a template, T7 or SP6 promoters, and NTPs as substrates, RNA polymerase is used to synthesize mRNA and then modify it. The mRNA capping process mainly adds 7-methylguanosine (m7G) to the first nucleotide at the 5' end of the mRNA, which can protect the mRNA from degradation, increase its reproducibility, and improve splicing efficiency. It is a key factor in promoting ribosome targeting to mRNA for translation. Capping is performed using a capping enzyme, S-adenosylmethionine (SAM), and GTP. The enzymatic method uses the vaccinia virus capping system with a natural cap. The system contains RNA triphosphatase, guanine methyltransferase and guanine methyltransferase, methyl donor SAM, GTP, and reaction buffer. This reaction converts Cap 0 mRNA into Cap 1 mRNA. The reaction time is short and the cap structure is added in the correct direction. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. It is generated by direct transcription in vitro by designing a sequence encoding a fixed length in the template.

[0200] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0201] The purity of the tested plasmid DNA was 98.7%.

[0202] Step 2: mRNA purification

[0203] The target mRNA solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-mPES150). The membrane material was mPES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.03 MPa, and the flux of tangential flow filtration was 35 L / (h·m 2 ) to remove free nucleotides, ribonucleoside triphosphates, immunogenic impurities and other small molecular impurities to obtain concentrated solution IV. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH = 3.5 acetic acid buffer and ultrapure water.

[0204] 5 g of DNA enzyme was added to the concentrated solution IV to fully dissolve the residual DNA fragments, and then 1 L of 0.3 mol / L sodium dodecyl sulfate solution was added. After mixing for 44 minutes, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.1-9.4. After mixing for 26 minutes, the enzyme and other proteins in the solution were salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES / SPES100). The membrane material was PES / SPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.25 MPa, and the flux of the tangential flow filtration was 25 L / (h·m 2 ) to obtain concentrated solution V. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0205] 1000 mL of 1 mol / L triisopropanolamine was added to the concentrated solution V and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 8.4-8.7. After mixing evenly for 38 minutes, the exogenous RNA in the solution was completely precipitated. The supernatant was extracted and the supernatant was dead-end filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-mPSF100). The membrane material was mPSF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.05 MPa, and the dead-end filtration flux was 45 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0206] 600 mL of 1.7 mol / L ethylene glycol was added to the concentrate VI and mixed thoroughly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 8.5-8.8. After mixing for 30 minutes, the dsRNA in the solution was precipitated. The supernatant was extracted and tangentially filtered through a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES100). The membrane material was mPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.15 MPa, and the flux of the tangential flow filtration was 34 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0207] At this time, the main substance in the concentrated solution VII is mRNA. For further purification, the following operation is adopted. 1500mL of 2.5mol / L ammonium bicarbonate is added to the concentrated solution VII, mixed evenly, and then 0.5mol / L hydrochloric acid buffer with pH=7.8 is added to adjust the pH of the solution to 9.2-9.4. After mixing evenly for 30min, the mRNA is salted out and dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PES100). The membrane material is PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 100KDa, the pressure is 0.08MPa, and the flux of the dead-end filtration is 28L / (h·m 2 ) to obtain a concentrate again; the operation was repeated 4 times according to the above parameters, and the concentrate was re-dissolved and filtered to obtain a high-purity target mRNA stock solution for LNP encapsulation / loading.

[0208] The assembly and formation of LNPs are driven by hydrophobic and electrostatic forces. Four lipids (dissociated lipids, saturated phospholipids, cholesterol lipids, and polyethylene glycol lipids) are first dissolved in ethanol. Two volumes of lipid-containing ethanol solution are typically mixed with five volumes of mRNA in an acetate aqueous buffer at pH 4. When the lipids come into contact with the aqueous buffer, they become insoluble in the 3:4 water / ethanol solvent, and the ionizable lipids become protonated and positively charged, which then drives them to electrostatically bind to the negatively charged mRNA phosphate backbone. At the same time, the lipids become insoluble, forming lipid particles that encapsulate the mRNA in the aqueous suspension. When the aqueous and lipid phases mix, the ionized lipids are protonated when the mixed phase pH approaches 5.8. When the mixed phase pH approaches 6.9, the mRNA is encapsulated. Subsequently, by dilution or dialysis to pH 7.8, uncharged LNP nanolipid particles are formed.

[0209] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0210] The purity of the tested mRNA was 97.5%.

[0211] Step 3: mRNA encapsulation / loading and purification (fluid exchange and concentration)

[0212] The encapsulated solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-PES100). The membrane material was PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.08 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2), removing small molecular impurities such as EDTA, sodium chloride, ethanol and sodium acetate to obtain concentrated solution VIII. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0213] 1000 mL of 1.5 mol / L hexadecyltrimethylammonium bromide was added to the concentrate VIII and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.5-9.8. After mixing evenly for 30 minutes, the unencapsulated or unloaded mRNA was salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 2, model UC-PAN150). The membrane material was PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.06 MPa, and the flux of the tangential flow filtration was 40 L / (h·m 2 ) to obtain highly purified encapsulated mRNA vaccine. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0214] The purity of the encapsulated mRNA was tested to be 99.1%.

