Rapid mRNA purification method and application thereof

Abstract

The invention discloses an mRNA (messenger ribonucleic acid) rapid purification method and application thereof. The method comprises the following steps: mixing, stirring and adsorbing polymer microspheres, a high-salt solution, an organic solvent, a buffer solution and an mRNA dissolving solution. According to the method, the mRNA is purified in a static mode of adsorption and elution of the polymer microspheres, the mRNA can be rapidly separated from the reaction liquid in a short time only through one-step purification, a substrate, related polymerase and other components can be effectively removed, the recovery rate can reach 90% or above, a solvent used in the process is safe and non-toxic, and the method is suitable for drug purification and production processes and has good application prospects. And compared with a column chromatography experiment, the purification is more flexible, and the steps are simple.

Description

Technical Field

[0001] The invention belongs to the technical field of drug purification and relates to a method for rapid purification of mRNA and application thereof. Background Art

[0002] As a vaccine, mRNA has natural advantages that traditional inactivated virus vaccines cannot match, such as high efficiency, safety, and controllable production quality. However, the mRNA production process, especially the purification link, is relatively challenging. Most therapeutic mRNAs are prepared by the in vitro reaction IVT method, and the mRNA prepared by this method contains a variety of impurities. These include 4 conventional base substrates, linearized template DNA, Escherichia coli HCP / HCD, polymerase, host cell protein for polymerase production, enzyme protector BSA, incomplete mRNA fragments, double-stranded RNA (dsRNA), etc. At present, the more common methods are precipitation (lithium chloride, ethanol, etc.) and chromatography (affinity, ion, hydrophobic, ultrafiltration, CHT, CORE700, etc.). However, the chromatography method has some effect on larger aggregates or smaller fragments, but it is helpless for some impurities of comparable size. At the same time, the industrialization of this method is difficult to scale up. The ultrafiltration method is time-consuming, complicated to operate, and has a low capture rate.

[0003] In summary, there is an urgent need to provide a rapid purification method for mRNA with high recovery rate and high throughput. Summary of the invention

[0004] In view of the deficiencies in the prior art and actual needs, the present invention provides a method for rapid purification of mRNA and its application. The static adsorption mode is adopted to quickly capture mRNA molecules after the IVT reaction, and the IVT reaction substrate and the polymerase and protective agent in the reaction process can be efficiently removed. The protein removal can reach 2 log values. The operation is simple and the cost is low. It is suitable for small-scale tests and industrial scale-up, and the recovery rate is higher than that of chromatography and other methods.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for rapid purification of mRNA, the method comprising:

[0007] The polymer microspheres, high salt solution, organic solvent, buffer solution and mRNA solution are mixed and stirred for adsorption.

[0008] The static method of purifying mRNA by polymethacrylate microsphere adsorption elution of the present invention can quickly separate mRNA from the reaction solution in a short time with only one step of purification, and can effectively remove components such as substrates and related polymerases, with a recovery rate of more than 90%. The solvent used in the process is safe and non-toxic, suitable for drug purification and production processes, and its purification is more flexible and simpler than column chromatography experiments. The method is fast and efficient, and can obtain mRNA of higher purity, solving the problems of high cost and long process time of conventional affinity chromatography.

[0009] The UniCore10CM microspheres in the present invention are non-porous, 10-micron-diameter monodispersed polystyrene microspheres with carboxyl functional groups bonded to the surface. The microsphere filler has good mechanical strength, high chemical stability, and is resistant to acids, alkalis, and basic chemical solvents. It can be used to enrich nucleic acids, especially mRNA, by static adsorption. It can achieve rapid capture of mRNA by static binding in one step, with protein removal close to 99% and a recovery rate greater than 90%.

[0010] The adsorption principle of UniCore10CM microspheres is that the appropriate ratio of PEG and sodium chloride concentrations, coupled with the cationic bridge formed by divalent metal ions, enables the mRNA to interact with the COO-functional group of the microspheres, thereby producing an adsorption effect.

[0011] Sodium chloride and PEG are used as chaotropic agents, which can promote the precipitation of mRNA or adsorption on the surface of microspheres during the adsorption process. The principle is that the water-stripping effect and the encapsulating molecules reduce the exposure of the charged surface of the molecules, which easily causes the mRNA molecules to bind and precipitate.

[0012] The 1000nt mRNA test depends on a certain concentration of sodium chloride and PEG, and both are positively correlated. When the concentration of NaCl is 0.5M and the concentration of PEG4000 is less than 10%, the mRNA binding efficiency is not high; increasing the concentration of NaCl or PEG4000 will increase the mRNA binding efficiency. When the concentration of NaCl is not less than 0.5M and the concentration of PEG4000 is greater than 10%, the mRNA binding efficiency is greater than 60%; when the concentration of NaCl is 2M and the concentration of PEG4000 is 10%, the mRNA binding efficiency is greater than 80%; when the concentration of PEG4000 is increased to 15%, the mRNA binding efficiency can also be greater than 80%.

[0013] Preferably, the source of the mRNA includes an in vitro transcription synthesis reaction solution of mRNA or a solution of a crude mRNA after precipitation.

