Eluent and application thereof in purification of long-chain mRNA

Abstract

The invention relates to the technical field of biological medicines, in particular to an eluent and application thereof in purification of long-chain RNA (Ribonucleic Acid). The eluent is formed by mixing an acidic buffer solution 1 with the pH value of 3.0-6.6 and a monobasic strong base solution 2 with the concentration of 0.2-4M in proportion. Experiments show that the eluent provided by the invention realizes anion exchange chromatography purification of large-fragment RNA (greater than 300nt) in a normal temperature / room temperature environment (10-37 DEG C). A chaotropic agent (such as urea) is added into a chromatographic system before the eluent is used for elution, or the chaotropic agent is further added into the eluent for elution, the purification method provided by the invention is stable and easy to amplify, and a foundation is laid for subsequent large-scale RNA drug development.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an eluent and its application in purifying long-chain RNA. Background Art

[0002] In vitro transcription (IVT) is a process of preparing mRNA using linear DNA as a template. The final IVT product contains various components, such as template DNA, protease, and dsRNA, etc. Therefore, it is necessary to purify the IVT product to obtain pure mRNA for subsequent LNP packaging. However, the existing affinity chromatography purification methods for different templates may result in the situation that impurities such as dsRNA in the final product exceed the standard. Therefore, it is sometimes particularly important to add a second-step chromatography purification (such as hydrophobic chromatography and ion exchange chromatography).

[0003] However, due to its large molecular weight and charge, ribonucleic acid (RNA) often has the difficulty that it is easy to bind but difficult to elute in anion exchange chromatography purification. Therefore, at present, there are few cases of using anion exchange chromatography purification for RNA purification, and it is mostly used for short-sequence purification. In this regard, the industry often uses combined conditions such as high temperature, organic co-solvents, and ion-pair salts to assist elution to solve the above problems. However, both high temperature and organic co-solvents have safety risks in industrialization. Ion-pair salts are generally non-conventional salts, which require additional quality control processes, and ion-pair salts often also require high-temperature conditions. Therefore, it is very necessary to develop a method for purifying long-chain RNA (>300 nt) by anion exchange chromatography using industrial conventional reagents at room temperature. Summary of the Invention

[0004] In view of this, the present invention provides an eluent and its application in purifying long-chain RNA. Using the eluent of the present invention, the purification of long-chain RNA can be achieved at room temperature, and the RNA recovery rate can reach more than 95%. There is no need to raise the temperature of the buffer system, sample, and column incubator during the purification process. The purification method provided by the present invention is stable and easy to scale up, laying a foundation for subsequent large-scale RNA drug development.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] An eluent, characterized in that it comprises Solution 1 and Solution 2, wherein Solution 1 is an acidic buffer solution with a pH of 3.0 - 6.6, and Solution 2 is a 0.2 - 4M monobasic strong base solution.

[0007] In some embodiments, Solution 1 and Solution 2 can be directly formulated according to a certain volume ratio; in some embodiments, Solution 1 and Solution 2 can be connected to different pipelines through AKTA Avant 150 or other systems with similar principles, and different ratios of Solution 1 and Solution 2 can be set according to the program settings.

[0008] In some embodiments, Solution 1 of the present invention further contains 0.6 - 3.5 M monovalent Cl-ion salts. In some embodiments, the concentration of the monovalent Cl-ion salt can specifically be 0.6 M, 1 M, 1.8 M, 2.1 M, 3 M, 3.5 M.

[0009] Furthermore, in some embodiments, the monovalent Cl-ion salt is NaCl and / or KCl.

[0010] In some embodiments, Solution 1 is a 20 mM - 100 mM Cit-Buffer with a pH of 3.0 - 6.6 containing 0.6 - 3.5 M monovalent Cl-ion salts.

[0011] In some embodiments, Solution 1 is a 20 mM - 100 mM Cit-Buffer with a pH of 3.0 - 6.6 containing 1 - 3.5 M monovalent Cl-ion salts.

[0012] In some embodiments, in Solution 1, the pH of the acidic buffer is 3.0 - 6.6, specifically 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.2; the concentration of the buffer is 12 mM - 100 mM, specifically 12 mM, 15 mM, 18 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 100 mM; the acidic buffer is a citric acid - sodium citrate system, sodium dihydrogen phosphate - citric acid system, citric acid - sodium hydroxide - hydrochloric acid system, acetic acid - sodium acetate system, or glycine - hydrochloric acid system.

[0013] In some embodiments, Solution 1 further includes 2 - 8 M chaotropic agent. That is, Solution 1 is a 20 mM - 100 mM Cit-Buffer with a pH of 3.0 - 6.6 containing 0.6 - 3.5 M monovalent Cl-ion salts and 2 - 8 M chaotropic agent. The concentration of the chaotropic agent can specifically be 2 M, 2.4 M, 3.2 M, 4 M, 4.2 M, 4.8 M, 5 M, 6 M, 7 M, 8 M; the chaotropic agent is lithium perchlorate or other perchlorates, guanidine chloride or other guanidine salts, urea, butanol, ethanol, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, or one or several of them; in some specific embodiments, the chaotropic agent is urea.

[0014] In the eluent provided by the present invention, in the solution 2, the concentration of the strong monobase is 0.2 - 4M, specifically it can be 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M.

[0015] In some embodiments, the monovalent salt of Cl ion mentioned above is NaCl or KCl, the strong monobase is NaOH and / or KOH, and the chaotropic agent is urea or guanidine hydrochloride.

[0016] The eluent of the present invention is prepared by mixing solution 1 and solution 2, and when preparing the solution 2, its volume percentage is 10% - 80%. In some specific embodiments, the volume percentage of the eluent 2 when preparing is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%.

[0017] The present invention also provides a solution combination, including a balance solution, a wash solution, a sample dilution solution and the eluent of the present invention;

[0018] At least one of the balance buffer solution, the sample dilution solution, the wash solution and the eluent contains 2 - 8M chaotropic agent.

[0019] In the present invention, the addition of the chaotropic agent (such as urea) is not limited to the eluent. As long as urea exists in the system environment before elution, that is, urea can be introduced into any system solution in the equilibration, loading stage (adding the chaotropic agent to the sample dilution solution) and elution stage (adding the chaotropic agent to the wash solution). For example, at least one of the balance solution, the wash solution and the sample dilution buffer solution contains urea. Specifically, the solution combination provided by the present invention includes two cases: 1. The buffer systems of the balance buffer solution, the sample dilution solution and the wash solution do not contain the chaotropic agent, and the solution 1 of the eluent contains the chaotropic agent; 2. At least one of the balance buffer solution, the sample dilution solution and the wash solution contains the chaotropic agent, and the solution 1 of the eluent may or may not contain the chaotropic agent.