[0215] Example 8:

[0216] Step 1: Purification of plasmid DNA

[0217] Take 1000g of the bacterial stock solution, add 1.5L of 1.3mol / L potassium ethanolate solution, mix thoroughly to fully lyse the bacterial cells, then add 0.5mol / L Tris-EDTA buffer with pH=5.5, adjust the pH of the solution to 8.3-8.8, mix evenly for 40 minutes, and the protein denatures and precipitates completely. The supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PVDF750) with tangential flow filtration. The membrane material is PVDF, the hollow fiber ultrafiltration membrane has a cut-off molecular weight of 750KDa, the pressure is 0.35MPa, and the flux of the tangential flow filtration is 50L / (h·m 2 ). Cell debris and denatured proteins were removed to obtain a concentrated solution I. After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0218] To the obtained concentrated solution I, 1.5 L of 0.08 mol / L PVP K15 reagent was added and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.3-9.6. After mixing evenly for 30 minutes, the host genomic DNA was completely precipitated. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 1.6, model UC-PAN500). The membrane material was PAN, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 500 KDa, the pressure was 0.8 MPa, and the flux of the tangential flow filtration was 50 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L acetic acid buffer solution with pH=3.5 and ultrapure water.

[0219] 1 L of 3.3 mol / L sodium chloride solution was added to the concentrated solution II and mixed well. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.1-9.5. After mixing well for 35 minutes, the RNA was precipitated. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1.5 mm, inner and outer diameter ratio of 2, model UC-mPSF300). The membrane material was mPSF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 300 KDa, the pressure was 0.03 MPa, and the flux of the tangential flow filtration was 40 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0220] At this point, the main substance in concentrated solution III is plasmid DNA. For further purification, the following procedure is used. 0.33 mol / L PEG60003L is added to concentrated solution III and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 is added to adjust the solution pH to 7.5-8.1. After mixing evenly for 38 minutes, the plasmid DNA is precipitated. Dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner / outer diameter ratio of 2, model UC-mPES300). The membrane material is mPES, the hollow fiber ultrafiltration membrane has a molecular weight cutoff of 300 kDa, and the pressure is 0.14 MPa. The dead-end filtration flux is 32 L / (h·m 2 Repeat the above process three times, re-dissolve the concentrate, and filter it to obtain a high-purity plasmid DNA stock solution for in vitro transcription, capping, and tailing to produce the target mRNA.

[0221] Using linearized plasmid DNA as a template, T7 or SP6 promoters, and NTPs as substrates, RNA polymerase is used to synthesize mRNA and then modify it. The mRNA capping process mainly adds 7-methylguanosine (m7G) to the first nucleotide at the 5' end of the mRNA, which can protect the mRNA from degradation, increase its reproducibility, and improve splicing efficiency. It is a key factor in promoting ribosome targeting to mRNA for translation. Capping is performed using a capping enzyme, S-adenosylmethionine (SAM), and GTP. The enzymatic method uses the vaccinia virus capping system with a natural cap. The system contains RNA triphosphatase, guanine methyltransferase and guanine methyltransferase, methyl donor SAM, GTP, and reaction buffer. This reaction converts Cap 0 mRNA into Cap 1 mRNA. The reaction time is short and the cap structure is added in the correct direction. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. It is generated by direct transcription in vitro by designing a sequence encoding a fixed length in the template.

[0222] After each batch of liquid treatment was completed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0223] The purity of the tested plasmid DNA was 98.4%.

[0224] Step 2: mRNA purification

[0225] The target mRNA solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-mPES150). The membrane material was mPES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.02 MPa, and the flow rate of the tangential flow filtration was 45 L / (h·m 2 ) to remove free nucleotides, ribonucleoside triphosphates, immunogenic impurities and other small molecular impurities to obtain concentrated solution IV. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH = 7.5 phosphate buffer and ultrapure water.