[0014] Preferably, the polymer microspheres include any one of polymethacrylate microspheres, polystyrene microspheres, polystyrene divinylbenzene microspheres or polyacrylate microspheres, or a combination of at least two thereof.

[0015] Preferably, the surface of the polymer microspheres is modified with carboxyl groups and / or sulfopropyl groups.

[0016] Preferably, the polymer microspheres include UniCore10CM.

[0017] Preferably, the in vitro transcription synthesis reaction solution of the mRNA contains any one or a combination of at least two of a nucleotide substrate, a template, a metal ion, a polymerase or a protein protective agent.

[0018] Preferably, the buffer solution comprises Tris-HCl.

[0019] Preferably, the organic solvent comprises polyethylene glycol.

[0020] Preferably, the high salt solution includes any one of sodium chloride solution, potassium chloride solution, lithium chloride solution, sodium sulfate solution or potassium sulfate solution, or a combination of at least two thereof.

[0021] Preferably, the concentration of the high salt solution is 0.5-2.5M.

[0022] The point values ​​in the above 0.5-2.5M can be specifically selected as 0.5M, 0.6M, 0.8M, 1.0M, 1.2M, 1.4M, 1.6M, 1.8M, 2.0M, 2.2M, 2.5M, etc.

[0023] Preferably, the buffer solution comprises Tris-HCl.

[0024] Preferably, the concentration of Tris-HCl is 15-25 mM.

[0025] The point values ​​in the above 15-25mM can specifically be selected as 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 2.5mM, etc.

[0026] Preferably, the pH of the Tris-HCl is 6.5-7.5.

[0027] The specific point values ​​among 6.5-7.5 mentioned above can be 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, etc.

[0028] Preferably, the buffer solution further comprises divalent metal ions.

[0029] Preferably, the divalent metal ions include Mg 2+ and / or Ca 2+ .

[0030] Preferably, the concentration of divalent metal ions in the buffer solution is 20-40 mM.

[0031] The point values ​​in the above 20-40mM can specifically be selected as 20mM, 22mM, 24mM, 26mM, 28mM, 30mM, 32mM, 34mM, 36mM, 38mM, 40mM, etc.

[0032] Preferably, the method further comprises rinsing.

[0033] Preferably, the solution used for rinsing comprises an ethanol solution.

[0034] Preferably, the volume percentage of the ethanol solution is 40%-60%.

[0035] The point values ​​between 40% and 60% mentioned above may specifically be 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60% and the like.

[0036] Preferably, the method further comprises eluting.

[0037] Preferably, the solution used for elution comprises Tris-HCl and / or EDTA.

[0038] Preferably, the pH of the solution used for elution is 6-8.

[0039] The specific point values ​​among 6-8 mentioned above can be 6.0, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, etc.

[0040] Preferably, the concentration of Tris-HCl in the solution used for elution is 15-25 mM.

[0041] The point values ​​in the above 15-25mM can specifically be selected as 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 2.5mM, etc.

[0042] Preferably, the concentration of EDTA in the solution used for elution is 3-10 mM.

[0043] The point values ​​in the above 3-10mM can specifically be selected from 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, etc.

[0044] In a second aspect, the present invention provides the use of the rapid mRNA purification method described in the first aspect in isolating and purifying mRNA.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The static method of purifying mRNA by polymethacrylate microsphere adsorption elution of the present invention can quickly separate mRNA from the reaction solution in a short time with only one step of purification, and can effectively remove components such as substrates and related polymerases, with a recovery rate of more than 90%, a purification yield of more than 90%, and a removal rate of impurity proteins of 100%. The solvent used in the process is safe and non-toxic, suitable for drug purification and production processes, and its purification is more flexible and has simple steps compared to column chromatography experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 A bar chart comparing the mRNA binding efficiencies of the DOE experimental design groups in Example 1 of the present invention;

[0048] Figure 2 It is a response interaction curve diagram of the effect of NaCl and PEG4000 concentrations on binding efficiency in Example 1 of the present invention;

[0049] Figure 3 It is a response curve diagram of the effect of NaCl and PEG4000 concentrations on binding efficiency in Example 1 of the present invention;

[0050] Figure 4 is a contour plot of the binding efficiency and the concentrations of PEG4000 and NaCl in Example 1 of the present invention;

[0051] Figure 5 This is a comparison diagram of mRNA recovery rates of elution buffers with different pH values ​​in Example 2 of the present invention;

[0052] Figure 6 Comparison of mRNA elution amounts of elution buffers with different pH values ​​in Example 2 of the present invention;

[0053] Figure 7 It is a line graph of mRNA loss under different concentrations of ethanol elution conditions in Example 3 of the present invention;

[0054] Figure 8 It is a line graph of mRNA recovery rate under different concentrations of ethanol elution conditions in Example 3 of the present invention;

[0055] Fig. 9 This is a trend diagram of the flow-through of mRNA when different divalent metal salt ions are statically adsorbed on mRNA in Unicore 10CM in Example 4 of the present invention;