[0020] The specific concentration of the chaotropic agent can be 2M, 2.4M, 3.2M, 4M, 4.2M, 4.8M, 5M, 6M, 7M, 8M. The chaotropic agent is one or several of lithium perchlorate or other perchlorates, guanidine chloride or other guanidine salts, urea, butanol, ethanol, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea.

[0021] The balance buffer solution, sample diluent, and washing solution are selected from any one of the citric acid-sodium citrate system, sodium dihydrogen phosphate-citric acid system, citric acid-sodium hydroxide-hydrochloric acid system, acetic acid-sodium acetate system, or glycine-hydrochloric acid system with a pH of 3.0 - 6.6. The concentration of the buffer system is 10 - 100 mM, specifically it can be 10 mM, 20 mM, 25 mM, 50 mM, 75 mM, or 100 mM.

[0022] The present invention also provides the use of the eluent and the solution combination in purifying long-chain RNA at room temperature.

[0023] In the above application, the length of the long-chain RNA > 300 nt. The present invention has no special limitation on the type of RNA, which can be mRNA or other types of RNA such as rRNA, etc.

[0024] The present invention also provides a method for purifying long-chain RNA at room temperature. Dilute the sample containing long-chain RNA with the sample diluent, load it onto an anion chromatography column after equilibration, wash it, elute with the eluent of the present invention, and collect the elution peak.

[0025] Among them, at least one of the balance buffer solution for equilibration, sample diluent, washing solution for rinsing, and eluent for elution contains 2 - 8 M chaotropic agent. The chaotropic agent is one or several of lithium perchlorate or other perchlorates, guanidine chloride or other guanidinium salts, urea, butanol, ethanol, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea; in some specific embodiments, the chaotropic agent is urea.

[0026] When the chromatography system before elution does not contain 2 - 8 M chaotropic agent, the eluent used for elution is the eluent of the present invention containing a chaotropic agent;

[0027] In the system before elution, if any one of the equilibration solution, sample diluent, or washing solution contains 2 - 8 M chaotropic agent, the eluent used for elution is the eluent of the present invention that may or may not contain a chaotropic agent.

[0028] In some embodiments, in the system before elution, if at least one of the equilibration solution, sample diluent, or washing solution contains 2 - 8 M chaotropic agent, the eluent used for elution is the eluent of the present invention that does not contain a chaotropic agent; in some embodiments, in the system before elution, if at least one of the equilibration solution, sample diluent, or washing solution contains 2 - 8 M chaotropic agent, the eluent used for elution is the eluent of the present invention that contains a chaotropic agent.

[0029] In some embodiments, the anion chromatography column includes a weak anion chromatography column and a strong ion chromatography column.

[0030] In some embodiments, the anion chromatography column includes, but is not limited to, a weak anion chromatography column containing a -CH2-CH2-N-(CH2-CH3)2 functional group, a strong anion chromatography containing a -CH2-N + -(CH3)3 functional group.

[0031] In some embodiments, the anion chromatography column may be MaXtar Q, MaXtar Q HR, QChromstar HP, POROS TM GoPure TM HQ, POROS TM XQ or NanoGel TM -50Q HC column filled with at least one of them; in some embodiments, the weak anion chromatography column includes at least one of UniGel-30DEAE, DEAE Chromstar FF, MaXtar DEAE, UniDEAE-50XS, UniDEAE-30S as the filler column; in some embodiments, the strong anion chromatography column is a 50HQ column.

[0032] In some embodiments, the sample diluent, the equilibration buffer, and the wash buffer are any one of acidic buffer solutions in a citric acid-sodium citrate system, a sodium dihydrogen phosphate-citric acid system, a citric acid-sodium hydroxide-hydrochloric acid system, an acetic acid-sodium acetate system, or a glycine-hydrochloric acid system with a pH of 3.0 - pH 6.6, which may or may not contain 2 - 8M urea.

[0033] In some embodiments, the elution is an isocratic elution. Specifically, when the elution rate is 1.0 - 1.5 ml / min, a constant ratio elution with any ratio of the pipeline volume placed in solution 2 being 10% - 80% is set. Specifically, the pipeline placed in solution 2 can be set to 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%. In some embodiments, the elution elutes at least 3 column volumes (CVs).

[0034] In a specific embodiment, the pipeline placed in solution 2 may be pipeline B, and a constant ratio of the volume ratio of pipeline B being 10% - 80% is set, and the elution is at least 3 column volumes (CVs).

[0035] In some embodiments, the elution is gradient elution. Specifically, when the elution rate is 1.0 - 1.5 ml / min, B is set to increase from 0% to any proportion within 10% - 80% within 25 minutes, such as increasing from 0% to 10%, 0% to 20%, 0% to 30%, 0% to 40%, 0% to 50%, 0% to 60%, 0% to 70%, or 0% to 80%.

[0036] The present invention provides an eluent and its application in purifying long-chain RNA. The eluent is a buffer solution formed by mixing an acidic buffer solution containing 0.6 - 3M monovalent Cl salt and 0.2 - 0.5M monobasic strong base in a ratio.