[0226] 5 g of DNA enzyme was added to the concentrated solution IV to fully degrade the residual DNA fragments, and then 2 L of 0.2 mol / L sodium hexadecylsulfonate solution was added. After mixing for 45 minutes, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.2-9.6. After mixing for 36 minutes, the enzyme and other proteins in the solution were salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES / SPES100). The membrane material was PES / SPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.25 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2 ) to obtain concentrated solution V. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0227] 600 mL of 1 mol / L triisopropanolamine was added to the concentrated solution V and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 8.5-8.9. After mixing evenly for 48 minutes, the exogenous RNA in the solution was precipitated. The supernatant was extracted and the supernatant was dead-end filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PSF100). The membrane material was PSF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.06 MPa, and the dead-end filtration flux was 35 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0228] 800 mL of 1.6 mol / L ethanol was added to the concentrated solution VI and mixed well. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 8.6-8.8. After mixing for 40 minutes, the dsRNA in the solution was precipitated. The supernatant was extracted and tangentially filtered through a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES100). The membrane material was mPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.28 MPa, and the flux of the tangential flow filtration was 38 L / (h·m 2 ) to remove dsRNA and obtain concentrated solution VII. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0229] At this time, the main substance in the concentrated solution VII is mRNA. For further purification, the following operation is adopted. 1000 mL of 2.6 mol / L ammonium bicarbonate is added to the obtained concentrated solution VII, mixed evenly, and then 0.5 mol / L hydrochloric acid buffer with pH = 7.8 is added to adjust the pH of the solution to 9.1-9.4. After mixing evenly for 30 minutes, the mRNA is salted out and dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES100). The membrane material is PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 100 KDa, the pressure is 0.08 MPa, and the flux of the dead-end filtration is 42 L / (h·m 2 ) to obtain a concentrate again; the operation was repeated 4 times according to the above parameters, and the concentrate was re-dissolved and filtered to obtain a high-purity target mRNA stock solution for LNP encapsulation / loading.

[0230] The assembly and formation of LNPs are driven by hydrophobic and electrostatic forces. Four lipids (dissociated lipids, saturated phospholipids, cholesterol lipids, and polyethylene glycol lipids) are first dissolved in ethanol. Two volumes of lipid-containing ethanol solution are typically mixed with five volumes of mRNA in an acetate aqueous buffer at pH 4. When the lipids come into contact with the aqueous buffer, they become insoluble in the 3:4 water / ethanol solvent, and the ionizable lipids become protonated and positively charged, which then drives them to electrostatically bind to the negatively charged mRNA phosphate backbone. At the same time, the lipids become insoluble, forming lipid particles that encapsulate the mRNA in the aqueous suspension. When the aqueous and lipid phases mix, the ionized lipids are protonated when the mixed phase pH approaches 5.8. When the mixed phase pH approaches 6.9, the mRNA is encapsulated. Subsequently, by dilution or dialysis to pH 7.8, uncharged LNP nanolipid particles are formed.

[0231] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0232] The purity of the tested mRNA was 98.5%.

[0233] Step 3: mRNA encapsulation / loading and purification (fluid exchange and concentration)

[0234] The encapsulated solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-PES100). The membrane material was PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.2 MPa, and the flux of the tangential flow filtration was 30 L / (h·m 2) to remove small molecular impurities such as EDTA, sodium chloride, ethanol and sodium acetate to obtain concentrated solution VIII. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0235] Add 2000 mL of 1.8 mol / L dodecyltrimethylammonium bromide to the concentrate VIII and mix well. Then add 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 to adjust the pH of the solution to 9.6-9.8. After mixing for 30 minutes, the unencapsulated or unloaded mRNA is salted out. The supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 2, model UC-PAN150) with tangential flow filtration. The membrane material is PAN, the molecular weight cutoff of the hollow fiber ultrafiltration membrane is 150 KDa, the pressure is 0.08 MPa, and the flux of the tangential flow filtration is 45 L / (h·m 2 ) to obtain highly purified encapsulated mRNA vaccine. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0236] The purity of the encapsulated mRNA was tested to be 99.2%.

[0237] Example 9:

[0238] Step 1: Purification of plasmid DNA

[0239] Take 1000g of bacterial stock solution, add 2L of 1.8mol / L sodium methoxide solution, mix thoroughly to fully lyse the bacterial cells, then add 0.5mol / L Tris-EDTA buffer with pH=5.5, adjust the pH of the solution to 8.6-8.8, mix evenly for 40 minutes, and the protein denatures and precipitates completely. The supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PVDF750) with tangential flow filtration. The membrane material is PVDF, the hollow fiber ultrafiltration membrane has a cut-off molecular weight of 750KDa, the pressure is 0.25MPa, and the flux of tangential flow filtration is 40L / (h·m 2 ), cell debris and endotoxin were removed to obtain concentrated solution I. After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0240] To the obtained concentrated solution I, 2.5 L of 0.06 mol / L PVP K60 was added and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.2-9.6. After mixing evenly for 50 minutes, the host genomic DNA was precipitated. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 1.6, model UC-PAN500). The membrane material was PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 500 KDa, the pressure was 0.6 MPa, and the flux of the tangential flow filtration was 40 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L acetic acid buffer solution with pH=3.5 and ultrapure water.

[0241] 1 L of 3.4 mol / L sodium chloride solution was added to the concentrated solution II and mixed well. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.2-9.5. After mixing well for 25 minutes, the RNA was precipitated. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1.5 mm, inner / outer diameter ratio of 2, model UC-PVDF300). The membrane material was PVDF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 300 KDa, the pressure was 0.04 MPa, and the flux of the tangential flow filtration was 50 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0242] At this point, the main substance in concentrated solution III is plasmid DNA. For further purification, the following procedure is used. 1 L of 0.43 mol / L PEG10000 is added to concentrated solution III and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 is added to adjust the solution pH to 7.8-8.1. After mixing evenly for 41 minutes, the plasmid DNA is precipitated. Dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES300). The membrane material is mPES, the molecular weight cutoff of the hollow fiber ultrafiltration membrane is 300 kDa, the pressure is 0.15 MPa, and the dead-end filtration flux is 30 L / (h·m 2 ), and the concentrate was obtained again. The operation was repeated three times according to the above parameters. The final concentrate was redissolved and filtered to obtain a high-purity plasmid DNA stock solution for in vitro transcription, capping, and tailing to produce the target mRNA.