[0056] Fig.10 This is a trend diagram of the elution amount of target mRNA when different divalent metal salt ions are statically adsorbed on mRNA by Unicore 10CM in Example 4 of the present invention;

[0057] Fig.11 This is the total electrophoresis diagram of the 5200 nucleic acid fragment analyzer of the Unicore 10CM statically adsorbed 1000nt mRNA in Example 5 of the present invention;

[0058] Fig. 12A This is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using Abuffer static adsorption of 1000nt mRNA in Unicore 10CM in Example 5 of the present invention;

[0059] Fig. 12B This is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using Abuffer static adsorption of 1000nt mRNA in Unicore 10CM in Example 5 of the present invention;

[0060] Fig. 12C This is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using Abuffer static adsorption of 1000nt mRNA in Unicore 10CM in Example 5 of the present invention;

[0061] Fig.13A This is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using B buffer to statically adsorb 1000nt mRNA using Unicore 10CM in Example 5 of the present invention;

[0062] Fig. 13B This is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using B buffer to statically adsorb 1000nt mRNA using Unicore 10CM in Example 5 of the present invention;

[0063] Fig. 13C This is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using B buffer to statically adsorb 1000nt mRNA using Unicore 10CM in Example 5 of the present invention;

[0064] Fig.14 This is the total electrophoresis diagram of the 5200 nucleic acid fragment analyzer of the Unicore 10CM statically adsorbed 4000nt mRNA in Example 5 of the present invention;

[0065] Fig.15A This is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using the A buffer to statically adsorb 4000nt mRNA using Unicore 10CM in Example 5 of the present invention;

[0066] Fig. 15BThis is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using the A buffer to statically adsorb 4000nt mRNA using Unicore 10CM in Example 5 of the present invention;

[0067] Fig. 15C This is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using the A buffer to statically adsorb 4000nt mRNA using Unicore 10CM in Example 5 of the present invention;

[0068] Fig.16A This is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using B buffer to statically adsorb 4000nt mRNA using Unicore 10CM in Example 5 of the present invention;

[0069] Fig. 16B This is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using B buffer to statically adsorb 4000nt mRNA using Unicore 10CM in Example 5 of the present invention;

[0070] Fig. 16C This is a purity and integrity analysis chart of the 5200 nucleic acid fragment analyzer using B buffer to statically adsorb 4000nt mRNA using Unicore 10CM in Example 5 of the present invention;

[0071] Fig.17 This is a BSA standard curve diagram for the quantitative analysis of NanoOrange protein in Example 5 of the present invention;

[0072] Fig.18 This is the experimental spectrum of the NanoGel dT20 column experiment in Example 6 of the present invention. DETAILED DESCRIPTION

[0073] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0074] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0075] Example 1

[0076] Sodium chloride and PEG concentration studies.

[0077] 1 Experimental parameters

[0078] 1.1 Buffer composition

[0079] 1.1.1 Binding buffer

[0080] Binding buffers with different concentrations of NaCl and PEG4000 were prepared according to Table 1.

[0081] Table 1

[0082]

[0083]

[0084] 1.1.2 Washing buffer

[0085] 60% EtOH

[0086] 1.1.3 Elution buffer

[0087] 20mM Tris-HCl, 5mM EDTA, pH 7.0.

[0088] 1.3 Sample processing

[0089] Sample mRNA (1000 nt, 0.5 mg / mL) was diluted according to Table 2.

[0090] Table 2

[0091]

[0092] 2 Experimental process:

[0093] (1) UniCore 10CM (manufactured by Suzhou Nanovita Technology Co., Ltd.), fixed volume to 1 mL, add 4 mL of ultrapure water, and mix in a centrifuge tube;

[0094] (2) Take the corresponding spin column, i.e., a 0.45 μm wwPTFE centrifugal device (PALL, catalog number: ODPTFE04C34), mark it, and dispense 100 μL of the mixed filler into each tube of the marked spin column, i.e., each column volume (CV) is 20 μL;

[0095] (3) After centrifugation, add 20CV Binding Buffer, mix well and balance for 10 min, centrifuge and discard the supernatant;

[0096] (4) Based on the concentration of the diluted mRNA sample, the sample volume is 20 μg, aspirated and mixed, let stand for 10 minutes, and centrifuged; the supernatant is collected and marked;

[0097] (5) Add 5CV of 60% ethanol solution for elution, mix well, let stand for 10 min, centrifuge; collect the supernatant and mark;

[0098] (6) Add 2.5 CV Elution Buffer for elution, mix well, let stand for 10 min, centrifuge; collect the supernatant and mark;

[0099] (7) FT, Wash, and Elu samples were collected using NanoDrop TM One / OneC micro-volume UV-Vis spectrophotometer (ThermoFisher, catalog number: 701-058112) was used to detect and record the reading of each tube (concentration, A260 / 280, A260 / 230).