[0037] According to the addition sequence of urea, the elution of the present invention can be refined into Schemes 1 - 6: A and B in the following schemes are pipeline A and pipeline B in the equipment. It is a concrete description of one pipeline and another pipeline for the convenience of writing and understanding, but it is not limited to pipeline A and pipeline B only. If it is a multi-pipeline system, the above pipeline A and pipeline B can be replaced with pipeline 1 and pipeline 2, pipeline B and pipeline A, pipeline A and pipeline C, pipeline C and pipeline D, etc. For the convenience of example, in the following, the acidic buffer solution is selected as Cit-Buffer, the pH is selected as 4.0, NaOH is selected as 0.5M, the Cl salt is selected as NaCl with a concentration of 3M, and the chaotropic agent is selected as urea with a concentration of 8M; however, in specific embodiments, the acidic buffer solution can be selected from various buffer systems such as citric acid - sodium citrate system, sodium dihydrogen phosphate - citric acid system, citric acid - sodium hydroxide - hydrochloric acid system, acetic acid - sodium acetate system, or glycine - hydrochloric acid system, etc., and the concentration can also be selected as any concentration within 20mM - 100mM, or can also be various concentrations such as 8mM, 10mM, 16mM, 120mM, 160mM, etc.; the pH can be any pH within 3.0 - 6.6; the concentration of NaOH can be any concentration within 0.5 - 4M; the Cl salt can be NaCl or KCl, with a concentration of any concentration within 0.6 - 3.5M, and the chaotropic agent can be lithium perchlorate or other perchlorates, guanidine chloride or other guanidine salts, urea, butanol, ethanol, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, etc., with a concentration of any concentration within 2 - 8M, and of course can also be various concentrations such as 1M, 1.5M, 10M, 12M, etc. The overall experiment is carried out at normal temperature or room temperature, and the temperature can be 10 - 37°C. Here, room temperature is taken as an example. Scheme 1:

[0038] Room temperature equilibration: 20mM Cit-Buffer pH4.0

[0039] Sample loading: Dilute the sample obtained by tangential flow diafiltration 5-fold with 20 mM Cit-Buffer pH 4.0 and load the sample (before loading, the RNA is replaced by tangential flow into the 20 mM Cit-Buffer pH 4.0 dilution buffer).

[0040] Impurity removal (rinsing): 20 mM Cit-Buffer pH 4.0

[0041] Elution: A: 20 mM Cit-Buffer pH 4.0 + 3 M NaCl + 8 M urea; B: 0.5 M NaOH; Linear elution: The volume ratio of solution B is 10% - 80%, completed in 25 minutes or set isocratic elution: 80% B (equivalent to 20% A + 80% B, different instrument settings are different, please set according to the specific equipment requirements) ≥ 3 CVs

[0042] Regeneration: Deionized water ≥ 3 CVs; 0.5 M NaOH ≥ 3 CVs;

[0043] Equilibration and storage: Deionized water, 20% ethanol

[0044] Protocol 2:

[0045] Room temperature equilibration: 20 mM Cit-Buffer pH 4.0 + 8 M urea

[0046] Sample loading: Dilute the sample 5-fold with 20 mM Cit-Buffer PH4.0 + 8 M urea and load the sample (before loading, the RNA is replaced by tangential flow into the 20 mM Cit-Buffer PH4.0 dilution buffer).

[0047] Impurity removal (rinsing): 20 mM Cit-Buffer pH 4.0 + 8 M urea

[0048] Elution: A: 20 mM Cit-Buffer PH4.0 + 3 M NaCl; B: 0.5 M NaOH; Isocratic elution: The volume ratio of solution B is 10% - 80%, completed in 25 minutes or set isocratic elution: 80% B (equivalent to 20% A + 80% B, different instrument settings are different, set according to the specific equipment requirements) ≥ 3 CVs

[0049] Regeneration: Deionized water ≥ 3 CVs; 0.5 M NaOH ≥ 3 CVs;

[0050] Equilibration and storage: Deionized water, 20% ethanol

[0051] Protocol 3:

[0052] Room temperature equilibration: 20 mM Cit-Buffer pH 4.0

[0053] Sample loading: Dilute the sample 5 times with 20 mM Cit-Buffer pH 4.0 and then load it (before loading, the RNA is replaced by tangential flow into the 20 mM Cit-Buffer pH 4.0 dilution buffer).

[0054] Impurity removal (rinsing): 20 mM Cit-Buffer pH 4.0 + 8 M urea

[0055] Elution: A: 20 mM Cit-Buffer pH 4.0 + 3 M NaCl; B: 0.5 M NaOH; Gradient elution: The volume ratio of solution B is 10% - 80%, completed in 25 minutes or set isocratic elution: 80% B (equivalent to 20% A + 80% B, different instrument settings are different, set according to specific equipment requirements) ≥ 3 CVs

[0056] Regeneration: Deionized water ≥ 3 CVs; 0.5 M NaOH ≥ 3 CVs;

[0057] Equilibration and storage: Deionized water, 20% ethanol

[0058] Protocol 4:

[0059] Room temperature equilibration: 20 mM Cit-Buffer pH 4.0

[0060] Sample loading: Dilute the sample 5 times with 20 mM Cit-Buffer pH 4.0 + 8 M urea and then load it (before loading, the RNA is replaced by tangential flow into the 20 mM Cit-Buffer pH 4.0 dilution buffer).

[0061] Impurity removal (rinsing): 20 mM Cit-Buffer pH 4.0

[0062] Elution: A: 20 mM Cit-Buffer pH 4.0 + 3 M NaCl; B: 0.5 M NaOH; Gradient elution: The volume ratio of solution B is 10% - 80%, completed in 25 minutes or set isocratic elution: 80% B (equivalent to 20% A + 80% B, different instrument settings are different, set according to specific equipment requirements) ≥ 3 CVs

[0063] Regeneration: Deionized water ≥ 3 CVs; 0.5 M NaOH ≥ 3 CVs;

[0064] Equilibration and storage: Deionized water, 20% ethanol

[0065] Protocol 5:

[0066] Room temperature equilibration: 20 mM Cit-Buffer pH 4.0

[0067] Sample loading: Dilute the sample 5-fold with 20 mM Cit-Buffer pH 4.0 + 8 M urea and then load the sample (before loading, the RNA is tangentially flow-displaced into the 20 mM Cit-Buffer pH 4.0 dilution buffer).

[0068] Impurity removal (rinsing): 20 mM Cit-Buffer pH 4.0 + 8 M urea

[0069] Elution: A: 20 mM Cit-Buffer pH 4.0 + 3 M NaCl; B: 0.5 M NaOH; Gradient elution: The volume ratio of solution B is 10% - 80%, completed in 25 minutes, or set isocratic elution: 80% B (equivalent to 20% A + 80% B, different instrument settings are different, set according to the specific equipment requirements) ≥ 3 CVs

[0070] Regeneration: Deionized water ≥ 3 CVs; 0.5 M NaOH ≥ 3 CVs;

[0071] Equilibration and storage: Deionized water, 20% ethanol

[0072] Protocol 6:

[0073] Room temperature equilibration: 20 mM Cit-Buffer pH 4.0

[0074] Sample loading: Dilute the sample 5-fold with 20 mM Cit-Buffer PH4.0 and then load the sample (before loading, the RNA is tangentially flow-displaced into the 20 mM Cit-Buffer PH4.0 dilution buffer).