[0243] Using linearized plasmid DNA as a template, T7 or SP6 promoters, and NTPs as substrates, RNA polymerase is used to synthesize mRNA and then modify it. The mRNA capping process mainly adds 7-methylguanosine (m7G) to the first nucleotide at the 5' end of the mRNA, which can protect the mRNA from degradation, increase its reproducibility, and improve splicing efficiency. It is a key factor in promoting ribosome targeting to mRNA for translation. Capping is performed using a capping enzyme, S-adenosylmethionine (SAM), and GTP. The enzymatic method uses the vaccinia virus capping system with a natural cap. The system contains RNA triphosphatase, guanine methyltransferase and guanine methyltransferase, methyl donor SAM, GTP, and reaction buffer. This reaction converts Cap 0 mRNA into Cap 1 mRNA. The reaction time is short and the cap structure is added in the correct direction. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. It is generated by direct transcription in vitro by designing a sequence encoding a fixed length in the template.

[0244] After each batch of liquid treatment was completed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0245] The purity of the tested plasmid DNA was 98.8%.

[0246] Step 2: mRNA purification

[0247] The target mRNA solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-mPES150). The membrane material was mPES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.03 MPa, and the flux of tangential flow filtration was 40 L / (h·m 2 ) to remove free nucleotides, ribonucleoside triphosphates, immunogenic impurities and other small molecular impurities to obtain concentrated solution IV. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH = 7.5 phosphate buffer and ultrapure water.

[0248] 5 g of DNA enzyme was added to the concentrated solution IV to fully degrade the residual DNA fragments, and then 3 L of 0.3 mol / L sodium hexadecylsulfonate solution was added. After mixing for 45 minutes, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.3-9.6. After mixing for 36 minutes, the enzyme and other proteins in the solution were salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES / SPES100). The membrane material was PES / SPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.35 MPa, and the flux of the tangential flow filtration was 40 L / (h·m 2 ) to obtain concentrated solution V. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0249] 600 mL of 1 mol / L triisopropanolamine was added to the concentrated solution V and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 8.5-8.8. After mixing evenly for 48 minutes, the exogenous RNA in the solution was precipitated. The supernatant was extracted and the supernatant was dead-end filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-mPSF100). The membrane material was mPSF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.08 MPa, and the dead-end filtration flux was 35 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0250] 800 mL of 2 mol / L ethylene glycol was added to the concentrate VI and mixed thoroughly. Then, 0.5 mol / L hydrochloric acid buffer (pH 7.8) was added to adjust the pH of the solution to 10.2-10.5. After mixing for 30 minutes, the dsRNA in the solution was precipitated. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES100). The membrane material was mPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 kDa, and the pressure was 0.18 MPa. The flux of the tangential flow filtration was 41 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH 7.5 phosphate buffer and ultrapure water.

[0251] At this time, the main substance in the concentrated solution VII is mRNA. For further purification, the following operation is adopted. 1000mL of 3.6mol / L ammonium chloride is added to the concentrated solution VII, mixed evenly, and then 0.5mol / L hydrochloric acid buffer with pH=7.8 is added to adjust the pH of the solution to 9.1-9.4. After mixing evenly for 40 minutes, the mRNA is salted out and dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PES100). The membrane material is PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 100KDa, the pressure is 0.06MPa, and the flux of the dead-end filtration is 40L / (h·m 2 ) to obtain a concentrate again; the operation was repeated 4 times according to the above parameters, and the concentrate was re-dissolved and filtered to obtain a high-purity target mRNA stock solution for LNP encapsulation / loading.

[0252] The assembly and formation of LNPs are driven by hydrophobic and electrostatic forces. Four lipids (dissociated lipids, saturated phospholipids, cholesterol lipids, and polyethylene glycol lipids) are first dissolved in ethanol. Two volumes of lipid-containing ethanol solution are typically mixed with five volumes of mRNA in an acetate aqueous buffer at pH 4. When the lipids come into contact with the aqueous buffer, they become insoluble in the 3:4 water / ethanol solvent, and the ionizable lipids become protonated and positively charged, which then drives them to electrostatically bind to the negatively charged mRNA phosphate backbone. At the same time, the lipids become insoluble, forming lipid particles that encapsulate the mRNA in the aqueous suspension. When the aqueous and lipid phases mix, the ionized lipids are protonated when the mixed phase pH approaches 5.8. When the mixed phase pH approaches 6.9, the mRNA is encapsulated. Subsequently, by dilution or dialysis to pH 7.8, uncharged LNP nanolipid particles are formed.