[0100] 3 Experimental results

[0101] DOE design grouping and the combined efficiency of each group are as follows Figure 1 As shown, when the NaCl concentration is 0.5M and the PEG 4000 concentration is 5%, the mRNA binding efficiency is not high. When the PEG 4000 concentration is increased to 10%, the mRNA binding efficiency does not increase significantly. When the PEG4000 concentration is increased to 15%, the mRNA binding efficiency increases significantly. When the PEG 4000 concentration remains unchanged and the NaCl concentration is increased to 1.25M, the mRNA binding efficiency decreases. When the NaCl concentration is 1.25M and the PEG 4000 concentration is 10%, the mRNA binding efficiency is greater than 70%, and the mRNA binding efficiency is significantly improved when the PEG 4000 concentration is increased to 10%. When the NaCl concentration is 2M and the PEG 4000 concentration is 5%, the mRNA binding efficiency can reach 50%, and the mRNA binding efficiency increases with the increase of the PEG 4000 concentration. The response interaction curve of the effect of NaCl and PEG4000 concentrations on the binding efficiency is shown in the figure. Figure 2 As shown in the figure, when the concentration of PEG 4000 is 5%, the concentration of NaCl increases from 0.5M to 2.5M, and the mRNA binding efficiency does not increase significantly; when the concentration of PEG 4000 is 20%, the concentration of NaCl increases from 0.5M to 2.5M, and the mRNA binding efficiency increases significantly; mRNA testing depends on a certain concentration of sodium chloride and PEG, and both are positively correlated. The effect of PEG 4000 concentration is more obvious, followed by NaCl. The response curve of the effect of NaCl and PEG4000 concentration on binding efficiency is shown in the figure. Figure 3As shown, the mRNA binding efficiency increases with the increase of NaCl concentration, and the mRNA binding efficiency increases with the increase of PEG 4000 concentration. There is an interaction between sodium chloride concentration and PEG concentration on the binding of mRNA to UniCore 10CM. The change of sodium chloride concentration under high PEG concentration brings a greater increase in binding capacity. The contour line diagram of binding efficiency and PEG4000 and NaCl concentration is shown in Figure 4 The mRNA binding efficiency shown is positively correlated with the concentration of NaCl and PEG 4000. When the concentration of PEG 4000 is greater than 12.5% ​​and the concentration of NaCl is greater than 1.5M, the mRNA binding efficiency is better, which is above 60%.

[0102] Example 2

[0103] Effect of pH on mRNA purification.

[0104] 1. Experimental parameters

[0105] 1.2 Buffer composition

[0106] 1.2.1Binding Buffer Recipe

[0107] 20mM Tris-HCl, 15mM MgCl 2 ,5mM EDTA, 1.5M NaCl, 12.5% ​​PEG4000, pH7.0.

[0108] Prepare elution buffers with different pH values ​​according to Table 3.

[0109] Table 3

[0110]

[0111]

[0112] 1.3 Sample processing

[0113] Sample mRNA (1000 nt, 0.5 mg / mL) was diluted according to Table 4.

[0114] Table 4

[0115] Components Dosage Final concentration Binding Buffer Make up to 100 μL mRNA (1000nt, 0.5mg / mL) 40μL 0.2mg / mL BSA (2 mg / mL) 10μL 0.2mg / mL

[0116] 2 Experimental operation

[0117] (1) UniCore 10CM (manufactured by Suzhou Nanovita Technology Co., Ltd.), fixed volume to 1 mL, add 4 mL of ultrapure water, and mix in a centrifuge tube;

[0118] (2) Take the corresponding spin column, i.e., a 0.45 μm wwPTFE centrifugal device (PALL, catalog number: ODPTFE04C34), mark it, and dispense 100 μL of the mixed filler into each tube of the marked spin column, i.e., each column volume (CV) is 20 μL;

[0119] (3) After centrifugation, add 20CV Binding Buffer, mix well and balance for 10 min, centrifuge and discard the supernatant;

[0120] (4) Based on the concentration of the diluted mRNA sample, the sample volume is 20 μg, aspirated and mixed, let stand for 10 minutes, and centrifuged; the supernatant is collected and marked;

[0121] (5) Add 2.5 CV Elution Buffer for elution, mix well, let stand for 10 min, centrifuge; collect the supernatant and mark;

[0122] (6) FT, Wash, and Elu samples were collected using NanoDrop TM One / OneC micro-volume UV-Vis spectrophotometer (ThermoFisher, catalog number: 701-058112) was used to detect and record the reading of each tube (concentration, A260 / 280, A260 / 230).

[0123] 3 Experimental results

[0124] The recovery rates of elution with different pH elution buffers are shown in Table 5.

[0125] Table 5

[0126]

[0127]

[0128] Comparison of mRNA recovery rates of elution buffers with different pH values Figure 5 As shown in Figure 2, the mRNA elution amount of elution buffers with different pH values ​​is compared as shown in Figure 2. Figure 6 As shown in the figure, the mRNA recovery rate of elution buffers with different pH values ​​was compared and it was found that the elution buffer was DEPC H 2 When the pH value is 0, the mRNA recovery rate is the highest, which can reach 72%, and the elution volume is the highest, which can reach 14.35μg, but the mRNA storage time in water is short. When the pH value of the eluent is 6, the recovery rate is the lowest, and the mRNA elution volume is the lowest. When the pH value of the eluent is 7 or 8, the recovery rate is similar, but when the pH value is 8, the eluent is alkaline and the mRNA is unstable. Therefore, 20mM Tris-HCl, 5mM EDTA, pH 7.0 are selected as the eluent.