[0075] Impurity removal (rinsing): 20 mM Cit-Buffer pH 4.0 + 8 M urea

[0076] Elution: A: 20 mM Cit-Buffer pH 4.0 + 3 M NaCl + 8 M urea; B: 0.5 M NaOH; Gradient elution: The volume ratio of solution B is 10% - 80%, completed in 25 minutes or set isocratic elution: 40% B (equivalent to 60% A + 40% B, different instrument settings are different, set according to the specific equipment requirements) ≥ 3 CVs

[0077] Regeneration: Deionized water ≥ 3 CVs; 0.5 M NaOH ≥ 3 CVs;

[0078] Equilibration and storage: Deionized water, 20% ethanol

[0079] In addition to the above-mentioned Solutions 1-6, urea can also be added to both the equilibration buffer and the eluted Buffer A, or urea can be added to the equilibration buffer, sample loading buffer, rinsing buffer, etc. That is, it is sufficient that at least one of the buffers in the equilibration, sample loading, rinsing, and elution steps contains urea. Of course, when at least one of the buffers in the equilibration, sample loading, rinsing, and elution steps contains urea, the eluate may or may not contain urea.

[0080] Using the eluate of the present invention to purify a sample containing long-chain RNA has the following advantages:

[0081] 1) The anion exchange chromatography purification method provided by the present invention realizes the purification of long-chain RNA at room temperature without heating the buffer system, sample, and column incubator during the purification process. Moreover, this method is stable and easy to scale up, laying a foundation for subsequent large-scale RNA drug development.

[0082] 2) The eluate combination provided by the present invention can achieve a recovery rate of greater than 95% when purifying large-fragment RNA.

[0083] 3) The eluate of the present invention changes the RNA charge by reducing the pH of the sample loading buffer system, changes the system conductivity by adding NaCl, and simultaneously introduces a combination of urea and NaOH, ultimately realizing the effective elution of long-chain RNA at room temperature, perfectly avoiding the previous elution methods that are not conducive to RNA stability, such as ion-pair salts (such as triethylamine, tetrabutylammonium hydroxide, tetrabutylammonium bromide, dodecyltrimethylammonium chloride, trifluoroacetic acid, pentanesulfonic acid sodium salt, hexanesulfonic acid sodium salt, heptanesulfonic acid sodium salt, octanesulfonic acid sodium salt, dodecylsulfonic acid sodium salt, sodium dodecyl sulfate, etc. ion-pair salts) and high temperature. Moreover, this method is stable and easy to scale up, laying a foundation for subsequent large-scale RNA drug development. Brief Description of the Drawings

[0084] Figure 1 Showing the AKTAAvant150 protein purification chromatography system;

[0085] Figure 2 Showing the R37 plasmid map, where the RNA transcription length is 2756 bp;

[0086] Figure 3 Showing the mRNA purification chromatogram in Example 1;

[0087] Figure 4 Showing the mRNA purification chromatogram in Example 2;

[0088] Figure 5 Showing the mRNA purification chromatogram in Example 3;

[0089] Figure 6 Showing the mRNA function verification in Example 3, whereFigure 6 A is for the integrity analysis of RNA using agarose gel electrophoresis, Figure 6 B is for RNA function verification;

[0090] Figure 7 showing the mRNA purification chromatograms of the eluents containing different concentrations of sodium hydroxide in Example 4;

[0091] Figure 8 showing the mRNA purification chromatogram of the eluent purified with the acidic buffer solution without NaCl in one of the eluent formulation solutions in Example 4B

[0092] Figure 9 showing the mRNA purification chromatogram of the eluent purified with the acidic buffer solution containing 1 M NaCl in one of the eluent formulation solutions in Example 4B;

[0093] Figure 10 showing the mRNA purification chromatogram of the eluent purified with the acidic buffer solution containing 3 M NaCl in one of the eluent formulation solutions in Example 4B

[0094] Figure 11 showing the mRNA purification chromatogram of the eluent purified with the acidic buffer solution containing 3.5 M NaCl in one of the eluent formulation solutions in Example 4B

[0095] Figure 12 showing the mRNA purification chromatogram of the eluent purified with the acidic buffer solution with a pH of 3.0 in one of the eluent formulation solutions in Example 4C

[0096] Figure 13 showing the mRNA purification chromatogram of the eluent purified with the acidic buffer solution with a pH of 4.0 in one of the eluent formulation solutions in Example 4C

[0097] Figure 14 showing the mRNA purification chromatogram of the eluent purified with the acidic buffer solution with a pH of 5.0 in one of the eluent formulation solutions in Example 4C

[0098] Figure 15 showing the mRNA purification chromatogram of the eluent purified with the acidic buffer solution with a pH of 6.2 in one of the eluent formulation solutions in Example 4C;

[0099] Figure 16 showing the mRNA purification chromatogram of the eluent purified with the acidic buffer solution containing 0 mM Cit-Buffer in one of the eluent formulation solutions in Example 4D (top: mAu; bottom: pH);

[0100] Figure 17Purification chromatogram of mRNA eluted with an eluent formulation containing 20 mM Cit-Buffer in one of the acidic buffer solutions in Example 4D (top: mAu; bottom: pH);

[0101] Figure 18 Purification chromatogram of mRNA eluted with an eluent formulation containing 50 mM Cit-Buffer in one of the acidic buffer solutions in Example 4D (top: mAu; bottom: pH);

[0102] Figure 19 Purification chromatogram of mRNA eluted with an eluent formulation containing 100 mM Cit-Buffer in one of the acidic buffer solutions in Example 4D (top: mAu; bottom: pH);

[0103] Figure 20 Purification chromatogram of mRNA eluted with an eluent formulation containing 0 M urea in one of the acidic buffer solutions in Example 4E;

[0104] Figure 21 Purification chromatogram of mRNA eluted with an eluent formulation containing 2 M urea in one of the acidic buffer solutions in Example 4E;

[0105] Figure 22 Purification chromatogram of mRNA eluted with an eluent formulation containing 4 M urea in one of the acidic buffer solutions in Example 4E;

[0106] Figure 23 Purification chromatogram of mRNA eluted with an eluent formulation containing 8 M urea in one of the acidic buffer solutions in Example 4E;