[0253] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0254] The purity of the tested mRNA was 99.3%.

[0255] Step 3: mRNA encapsulation / loading and purification (fluid exchange and concentration)

[0256] The encapsulated solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-PES100). The membrane material was PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.2 MPa, and the flux of tangential flow filtration was 40 L / (h·m 2) to remove small molecular impurities such as EDTA, sodium chloride, ethanol and sodium acetate to obtain concentrated solution VIII. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0257] 1000 mL of 1.6 mol / L tetramethylammonium chloride was added to the concentrate VIII and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.4-9.7. After mixing evenly for 30 minutes, the unencapsulated or unloaded mRNA was salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 2, model UC-PAN150). The membrane material was PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.07 MPa, and the flux of the tangential flow filtration was 35 L / (h·m 2 ) to obtain highly purified encapsulated mRNA vaccine. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH = 7.5 phosphate buffer and ultrapure water.

[0258] The purity of the encapsulated mRNA was tested to be 99.2%.

[0259] Example 10:

[0260] Step 1: Purification of plasmid DNA

[0261] Take 1000g of bacterial stock solution, add 4L of 1.8mol / L potassium ethanolate solution, mix thoroughly to fully lyse the bacterial cells, then add 0.5mol / L Tris-EDTA buffer with pH=5.5, adjust the pH of the solution to 8.3-8.5, mix evenly for 30 minutes, and the protein denatures and precipitates completely. The supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1mm, inner and outer diameter ratio of 2, model UC-PVDF750) with tangential flow filtration. The membrane material is PVDF, the hollow fiber ultrafiltration membrane has a cut-off molecular weight of 750KDa, the pressure is 0.05MPa, and the flux of tangential flow filtration is 26L / (h·m 2 ). Cell debris and endotoxins were removed to obtain a concentrated solution I. After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0262] To the concentrated solution I, 0.04 mol / L PVP K903L reagent was added and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.1-9.4. After mixing evenly for 50 minutes, the host genomic DNA was completely precipitated. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 1.6, model UC-PAN500). The membrane material was PAN, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 500 KDa, the pressure was 0.4 MPa, and the flux of the tangential flow filtration was 45 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L acetic acid buffer solution with pH=3.5 and ultrapure water.

[0263] 1 L of 3.5 mol / L sodium chloride solution was added to the concentrated solution II and mixed well. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.3-9.6. After mixing well for 35 minutes, the RNA was completely precipitated. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1.5 mm, inner and outer diameter ratio of 2, model UC-PVDF300). The membrane material was PVDF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 300 KDa, the pressure was 0.03 MPa, and the flux of the tangential flow filtration was 60 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0264] At this point, the main substance in concentrated solution III is plasmid DNA. For further purification, the following procedure is used. 1 L of 0.45 mol / L PEG20000 is added to concentrated solution III and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 is added to adjust the solution pH to 7.8-8.3. After mixing evenly for 45 minutes, the plasmid DNA is precipitated. Dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES300). The membrane material is mPES, the molecular weight cutoff of the hollow fiber ultrafiltration membrane is 300 kDa, the pressure is 0.25 MPa, and the dead-end filtration flux is 35 L / (h·m 2 Repeat the above process three times, re-dissolve the final concentrate and filter it to obtain a high-purity plasmid DNA stock solution for in vitro transcription, capping, and tailing to produce the target mRNA.

[0265] Using linearized plasmid DNA as a template, T7 or SP6 promoters, and NTPs as substrates, RNA polymerase is used to synthesize mRNA and then modify it. The mRNA capping process mainly adds 7-methylguanosine (m7G) to the first nucleotide at the 5' end of the mRNA, which can protect the mRNA from degradation, increase its reproducibility, and improve splicing efficiency. It is a key factor in promoting ribosome targeting to mRNA for translation. Capping is performed using a capping enzyme, S-adenosylmethionine (SAM), and GTP. The enzymatic method uses the vaccinia virus capping system with a natural cap. The system contains RNA triphosphatase, guanine methyltransferase and guanine methyltransferase, methyl donor SAM, GTP, and reaction buffer. This reaction converts Cap 0 mRNA into Cap 1 mRNA. The reaction time is short and the cap structure is added in the correct direction. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. The PolyA tail of mRNA has the function of facilitating the transport of mRNA from the nucleus to the cytoplasm, reducing the degradation of mRNA by ribozymes, and enhancing the stability of mRNA. It is generated by direct transcription in vitro by designing a sequence encoding a fixed length in the template.

[0266] After each batch of liquid treatment was completed, the ultrafiltration membrane was backwashed with 0.5 mol / L, pH=3.5 acetic acid buffer and ultrapure water.

[0267] The purity of the tested plasmid DNA was 97.8%.

[0268] Step 2: mRNA purification

[0269] The target mRNA solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-mPES150). The membrane material was mPES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.02 MPa, and the flux of tangential flow filtration was 20 L / (h·m 2 ) to remove free nucleotides, ribonucleoside triphosphates, immunogenic impurities and other small molecular impurities to obtain concentrated solution IV. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH = 7.5 phosphate buffer and ultrapure water.