[0129] Example 3

[0130] Effect of eluent ethanol concentration on purification.

[0131] 1 Experimental parameters

[0132] 1.1 Buffer composition

[0133] 1.1.1Binding Buffer

[0134] 20mM Tris-HCl, 15mM MgCl 2 ,1.5M NaCl, 12.5% ​​PEG4000, pH7.0.

[0135] 1.1.2Washing Buffer

[0136] ①0% EtOH; ②10% EtOH; ③20% EtOH; ④30% EtOH; ⑤40% EtOH; ⑥60% EtOH

[0137] 1.1.3Elution Buffer

[0138] DEPC H 2 0

[0139] 1.2 Sample processing:

[0140] Sample mRNA (1000 nt, 0.5 mg / mL) was diluted according to Table 6.

[0141] Table 6

[0142] Components Dosage Final concentration Binding Buffer Make up to 100 μL / mRNA (1000nt, 0.5mg / mL) 40μL 0.2mg / mL BSA (2 mg / mL) 10μL 0.2mg / mL

[0143] 2 Experimental operation:

[0144] (1) UniCore 10CM (produced by Suzhou Nanovita Technology Co., Ltd.), adjust the volume to 1 ml, add 4 ml of ultrapure water, and mix in a centrifuge tube.

[0145] (2) Take the corresponding spin column, i.e., a 0.45 μm wwPTFE centrifugal device (PALL, catalog number: ODPTFE04C34), mark it, and dispense 100 μL of the mixed filler into each tube of the marked spin column, i.e., the volume (CV) of each tube is 20 μL.

[0146] (3) After centrifugation, add 20CV Binding Buffer, mix well, equilibrate for 10 min, and centrifuge to discard the supernatant.

[0147] (4) Based on the concentration of the diluted mRNA sample, the sample loading amount is 20 μg. After aspiration and mixing, let it stand for 10 minutes and centrifuge. Collect the supernatant and mark it.

[0148] (5) Add 5CV of ethanol solution of the corresponding test concentration for elution, mix well, let stand for 10 minutes, and centrifuge; collect the supernatant and mark it.

[0149] (6) Add 2.5 CV Elution Buffer for elution, mix well, let stand for 10 min, and centrifuge; collect the supernatant and mark it.

[0150] (7) FT, Wash, and Elu samples were collected using NanoDrop TM One / OneC micro-volume UV-Vis spectrophotometer (ThermoFisher, catalog number: 701-058112) was used to detect and record the reading of each tube (concentration, A260 / 280, A260 / 230)

[0151] 3 Experimental results

[0152] Conclusion: When the ethanol concentration of the washing buffer is above 60%, there is little loss of mRNA

[0153] The line graph of mRNA loss under different ethanol concentrations is shown in Figure 7 As shown in the figure, the mRNA loss under different concentrations of ethanol elution conditions is compared and it is found that as the ethanol concentration increases, the mRNA loss decreases accordingly, and when the ethanol concentration reaches more than 60%, there is basically no loss of mRNA. The mRNA recovery rate under different concentrations of ethanol elution conditions is shown in the figure. Figure 8 As shown, by comparing the mRNA recovery rates under different ethanol elution conditions, it was found that as the ethanol concentration increased, the mRNA recovery rate increased accordingly, and when the ethanol concentration reached more than 60%, there was basically no loss of mRNA.

[0154] Example 4

[0155] Effects of metal ions on mRNA binding efficiency.

[0156] 1 Experimental parameters

[0157] 1.1 Buffer composition

[0158] 1.1.1Binding Buffer

[0159] Prepare Binding Buffer stock solution according to Table 7

[0160] Table 7

[0161]

[0162] Dilute to Mg 2+ , Ca 2+ To 0mM, 5mM, 10mM, 15mM, 20mM, 30mM, 50mM, 70mM, 100mM.

[0163] 1.1.2Washing Buffer

[0164] 60% EtOH

[0165] 1.1.3Elution Buffer

[0166] 20M Tris-HCl, 5mM EDTA, pH 7.0

[0167] 1.2 Sample processing

[0168] Sample mRNA (1000 nt, 0.5 mg / ml) was diluted according to Table 8.