[0107] Figure 24 Purification chromatogram of mRNA after MgCl2 replaced NaCl in Example 5;

[0108] Figure 25 Purification chromatogram of mRNA after KCl replaced NaCl in Example 5;

[0109] Figure 26 Purification chromatogram of mRNA after (NH4)2SO4 replaced NaCl in Example 5;

[0110] Figure 27 Purification chromatogram of mRNA after 20 mM acetic acid-sodium acetate replaced Cit-Buffer;

[0111] Figure 28 Purification chromatogram of mRNA after 20 mM glycine-hydrochloric acid replaced 20 mM Cit-Buffer;

[0112] Figure 29 Chromatogram of mRNA purification after replacing NaOH with disodium hydrogen phosphate

[0113] Figure 30 Chromatogram of mRNA purification after replacing NaOH with Tris;

[0114] Figure 31 Chromatogram of mRNA purification after replacing NaOH with KOH;

[0115] Figure 32 Chromatogram of mRNA purification after replacing urea with guanidine hydrochloride;

[0116] Figure 33 Chromatogram of mRNA purification using anion chromatography column 50HQ.

[0117] Figure 34 Verification of 10% elution volume ratio of 1.0M NaOH in Example 44A2;

[0118] Figure 35 Verification of 20% elution volume ratio of 1.0M NaOH in Example 44A2;

[0119] Figure 36 Verification of 50% elution volume ratio of 1.0M NaOH in Example 44A2;

[0120] Figure 37 Verification of 70% elution volume ratio of 1.0M NaOH in Example 44A2;

[0121] Figure 38 RNA integrity analysis of elution with 1.0M NaOH ratio in Example 44A2.

[0122] Figure 39 Verification of 20% elution volume ratio of 2.0M NaOH in Example 44A3;

[0123] Figure 40 Verification of 40% elution volume ratio of 2.0M NaOH in Example 44A3;

[0124] Figure 41 RNA integrity analysis of elution with 2.0M NaOH ratio in Example 44A3.

[0125] Figure 42 RNA integrity analysis of elution with 3.0M NaOH ratio in Example 44A4.

[0126] Figure 43 A shows verification of 5% or 10% elution volume ratio of 4.0M NaOH in Example 44A5;

[0127] Figure 43 Verification of elution with 4.0 M NaOH at a volume ratio of 50% in Example 44A5;

[0128] Figure 44 RNA integrity analysis of elution with 4.0 M NaOH in Example 44A5;

[0129] Figure 45 Verification of elution with 3.0 M NaOH at a volume ratio of 10% in Example 44A4;

[0130] Figure 46 Functional verification of RNA purified with eluents prepared by mixing different molar concentrations of NaOH in different ratios in Example 4. Detailed implementation mode

[0131] The present invention provides an eluent and its application in purifying long-chain RNA. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0132] The reagents and consumables used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0133] The method for purifying long-chain RNA at room temperature provided by the present invention includes: diluting a sample containing long-chain RNA with a sample diluent, loading and equilibrating an anion chromatography column, rinsing, eluting with the eluent of the present invention, and collecting the elution peak;

[0134] At least one of the equilibrium buffer for equilibration, the sample diluent, the rinse solution for rinsing, and the eluent for elution contains 2-8 M urea.

[0135] This method combines the eluent of the present invention with urea, or uses the urea-containing eluent of the present invention to purify long-chain RNA on an anion chromatography column, and finally achieves an RNA recovery rate of more than 95% at room temperature.

[0136] 1.1. mRNA purification reagents and consumables

[0137] Table 1

[0138]

[0139]

[0140] 1.2. Reagent preparation: The Cit-Buffer in this part is a citric acid-sodium citrate system. For the preparation ratios of the sodium dihydrogen phosphate-citric acid system, citric acid-sodium hydroxide-hydrochloric acid system, acetic acid-sodium acetate system, or glycine-hydrochloric acid system, refer to Cit-Buffer and replace the corresponding acids. For the citric acid-sodium hydroxide-hydrochloric acid system and citric acid-sodium citrate system, the concentration of citrate is 20 mM (20 mmol / L); for the acetic acid-sodium acetate system, the concentration of acetate is 20 mM (20 mmol / L); for the sodium dihydrogen phosphate-citric acid system, the concentration of phosphate is 20 mM (20 mmol / L); for the glycine-hydrochloric acid system, the concentration of glycine is 20 mM (20 mmol / L). The same replacement scheme applies to other concentrations.

[0141] 20 mM Cit-Buffer pH 4.0 3 L: Weigh 12.585 g of anhydrous citric acid and 10.1475 g of trisodium citrate dihydrate, place them in approximately 3 L of water for injection, and quantitatively dilute to 3 L with a volumetric flask. Filter through a 0.45 μm PVDF membrane for standby.

[0142] 25 mM Cit-Buffer pH 6.6 10 L: Weigh 3.3625 g of anhydrous citric acid and 68.3775 g of trisodium citrate dihydrate, place them in approximately 3 L of water for injection, and quantitatively dilute to 10 L with a volumetric flask. Filter through a 0.45 μm PVDF membrane for standby.

[0143] 25 mM Cit-Buf + 3 M NaCl pH 6.6 0.5 L: Weigh 86.77 g of NaCl, dissolve and mix it with 50 mM Cit-Buf until the total volume reaches 250 ml, then add 25 ml of WFI and quantitatively dilute to 500 ml. Filter through a 0.45 μm PVDF membrane for standby.

[0144] 20 mM Cit-Buf + 3 M NaCl pH 4.0 0.5 L: Weigh 86.77 g of NaCl, dissolve and mix it with 20 mM Cit-Buf, quantitatively dilute to 500 ml, and then filter through a 0.45 μm PVDF membrane for standby.

[0145] 20 mM Cit-Buf + 3 M NaCl + 8 M Urea pH 4.0 0.5 L: Weigh 86.77 g of NaCl and 240.24 g of urea, dissolve and mix them with 20 mM Cit-Buf ml, quantitatively dilute to 500 ml, and then filter through a 0.45 μm PVDF membrane for standby.

[0146] 20 mM Cit - Buf + 3 M NaCl + 8 M Urea pH4.0 + 0.01 NaOH 0.5 L: Weigh 86.77 g of NaCl, 240.24 g of urea and add 11.1 ml of 0.5 M NaOH. Then dissolve and mix well with 20 mM Cit - Buf, make up to 500 ml and filter through a 0.45 μm PVDF membrane for standby.