[0270] 5 g of DNA enzyme was added to the concentrated solution V to fully dissolve the residual DNA fragments, and then 2 L of 0.2 mol / L sodium dodecyl sulfate solution was added. After mixing for 30 minutes, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.1-9.4. After mixing for 30 minutes, the enzyme and other proteins in the solution were salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PAN150). The membrane material was PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.25 MPa, and the flux of the tangential flow filtration was 20 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0271] 600 mL of 1 mol / L tert-butylamine was added to the concentrated solution V and mixed well. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 8.5-8.8. After mixing for 48 minutes, the exogenous RNA in the solution was precipitated. The supernatant was extracted and the supernatant was dead-end filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC_mPSF100). The membrane material was mPSF, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.1 MPa, and the dead-end filtration flux was 35 L / (h·m 2 After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0272] 1000 mL of 1 mol / L glycerol was added to the concentrate VI and mixed thoroughly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 10.2-10.5. After mixing for 30 minutes, the dsRNA in the solution was precipitated. The supernatant was extracted and tangentially filtered through a gradient pore structure hollow fiber ultrafiltration membrane (1 mm diameter, inner / outer diameter ratio of 2, model UC-mPES100). The membrane material was mPES, the hollow fiber ultrafiltration membrane had a molecular weight cutoff of 100 KDa, the pressure was 0.2 MPa, and the flux of the tangential flow filtration was 35 L / (h·m 2 After each batch of liquid was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH 7.5 phosphate buffer and ultrapure water.

[0273] At this time, the main substance in the concentrated solution VII is mRNA. For further purification, the following operation is adopted. 2000 mL of 2.6 mol / L ammonium nitrate is added to the concentrated solution VII, mixed evenly, and then 0.5 mol / L hydrochloric acid buffer with pH = 7.8 is added to adjust the pH of the solution to 9.2-9.4. After mixing evenly for 30 minutes, the mRNA is salted out and dead-end filtration is performed using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 1 mm, inner and outer diameter ratio of 2, model UC-PES100). The membrane material is PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane is 100 KDa, the pressure is 0.04 MPa, and the flux of the dead-end filtration is 40 L / (h·m 2 ) to obtain a concentrated solution again; the operation was repeated 4 times according to the above parameters, and the concentrated solution was re-dissolved and filtered to obtain a high-purity target mRNA stock solution for LNP encapsulation / loading.

[0274] The assembly and formation of LNPs are driven by hydrophobic and electrostatic forces. Four lipids (dissociated lipids, saturated phospholipids, cholesterol lipids, and polyethylene glycol lipids) are first dissolved in ethanol. Two volumes of lipid-containing ethanol solution are typically mixed with five volumes of mRNA in an acetate aqueous buffer at pH 4. When the lipids come into contact with the aqueous buffer, they become insoluble in the 3:4 water / ethanol solvent, and the ionizable lipids become protonated and positively charged, which then drives them to electrostatically bind to the negatively charged mRNA phosphate backbone. At the same time, the lipids become insoluble, forming lipid particles that encapsulate the mRNA in the aqueous suspension. When the aqueous and lipid phases mix, the ionized lipids are protonated when the mixed phase pH approaches 5.8. When the mixed phase pH approaches 6.9, the mRNA is encapsulated. Subsequently, by dilution or dialysis to pH 7.8, uncharged LNP nanolipid particles are formed.

[0275] After each batch of liquid treatment is completed, the ultrafiltration membrane is backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0276] The purity of the tested mRNA was 98.8%.

[0277] Step 3: mRNA encapsulation / loading and purification (fluid exchange and concentration)

[0278] The encapsulated solution was directly filtered by tangential flow filtration using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.5 mm, inner and outer diameter ratio of 1.2, model UC-PES100). The membrane material was PES, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 100 KDa, the pressure was 0.3 MPa, and the flux of the tangential flow filtration was 50 L / (h·m 2) to remove small molecular impurities such as EDTA, sodium chloride, ethanol and sodium acetate to obtain concentrated solution VIII. After each batch of feed solution was processed, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH=7.5 phosphate buffer and ultrapure water.

[0279] 1000 mL of 1.8 mol / L spermidine was added to the concentrate VIII and mixed evenly. Then, 0.5 mol / L hydrochloric acid buffer with a pH of 7.8 was added to adjust the pH of the solution to 9.5-9.7. After mixing evenly for 20 minutes, the unencapsulated or unloaded mRNA was salted out. The supernatant was extracted and tangentially filtered using a gradient pore structure hollow fiber ultrafiltration membrane (diameter 0.8 mm, inner and outer diameter ratio of 2, model UC-PAN150). The membrane material was PAN, the cut-off molecular weight of the hollow fiber ultrafiltration membrane was 150 KDa, the pressure was 0.08 MPa, and the flux of the tangential flow filtration was 40 L / (h·m 2 ) to obtain highly purified encapsulated mRNA vaccine. After each batch of liquid treatment, the ultrafiltration membrane was backwashed with 0.2 mol / L, pH = 7.5 phosphate buffer and ultrapure water.