[0169] Table 8

[0170] Components Dosage Final concentration Binding Buffer Make up to 100 μL / mRNA (1000nt, 0.5mg / mL) 40μL 0.2mg / mL BSA (2 mg / mL) 10μL 0.2mg / mL

[0171] 2 Experimental operation

[0172] (1) UniCore 10CM (manufactured by Suzhou Nanovita Technology Co., Ltd.), fixed volume to 1 mL, add 4 mL of ultrapure water, and mix in a centrifuge tube;

[0173] (2) Take the corresponding spin column, i.e., a 0.45 μm wwPTFE centrifugal device (PALL, catalog number: ODPTFE04C34), mark it, and dispense 100 μL of the mixed filler into each tube of the marked spin column, i.e., each column volume (CV) is 20 μL;

[0174] (3) After centrifugation, add 20CV Binding Buffer, mix well and balance for 10 min, centrifuge and discard the supernatant;

[0175] (4) Based on the concentration of the diluted mRNA sample, the sample volume is 20 μg, aspirated and mixed, let stand for 10 minutes, and centrifuged; the supernatant is collected and marked;

[0176] (5) Add 5CV of 60% ethanol solution for elution, mix well, let stand for 10 min, centrifuge; collect the supernatant and mark;

[0177] (6) Add 2.5 CV Elution Buffer for elution, mix well, let stand for 10 min, centrifuge; collect the supernatant and mark;

[0178] (7) FT, Wash, and Elu samples were collected using NanoDrop TM One / OneC micro-volume UV-Vis spectrophotometer (ThermoFisher, catalog number: 701-058112) was used to detect and record the reading of each tube (concentration, A260 / 280, A260 / 230).

[0179] 3 Experimental results

[0180] The mRNA flux trend diagram when different divalent metal salt ions are statically adsorbed on Unicore 10CM is shown in the figure below. Fig. 9 As shown in the figure, the mRNA flow-through amount when different divalent metal salt ions were statically adsorbed on Unicore 10CM was compared. The mRNA flow-through amount decreased with the increase of divalent metal salt ion concentration. When the divalent metal salt ion concentration reached 30mM, the mRNA basically did not flow through. The trend graph of the elution amount of the target mRNA when different divalent metal salt ions were statically adsorbed on Unicore 10CM is shown in the figure. Fig.10 As shown, the elution amount of target mRNA when different divalent metal salt ions were statically adsorbed on Unicore 10CM was compared. The elution amount of mRNA increased with the increase of divalent metal salt ion concentration. When the divalent metal salt ion concentration reached 30mM, mRNA basically did not flow through.

[0181] Example 5

[0182] mRNAs of different lengths were tested under the optimal combination of parameters.

[0183] 1 Experimental parameters

[0184] 1.1 Buffer composition

[0185] 1.1.1Binding Buffer

[0186] Prepare two Binding Buffers according to Table 9, and the components are selected according to the response curves of NaCl and PEG4000 in the DOE design.

[0187] Table 9

[0188] Serial number Binding Buffer Recipe A <![CDATA[20mM Tris-HCl,30mM MgCl 2 ,1.0M NaCl,15%PEG4000,pH7.0]]> B <![CDATA[20mM Tris-HCl,30mM MgCl 2 ,2.0M NaCl,12.5%PEG4000,pH7.0]]>

[0189] 1.1.2Washing Buffer

[0190] 60% EtOH

[0191] 1.1.3Elution Buffer

[0192] 20M Tris-HCl, 5mM EDTA, pH 7.0

[0193] 1.2 Sample processing

[0194] 1.2.11000nt mRNA

[0195] Sample mRNA (1000 nt, 0.5 mg / mL) was diluted according to Table 10.

[0196] Table 10

[0197] Components Dosage Final concentration Binding Buffer Make up to 100 μL / mRNA (1000nt, 0.5mg / mL) 40μL 0.2mg / mL BSA (2 mg / mL) 10μL 0.2mg / mL

[0198] 1.2.24000nt mRNA

[0199] Sample mRNA (4000 nt, 1.9 mg / mL) was diluted according to Table 11.

[0200] Table 11

[0201] Components Dosage Final concentration Binding Buffer Make up to 100 μL / mRNA (4000 nt, 1.9 mg / mL) 10.5μL 0.2mg / mL BSA (2 mg / mL) 10μL 0.2mg / mL

[0202] 2 Experimental operation:

[0203] (1) UniCore 10CM (manufactured by Suzhou Nanovita Technology Co., Ltd.), fixed volume to 1 mL, add 4 mL of ultrapure water, and mix in a centrifuge tube;

[0204] (2) Take the corresponding spin column, i.e., a 0.45 μm wwPTFE centrifugal device (PALL, catalog number: ODPTFE04C34), mark it, and dispense 100 μL of the mixed filler into each tube of the marked spin column, i.e., each column volume (CV) is 20 μL;

[0205] (3) After centrifugation, add 20CV Binding Buffer, mix well and balance for 10 min, centrifuge and discard the supernatant;

[0206] (4) Based on the concentration of the diluted mRNA sample, the sample volume is 20 μg, aspirated and mixed, let stand for 10 minutes, and centrifuged; the supernatant is collected and marked;

[0207] (5) Add 5CV of 60% ethanol solution for elution, mix well, let stand for 10 min, centrifuge; collect the supernatant and mark;

[0208] (6) Add 2.5 CV Elution Buffer for elution, mix well, let stand for 10 min, centrifuge; collect the supernatant and mark;

[0209] (7) FT, Wash, and Elu samples were collected using NanoDrop TM One / OneC micro-volume UV-Vis spectrophotometer (ThermoFisher, catalog number: 701-058112) was used to detect and record the reading of each tube (concentration, A260 / 280, A260 / 230);

[0210] (8) The samples were tested by 5200 fragment analyzer (Agilent, catalog number: M5310AA), BCA protein quantification (Yisheng Bio, catalog number: 20201ES76) and NanoOrange TM The BSA removal efficiency was determined using a protein quantification kit (Invitrogen, catalog number: N6666).