[0147] 20 mM Cit - Buf + 8 M Urea pH4.0 0.5 L: Weigh 240.24 g of urea, dissolve and mix well with 20 mM Cit - Buf, make up to 500 ml and filter through a 0.45 μm PVDF membrane for standby.

[0148] 0.5 M NaOH 0.5 L: Weigh 10 g of NaOH, dissolve and mix well with deionized water, make up to 500 ml and filter through a 0.45 μm PVDF membrane for standby.

[0149] 1.3 Ion Exchange Chromatography (IEX) Concepts and Applications

[0150] 1.3.1 Ion Exchange Chromatography Concepts

[0151] Ion Exchange Chromatography (IEX): It uses an ion exchanger as the stationary phase and separates based on the difference in the binding force when the component ions in the mobile phase reversibly exchange with the equilibrium ions on the exchanger. Ion exchange chromatography has wide applicability, high capacity, simplicity, and high resolution, making it a commonly used separation method. This technique is mainly used for separating and purifying charged biomolecules such as polypeptides, proteins, polynucleotides, and nucleic acids. Its ion exchange chromatography is mainly used in: applications in life sciences, genomics, and proteomics, such as the separation and purification of blood components like albumin, recombinant growth factors, enzymes, etc.; biotechnological analysis applications, for example, quality control and process monitoring; food and clinical research: studying wheat varieties and the correlation between proteinuria and different kidney diseases; fermentation: cation exchange resins are used to monitor the fermentation process during the production of β - galactosidase.

[0152] Ion exchanger: The stationary phase of ion exchange chromatography is an ion exchanger, which is formed by covalently binding a certain charge group to an insoluble inert high - molecular polymer matrix through a chemical reaction. An ion exchanger can be divided into three parts: a high - molecular polymer matrix, a charge group, and an equilibrium ion. The charge group is covalently bound to the high - molecular polymer to form a charged group that can undergo ion exchange. The equilibrium ion is the opposite ion bound to the charge group, and it can undergo a reversible exchange reaction with other ion groups in the solution.

[0153] Cation exchanger: An ion exchanger with a positively charged counter ion can exchange with positively charged ionic groups and is called a cation exchanger.

[0154] Anion exchanger: An ion exchanger with a negatively charged counter ion exchanges with negatively charged ionic groups and is called an anion exchanger.

[0155] Ion exchange adsorbents are porous media and can use various different particles and pore sizes. The particle sizes can include standard sizes used in chromatography, including less than 1 μm or 1 to 500 μm (e.g., 5, 10, 20, 50, 75, 100, 150...). The particles can include small silica beads or other types of particles. The pore sizes can include greater than 500 Å, or greater than or equal to 600, 700, 800, 900, 1,000, 2,000, 4,000 or 8,000 Å, and smaller pore sizes can also be used, such as 1 to 500 Å.

[0156] Ion exchange adsorbents can be polystyrene divinylbenzene, polymethacrylate, cross-linked agarose, silica or allyl dextran with NN - bisacrylamide, as well as various other adsorbents. Ion exchange adsorbents can also be non-porous media, such as monolithic columns. Anion chromatography columns can be weak anion chromatography columns including those with -CH2-CH2-N-(CH2-CH3)2 functional groups, or strong anion chromatography columns with -CH2-N + -(CH3)3 functional groups.

[0157] In some applications, membrane-based ion exchangers can be used, such as MILLIPORE CHROMASORB TM or SARTORIUS SARTOBIND TM , and in addition, mixed-mode or combinations of ion exchangers can also be used.

[0158] Common ion exchange adsorbents can contain a variety of different positively charged functional groups, such as quaternary amines, polyethyleneimine, diethylaminomethyl and dimethylaminopropyl. Common strong anion exchangers contain negatively charged functional groups, such as quaternary amines. Weak anion exchangers can contain a variety of different negatively charged functional groups, such as polyethyleneimine, diethylaminomethyl, dimethylaminopropyl or polyallylamine.

[0159] Anion Exchange Chromatography (AEX): A purification method that utilizes the ionic interaction between a positively charged adsorbent and negatively charged molecules. AEX adsorbents typically contain charged functional groups crosslinked to a solid-phase medium. There are two types of anion exchange media, "strong" and "weak" exchangers or exchange groups. The classification of strong and weak anion exchangers refers to the ionization of the charged functional groups in response to changes in pH. Anion exchange adsorbents or resins facilitate RNA capture due to their interaction with the negatively charged phosphate backbone of RNA transcripts, providing an effective separation mode.

[0160] Due to the relevant charge density of the molecules, large RNA transcripts are difficult to elute from the separation matrix. The interaction between large RNA transcripts and ion exchange adsorbents is much stronger than that of small RNA transcripts. In addition, RNA can form highly complex structured macromolecules, and they are capable of folding into many unique structures and conformations. Moreover, RNA transcripts can bind to other RNA transcripts and form higher-order structures. These phenomena may contribute to very strong interactions with the adsorbent. In this example, a weak anion exchanger containing Diethylaminoethyl (DEAE) (such as UniGel-30DEAE) is used for RNA purification. Other fillers such as DEAE Chromstar FF, MaXtar DEAE, UniDEAE-50XS, UniDEAE-30S, -DEAE and other DEAE-containing anion exchangers can be used to replace UniGel-30DEAE in this example. In addition, weak anion exchanger fillers containing Diethylaminopropyl (ANX) (such as ANX Focurose 6BB, ANX Focurose HPL, ANX Focurose 6HF, ANX Sepharose 4 Fast Flow) can also be used for RNA purification.

[0161] Table 2 Common Ion Exchange Classification, Functional Groups and Commercial Fillers

[0162]

[0163]

[0164] 1.3.2 Steps of Ion Exchange Chromatography (IEX)

[0165] In this embodiment, the principle of purifying RNA samples using AEX is that RNA transcripts bind to the positively charged functional groups of the ion exchange adsorbent. Impurities or at least a portion of the impurities can also pass through the ion exchange adsorbent without being adsorbed during sample loading (i.e., flow-through, referring to substances that do not bind to the chromatography column when the sample flows through the column during sample loading, including unbound impurities and target binding samples that flow out of the column due to exceeding the binding capacity of the column). This achieves the purification of RNA transcripts from a sample containing RNA transcripts and impurities. The RNA transcripts can be large RNA transcripts or chemically modified RNA transcripts. The ion exchange chromatography steps include: equilibration, sample loading, impurity removal (washing), elution, and regeneration and preservation.