[0280] The purity of the encapsulated mRNA was tested to be 99.4%.

[0281] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0282] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be construed in the widest possible manner consistent with the principles and novel features disclosed herein.

Claims

1. A process for large-scale purification of mRNA vaccines using a gradient pore structure hollow fiber ultrafiltration membrane, characterized in that: The steps include: (1) Plasmid DNA purification: The cultured bacterial solution is subjected to cell lysis, and after lysis, precipitation and / or salting-out treatment is performed in combination with filtration through a hollow fiber ultrafiltration membrane with a gradient pore structure to obtain purified plasmid DNA; (2) mRNA purification: Purified plasmid DNA is transcribed in vitro, capped, and tailed to obtain target mRNA, followed by the addition of DNase, and then subjected to precipitation and / or salting-out treatment combined with filtration through a gradient pore structure hollow fiber ultrafiltration membrane to obtain purified mRNA; (3) Purification after mRNA encapsulation or loading: After the purified mRNA is encapsulated or loaded, precipitation and / or salting-out treatment is performed in conjunction with filtration through a hollow fiber ultrafiltration membrane with a gradient pore structure to obtain the mRNA vaccine; In steps (1)-(3), The gradient pore structure hollow fiber ultrafiltration membrane is made of at least one of polyvinylidene fluoride PVDF, polyethersulfone PES, polysulfone PSF, sulfonated polyethersulfone SPES, modified polyethersulfone mPES, modified polysulfone mPSF, and polyacrylonitrile PAN, and the cut-off molecular weight range of the gradient pore structure hollow fiber ultrafiltration membrane is 50KDa-750KDa; In steps (1)-(3), Internal pressure hollow fiber membrane modules are used for filtration. The diameter of the gradient pore structure hollow fiber ultrafiltration membrane in the module is 0.1-4 mm, and the ratio of the inner and outer diameters is 1.2-4. The filtration method is tangential flow filtration or dead-end filtration; Dead-end filtration pressure is 0.05-0.3MPa, tangential flow filtration flow rate is 15-150L / (h﹒m 2 ); After filtering in any of steps (1) to (3), backwashing is performed using reagent I; The reagent I used for backwashing includes at least one of hydrochloric acid buffer Tris-HCl, phosphate buffer Tris-H3PO4, ethylenediaminetetraacetic acid buffer Tris-EDTA, and acetate buffer Tris-CH3COOH. The concentration range of the reagent I is 0.1 mol / L to 1 mol / L, and the pH range is 7-10.

2. The process for large-scale purification of mRNA vaccines using a gradient pore structure hollow fiber ultrafiltration membrane according to claim 1, characterized in that: In step (1), precipitation and / or salting-out treatment is combined with filtration through a gradient pore structure hollow fiber ultrafiltration membrane to remove cell debris, proteins, host genomic DNA, RNA, and endotoxins; In step (2), precipitation and / or salting-out treatment is combined with filtration through a gradient pore structure hollow fiber ultrafiltration membrane to remove enzymes, free nucleotides, ribonucleoside triphosphates, immunogenic impurities in the reaction system, exogenous RNA fragments, dsRNA, and small DNA fragments; In step (3), precipitation and / or salting-out treatment is combined with filtration through a gradient pore structure hollow fiber ultrafiltration membrane to remove unencapsulated or unloaded mRNA and other impurities.

3. The process for large-scale purification of mRNA vaccines using a gradient pore structure hollow fiber ultrafiltration membrane according to claim 2, characterized in that: In step (1), After cell lysis, the protein is denatured and precipitated, and the supernatant is extracted and filtered through a gradient pore structure hollow fiber ultrafiltration membrane to remove cell debris and endotoxins to obtain concentrated solution I; The host genomic DNA in the concentrated solution I is precipitated, and the supernatant is filtered through a gradient pore structure hollow fiber ultrafiltration membrane to obtain concentrated solution II; The RNA in the concentrated solution II was precipitated, and the supernatant was filtered through a gradient pore structure hollow fiber ultrafiltration membrane to obtain concentrated solution III; The plasmid DNA in the concentrated solution III is precipitated, the precipitated plasmid DNA is redissolved and filtered 2-3 times, and filtered using a gradient pore structure hollow fiber ultrafiltration membrane to obtain the final concentrated solution to obtain purified plasmid DNA.