[0211] 3 Experimental results

[0212] The binding efficiency of Nano Drop in detecting 1000nt mRNA is shown in Table 12.

[0213] Table 12

[0214]

[0215]

[0216] The binding efficiency of Nano Drop in detecting 4000nt mRNA is shown in Table 13.

[0217] Table 13

[0218]

[0219] The total electrophoresis of Unicore 10CM static adsorption of 1000nt mRNA 5200 nucleic acid fragment analyzer is as follows Fig.11 As shown, C11000nt-mRNA sample; C21000nt-Abuffer (ABuffer formula see Table 10) loading solution; C31000nt-Abuffer FT sample; C41000nt-Abuffer Wash sample; C5&C61000nt-Abuffer elution sample; C71000nt-Bbuffer (B Buffer formula see Table 10) loading solution; C81000nt-B buffer FT sample; C91000nt-B bufferWash sample; C10&C111000nt-B buffer elution sample.

[0220] Unicore 10CM static adsorption of 1000nt mRNA 5200 nucleic acid fragment analyzer purity and integrity analysis chart - Abuffer (20mM Tris-HCl, 30mM MgCl 2 ,1.0M NaCl,15%PEG4000,pH7.0) as shown in Figure 12, the left one is the original mRNA with a purity of 51.7%; the middle and right ones are eluted samples with purities of 63.2% and 63.7% respectively.

[0221] The purity and integrity analysis chart of Unicore 10CM static adsorption of 1000nt mRNA on 5200 nucleic acid fragment analyzer - B buffer (20mM Tris-HCl, 30mM MgCl2, 1.0M NaCl, 15% PEG4000, pH7.0) is shown in Figure 13. The left one is the original mRNA with a purity of 51.7%; the middle and right ones are eluted samples with purities of 65.8% and 60.7% respectively.

[0222] The total electrophoresis diagram of Unicore 10CM static adsorption of 4000nt mRNA 5200 nucleic acid fragment analyzer is as follows Fig.14 As shown, D1 4000nt-mRNA sample; D2 4000nt-Abuffer (ABuffer formula see Table 10) loading solution; D3 4000nt-Abuffer FT sample; D4 4000nt-Abuffer Wash sample; D5 & D6 4000nt-Abuffer elution sample; D7 4000nt-B buffer (BBuffer formula see Table 10) loading solution; D8 4000nt-B buffer FT sample; D9 4000nt-B buffer Wash sample; D10 & D11 4000nt-B buffer elution sample,

[0223] Unicore 10CM static adsorption of 4000nt mRNA 5200 nucleic acid fragment analyzer purity and integrity analysis chart - Abuffer (20mM Tris-HCl, 30mM MgCl 2 , 1.0M NaCl, 15% PEG4000, pH7.0) as shown in Figure 15, the left one is the original sample of 4000nt mRNA, with a purity of 43.5%; the middle and right ones are eluted samples, with purities of 63.7% and 51.8% respectively.

[0224] Unicore 10CM static adsorption of 4000nt mRNA 5200 nucleic acid fragment analyzer purity and integrity analysis chart - Bbuffer (20mM Tris-HCl, 30mM MgCl 2, 2.0M NaCl, 12.5% ​​PEG4000, pH7.0) as shown in Figure 16, the left one is the original sample of 4000nt mRNA, with a purity of 43.5%; the middle and right ones are eluted samples, with purities of 54.6% and 48.2% respectively.

[0225] NanoOrange protein quantification BSA standard curve Fig.17 shown.

[0226] The results of NanoOrange protein quantification of 1000nt mRNA are shown in Table 14.

[0227] Table 14

[0228]

[0229] ABuffer (20mM Tris-HCl, 30mM MgCl 2 , 1.0M NaCl, 15% PEG4000, pH7.0), the BSA removal efficiency was 81% in the flow-through step, and 19% in the ethanol elution condition; B Buffer (20mM Tris-HCl, 30mM MgCl 2 , 2.0 M NaCl, 12.5% ​​PEG4000, pH 7.0), the BSA removal efficiency was 82% in the flow-through step, and the removal efficiency was 18% under ethanol elution conditions.

[0230] The results of NanoOrange protein quantification of 4000nt mRNA are shown in Table 15.

[0231] Table 15

[0232]

[0233]

[0234] ABuffer (20mM Tris-HCl, 30mM MgCl 2 , 1.0M NaCl, 15% PEG4000, pH7.0), the BSA removal efficiency in the flow-through step was 68%, and the removal efficiency under ethanol elution conditions was 32%; B Buffer (20mM Tris-HCl, 30mMMgCl 2 , 2.0 M NaCl, 12.5% ​​PEG4000, pH 7.0), the BSA removal efficiency was 71% in the flow-through step, and the removal efficiency was 29% under ethanol elution conditions.