[0166] Equilibration: Equilibrate the chromatography column to the starting conditions required to bind the target sample

[0167] The first step in ion exchange chromatography purification is to equilibrate the stationary phase to the required starting conditions. When equilibrium is reached, all the charged groups of the stationary phase are bound to exchangeable counterions, such as chloride or sodium ions. The pH value and the ionic strength of the starting buffer are selected to ensure that when the sample is loaded, the protein of interest binds to the sample binding medium and as many impurities as possible do not bind.

[0168] Sample loading: The step of flowing the mobile phase containing the sample into the chromatography column and allowing the sample to be purified to bind to the chromatography column

[0169] The second step is sample loading. The goal of this step is to bind the target molecule and remove all unbound impurities. The sample is delivered through the mobile phase (e.g., Tris-HCl buffer or other mobile phases), including the selection of wash solutions for further separation of impurity components in the sample after sample loading. The mobile composition can include any Hofmeister ion, salts (such as chlorides, bromides, citrates, iodides, sulfates, phosphates, perchlorates, etc.), and other mobile phase components. Mobile phase additives / modifiers can include organic reagents, such as ethanol, acetonitrile, isopropyl alcohol (IPA), DMSO, surfactant modifiers (such as polyethylene glycol), etc. The loading conditions for AEX chromatography can be carried out under denaturing conditions, partially denaturing conditions, or non-denaturing conditions. Denaturing conditions can include conditions where the RNA transcript is denatured due to temperature (e.g., 35 to 85 degrees Celsius), chaotropic agents (including lithium perchlorate and other perchlorates, guanidine chloride and other guanidinium salts, urea, butanol, ethanol, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, and various detergents, etc.), organic reagents (such as dimethyl sulfoxide (DMSO), acetonitrile, ethanol, and glyoxal), and other denaturing mechanisms. Denaturing conditions can include a combination of two or more of these types of denaturing conditions.

[0170] Column wash: Remove unbound or loosely bound non-target products during the loading process

[0171] The wash buffer is the loading or elution buffer, or a gradient percentage of the loading or elution buffer. In cases where impurities are captured by the ion exchange adsorbent, the wash step can cause the impurities to be released from the ion exchange adsorbent as the eluate. In cases where the impurities or a portion of the impurities pass through the ion exchange adsorbent during the loading step, the wash can cause any remaining portion of the impurities to be further released from the ion exchange adsorbent. The wash step can be carried out under any of the above denaturing conditions, or under partially denaturing or non-denaturing conditions. The wash step can be carried out at an elevated temperature or also at ambient temperature. The wash buffer used can include Tris, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phosphate, etc. The wash buffer is selected to release the captured impurities from the adsorbent, or to wash away any remaining impurities from the adsorbent, where the impurities typically pass through the adsorbent during loading.

[0172] Elution: The process of releasing the target product bound to the chromatography column

[0173] After all the target samples are bound to the chromatography column, the column is washed with the starting buffer to allow all unbound proteins to flow through the column, and finally the conditions are changed to elute the bound target protein. This is carried out with the elution buffer flowing into the ion exchange adsorbent. The elution step can be carried out under any of the above denaturing conditions, or under partially denaturing or non-denaturing conditions.

[0174] Sanitization: The process of releasing all bound residual samples in the chromatography column using a strong denaturing agent, then replacing the denaturing agent with deionized water and finally storing it with 20% ethanol to ensure that the chromatography column can be used for the next purification

[0175] The column is regenerated and any remaining bound molecules are removed by a final wash with a high ionic strength buffer (such as 0.5 M NaOH). This ensures that the full capacity of the stationary phase is available for the next experiment. Then, the column is re-equilibrated in the equilibration buffer before starting the next run. The regenerated chromatography column is generally stored with 20% ethanol.

[0176] 1.4 Purification chromatography

[0177] The simple procedure for purification in this research protocol is to flow the acidic buffer or the mixture of acidic buffer + urea required for equilibration into and out of an anion-exchange packing chromatography column from one end; after equilibration, the RNA sample enters the chromatography column from one end and binds to the packing, and the unbound impurities flow out from one end; similarly, the buffer system required for impurity removal also enters the chromatography column from one end and flows out from the other end to further elute the unbound impurities; finally, in the elution stage, acidic buffer A containing urea and NaCl is mixed with buffer B: NaOH (it can be mixed in a fixed ratio or NaOH can be introduced at a certain flow rate at the end of buffer A under the condition of being filled with solution A), and is introduced into the chromatography column in the same way for elution, and finally the sample flowing out of the chromatography column is collected.

[0178] Specifically, this research protocol can be as follows: flow the acidic buffer of 20 mM Cit-Buffer with a pH of 4.0 required for equilibration, with or without urea, into and out of an anion-exchange packing chromatography column from one end; after equilibration, the RNA sample enters the chromatography column from one end and binds to the packing, and the unbound impurities flow out from the other end; similarly, the buffer system required for impurity removal also enters the chromatography column from one end and flows out from the other end to further wash the unbound impurities; finally, in the elution stage, an elution buffer formed by mixing acidic buffer A containing urea + NaCl with buffer B: NaOH in a ratio is introduced into the chromatography column in the same way for elution, and finally the sample flowing out of the chromatography column is collected. The urea here can be introduced in the initial acidic buffer or in any one or more steps before and including the eluent, as long as it is ensured that during the elution process, the entire ion column and eluent environment contain urea components. Specifically, this research protocol can also be as follows: flow the acidic buffer of 10 - 100 mM Cit-Buffer with a pH of 3.0 - 6.6, with or without 2 - 8 M urea, required for equilibration into and out of an anion-exchange packing chromatography column from one end; after equilibration, the RNA sample enters the chromatography column from one end and binds to the packing, and the unbound impurities flow out from the other end; similarly, the buffer system required for impurity removal also enters the chromatography column from one end and flows out from the other end to further wash the unbound impurities; finally, in the elution stage, an elution buffer formed by mixing acidic buffer A containing 2 - 8 M urea + 1 - 3.5 M NaCl with 0.2 - 4 M NaOH buffer B is used as the eluent and introduced into the chromatography column in the same way for elution, and finally the sample flowing out of the chromatography column is collected. The urea here can be introduced in the initial acidic buffer or in any step before including the eluent, as long as it is ensured that during the elution process, the eluent environment contains 4 - 8 M urea components.