4. The process for large-scale purification of mRNA vaccines using a gradient pore structure hollow fiber ultrafiltration membrane according to claim 3, characterized in that: In step (1), The reagent used for cell lysis is reagent II, including at least one of potassium hydroxide KOH, sodium hydroxide NaOH, sodium citrate, sodium methoxide CH3ONa, and potassium ethoxide CH3COOK. The concentration of reagent II is 0.01-5 mol / L, and the treatment time is 5-200 min. The reagents used for protein precipitation include reagent I, which includes at least one of a hydrochloric acid buffer Tris-HCl, a phosphate buffer Tris-H3PO4, an ethylenediaminetetraacetic acid buffer Tris-EDTA, and an acetate buffer Tris-CH3COOH, wherein the pH range of reagent I is 7-10 and the treatment time is 5-120 minutes; The reagent used to precipitate the host genomic DNA includes reagent III, which includes at least one of PVPK15, PVPK30, PVPK60, PVPK90, PVPK120, polyvinyl alcohol (PVA), and ethylene glycol. The concentration of reagent III is 0.001-1 mol / L, and the treatment time of reagent III is 10-60 min. The reagent used for precipitating RNA includes reagent IV, which includes at least one of calcium chloride (CaCl2), sodium chloride (NaCl), sodium nitrate (NaNO3), barium chloride (BaCl2), potassium nitrate (KNO3), and calcium nitrate (Ca(NO3)2). The concentration of reagent IV is 0.01-5 mol / L, and the treatment time of reagent IV is 10-180 min. The reagent used for precipitating plasmid DNA with concentrated solution III includes reagent V, which includes at least one of PEG200, PEG400, PEG800, PEG1000, PEG2000, PEG4000, PEG8000, PEG10000, and PEG20000, with a concentration of 0.01-5 mol / L and a treatment time of 10-60 min.

5. The process for large-scale purification of mRNA vaccines using a gradient pore structure hollow fiber ultrafiltration membrane according to claim 2, characterized in that: In step (2), The target mRNA solution is directly filtered through a gradient pore structure hollow fiber ultrafiltration membrane to remove free nucleotides, ribonucleoside triphosphates, and immunogenic impurities in the reaction system to obtain concentrated solution IV; The obtained concentrated solution IV is treated with DNA enzyme, and then the enzyme and protein are salted out. After salting out, the supernatant is filtered through a hollow fiber ultrafiltration membrane with a gradient pore structure to obtain concentrated solution V; The exogenous RNA in the concentrated solution V is precipitated, and the supernatant is filtered through a gradient pore structure hollow fiber ultrafiltration membrane to obtain a concentrated solution VI; The dsRNA in the concentrated solution VI is precipitated, and the supernatant is filtered through a gradient pore structure hollow fiber ultrafiltration membrane to obtain concentrated solution VII; The mRNA in the concentrated solution VII is subjected to salt precipitation and directly filtered through a gradient pore structure hollow fiber ultrafiltration membrane to obtain a concentrated solution. The mRNA in the concentrated solution is redissolved and filtered 2-3 times to obtain the final concentrated solution to obtain purified mRNA.

6. The process for large-scale purification of mRNA vaccines using a gradient pore structure hollow fiber ultrafiltration membrane according to claim 5, characterized in that: In step (2), The reagent used for the salting-out enzyme includes reagent VI, which includes at least one of sodium dodecylsulfonate, sodium dodecylbenzenesulfonate, sodium hexadecylsulfonate, sodium hexadecylbenzenesulfonate, and sodium ligninsulfonate. The concentration of reagent VI is 0.01-5 mol / L, and the treatment time of reagent VI is 10-60 min. The reagent used to precipitate exogenous RNA includes reagent VII, which includes at least one of colchicine, 2-hydroxyethylamine, triisopropanolamine, tert-butylamine, and isobutylamine. The concentration of reagent VII is 0.01-2 mol / L, and the treatment time of reagent VII is 10-40 min. The reagent used for precipitating dsRNA includes reagent VIII, which includes at least one of ethylene glycol, ethanol, n-butanol, glycerol, acetone, and propanol. The concentration of reagent VIII is 0.01-5 mol / L, and the treatment time of reagent VIII is 10-100 minutes. The reagent used for salting out mRNA includes reagent IX, which includes at least one of ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, ammonium carbonate, and ammonium bicarbonate. The concentration of reagent IX is 0.01-5 mol / L, and the treatment time of reagent IX is 10-180 min.

7. The process for large-scale purification of mRNA vaccines using a gradient pore structure hollow fiber ultrafiltration membrane according to claim 2, characterized in that: In step (3), The mRNA encapsulated or loaded solution is directly filtered through a gradient pore structure hollow fiber ultrafiltration membrane to remove other small molecular impurities to obtain concentrated solution VIII; The unencapsulated or unloaded mRNA in the concentrate VIII is subjected to salting out. After salting out, the supernatant is filtered using a gradient pore structure hollow fiber ultrafiltration membrane to obtain a concentrate, which is the purified encapsulated / transferred mRNA.

8. The process for large-scale purification of mRNA vaccines using a gradient pore structure hollow fiber ultrafiltration membrane according to claim 7, characterized in that: In step (3), The reagent used for salting out unencapsulated or unloaded mRNA includes reagent X, which includes at least one of tetramethylammonium chloride, dodecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, and spermidine. The concentration of reagent X is 0.01-5 mol / L, and the treatment time of reagent X is 10-100 min.

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