[0235] Comparative Example 1

[0236] NanoGel dT20 on-column experiment

[0237] 1 Experimental parameters

[0238] 1.1 Buffer composition

[0239] 1.1.1 Balance solution

[0240] 10mM Tris-HCl+0.5M NaCl+1mM EDTApH7.4

[0241] 1.1.2 Eluent

[0242] DI water

[0243] 1.1.2 Regeneration fluid

[0244] 0.1M NaOH

[0245] 1.2 Sample processing

[0246] Sample mRNA (1000 nt, 0.5 mg / mL) was diluted according to Table 12.

[0247] Table 12

[0248]

[0249]

[0250] 1.3 Materials and instruments

[0251] 1.3.1 Instrument: AKTAexplorer

[0252] 1.3.2 Filler: NanoGel dT20 prepacked column 1mL

[0253] 2 Experimental operation

[0254] (1) First, equilibrate the chromatography column with 5CV equilibration buffer, then load the protein, collect the sample solution, and mark it;

[0255] (2) After loading, wash with 5 CV of equilibration buffer, collect the eluate, and mark it;

[0256] (3) Then elute with 5CV elution buffer, collect the eluate, and label it;

[0257] (4) The column was regenerated with 0.1 M NaOH for 5 CV and then equilibrated with 5 CV of equilibration buffer. Fig.18 ;

[0258] (5) FT, Wash, and Elu samples were collected using NanoDrop TMOne / OneC micro-volume UV-Vis spectrophotometer (ThermoFisher, catalog number: 701-058112) was used to detect and record the reading of each tube (concentration, A260 / 280, A260 / 230);

[0259] (6) The samples were tested by 5200 fragment analyzer (Agilent, catalog number: M5310AA), BCA protein quantification (Yisheng Bio, catalog number: 20201ES76) and NanoOrange TM The BSA removal efficiency was determined using a protein quantification kit (Invitrogen, catalog number: N6666).

[0260] 3 Experimental results

[0261] The recovery rate of the NanoGel dT20 column experiment was 78%. Compared with the static adsorption experiment, it was found that the static adsorption method was fast and efficient, and could obtain mRNA with higher purity, solving the problem of high cost and long process time of conventional affinity chromatography.

[0262] In summary, the static method of purifying mRNA by polymethacrylate microsphere adsorption elution of the present invention can quickly separate mRNA from the reaction solution in a short time with only one purification step, and can effectively remove components such as substrates and related polymerases, with a recovery rate of more than 90%. The solvent used in the process is safe and non-toxic, and is suitable for drug purification and production processes. Compared with column chromatography experiments, its purification is more flexible and the steps are simple.

[0263] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for rapid purification of mRNA, characterized in that: The method comprises: The polymer microspheres, high salt solution, organic solvent, buffer solution and mRNA solution are mixed and stirred for adsorption.

2. The method according to claim 1, characterized in that The source of the mRNA includes an in vitro transcription synthesis reaction solution of mRNA or a solution of a crude mRNA after precipitation; Preferably, the polymer microspheres include any one or a combination of at least two of polymethacrylate microspheres, polystyrene microspheres, polystyrene divinylbenzene microspheres or polyacrylate microspheres; Preferably, the surface of the polymer microspheres is modified with carboxyl groups and / or sulfopropyl groups; Preferably, the polymer microspheres include UniCore10CM.

3. The method according to claim 2, characterized in that The in vitro transcription synthesis reaction solution of the mRNA contains any one of nucleotide substrates, templates, metal ions, polymerase or protein protective agents, or a combination of at least two of them.

4. The method according to any one of claims 1 to 3, characterized in that: The organic solvent includes polyethylene glycol.

5. The method according to any one of claims 1 to 4, characterized in that: The high salt solution includes any one of sodium chloride solution, potassium chloride solution, lithium chloride solution, sodium sulfate solution or potassium sulfate solution, or a combination of at least two thereof; Preferably, the concentration of the high salt solution is 0.5-2.5M.

6. The method according to any one of claims 1 to 5, characterized in that: The buffer solution includes Tris-HCl; Preferably, the concentration of Tris-HCl is 15-25 mM; Preferably, the pH of the Tris-HCl is 6.5-7.

5.

7. The method according to any one of claims 1 to 6, characterized in that: The buffer solution also includes divalent metal ions; Preferably, the divalent metal ions include Mg 2+ and / or Ca 2+ ; Preferably, the concentration of divalent metal ions in the buffer solution is 20-40 mM.

8. The method according to any one of claims 1 to 7, characterized in that: The method further comprises rinsing; Preferably, the solution used for elution includes an ethanol solution; Preferably, the volume percentage of the ethanol solution is 40%-60%.

9. The method according to any one of claims 1 to 8, characterized in that: The method further comprises eluting; Preferably, the solution used for elution comprises Tris-HCl and / or EDTA; Preferably, the pH of the solution used for elution is 6-8; Preferably, the concentration of Tris-HCl in the solution used for elution is 15-25 mM; Preferably, the concentration of EDTA in the solution used for elution is 3-10 mM.

10. Use of the mRNA rapid purification method according to any one of claims 1 to 9 in separating and purifying mRNA.

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