[0179] Specifically, this research protocol can also: flow the 20 mM Cit-Buffer with or without 8 M urea for balancing the pH of the required acidic buffer at pH 4.0 from one end of the anion packing chromatography column and out from the other end; after balancing, the RNA sample enters the chromatography column from one end and binds to the packing, and the unbound impurities flow out from the other end; similarly, the buffer system required for impurity removal also enters from one end of the chromatography column and flows out from the other end to further elute the unbound impurities; finally, at the elution stage, the elution buffer formed by mixing the acidic buffer A containing 8 M urea + 3 M NaCl and the 0.5 M NaOH buffer B in a ratio is introduced into the chromatography column in the same way for elution, and finally the sample flowing out of the chromatography column is collected. The urea here can be introduced in the acidic buffer at the very beginning or at any step before the elution buffer and its previous steps, as long as it is ensured that during the elution process, the entire ion column and the elution buffer environment contain 8 M urea component.

[0180] In addition, other chromatography systems with the same or similar principles are applicable to this protocol. Common methods include using gravity columns based on manual operation and chromatography systems covering the entire process from laboratory scale to process scale-up and production scale. For example go protein purification system, plus, start, pure, avant chromatography system, pcc and BioProcess pcc, pilot 600, process TM chromatography system, ready TM 450 system, ready, ready XL, BioProcess modular chromatography system, STP protein chromatography system, SDA protein purification series, SCG-P protein purification series, SCG-Phf protein purification series, SDL protein chromatography system, protein purification system SCG series, SCG-D dual-column protein chromatography system, SCG-Q four-column protein chromatography system, etc.

[0181] Taking the AKTA Avant150 (Cytiva) chromatography system used in this example as an example: The chromatography system used in this research has the ability to pass the acidic buffer required for balancing or the mixture of acidic buffer + urea through the system pump and pipeline system (such as Figure 1One or a combination of Quaternary Valve, Inlet Valve A, and Inlet Valve B) flows into the device, forms a mixed solution in the Mixer, enters the Column Valve and the Column through the Injection Valve and the system pipeline, and finally flows through the UV Monitor, Conductivity Monitor, pH Valve, and pH Monitor in sequence, and then flows out of the system through the Outlet Valve and Wash to balance the entire system. After balancing, the RNA sample is introduced through the Sample loop into the subsequent pipeline of the Injection Valve, and the flow path is the same as that during balancing. The buffer system required for impurity removal is introduced into the chromatography column in the same way as the system flow path during balancing; finally, in the elution stage, an acidic buffer solution A containing urea and NaCl and a NaOH buffer solution B are introduced into the chromatography column through the mixing tank in a certain ratio in the same system flow path as during balancing to achieve the purpose of eluting the sample, and the eluted sample is finally detected by pH, conductivity, and UV detectors and flows into the collector. It should be noted that during the purification loading process, the sample can be loaded through Inlet Valve A, Inlet Valve B, Sample Inlet Valve, and Injection Valve (Loop ring). In the examples of this article, if there is no other description of sample loading, a syringe is used to load the sample through the Injection Valve (Loop ring). In addition to using a syringe for loading through the Injection Valve (Loop ring), an ALIAS Bio autosampler, ALIAS Bio Cool autosampler, ALIAS Bio PREP autosampler, and ALIAS Bio PREP Cool autosampler can also be used.

[0182] This study used systems and packings commonly used in the art: protein purification chromatography system: AKTA Avant150 (Cytiva) (see Figure 1 ), chromatography packing: -30S (NanoChrom Tech), for research and verification. The RNA sample used was R37 mRNA (8458-230223, Figure 2As shown, the fragment size is 2756 bp. In addition, in the figures shown in the results of the examples, the curve marked as Conc B represents the real-time curve of the proportion of the InletValve B solution of the purifier, which is a built-in curve of the system operation and does not affect the result evaluation. The curve marked as Cond represents the real-time conductivity curve of the system pipeline after passing through the chromatography column during the purification process, which is a built-in curve of the system operation and does not affect the result evaluation. The curve marked as UV represents the absorption monitoring curve of the R sample during the purification process. The peak-shaped line appearing in the UV region is the elution peak, representing that the target purified RNA is eluted, which is the target analysis result. The curve marked as pH represents the real-time pH monitoring curve of the liquid in the chromatography column and the system pipeline. The elution collection peak of the RNA during the elution process in the figure needs to be determined by the UV curve, and the pH curve can be combined for condition optimization. Keeping the pH within a certain range (slightly acidic to neutral) helps to stabilize the RNA. Otherwise, the alkaline environment is likely to degrade the RNA (such as the straight upward situation of the pH curve in the example resulting in the elution collection solution being alkaline). In short: during the process of the UV rising and then falling, the pH curve must be kept within the range of slightly acidic to neutral, and there should be no straight upward situation.

[0183] In chromatography purification, isocratic elution and gradient elution are often used. Isocratic elution: It refers to an elution method in which the composition ratio and flow rate of the mobile phase remain constant during the chromatographic operation. Gradient elution: Also known as gradient elution, programmed elution or linear elution. It refers to continuously changing the concentration ratio of the mobile phase to a certain extent within the same analysis cycle, which is called gradient elution.

[0184] The present invention will be further described below in conjunction with the examples:

[0185] Example 1 NaOH is beneficial to improving the elution efficiency of mRNA

[0186] Reagent 1: 25 mM Cit-Buf pH 6.6

[0187] Reagent 2: 25 mM Cit-Buf + 1 M NaCl pH 6.6

[0188] Reagent 3: 25 mM Cit-Buf + 3 M NaCl pH 6.6

[0189] 1. Equilibration: Place the required A1 / B1 injection pipelines into the bottles containing Reagent 1 / Reagent 2 respectively. In the chromatography software, click to select the A1 / B1 pump positions, and click "Start A Wash Pump / Start B Wash Pump" to start the Wash by the system. Then set the system B1 to 0% (i.e., 100% A1 + 0% B1) and equilibrate at a flow rate of 1.0 ml / min until the conductivity / pH is stable.

[0190] 2. Sample Loading / Washing: Pause the system, install a 5 ml loop, use a syringe to aspirate approximately 20 ml of WFI to wash the loop, and also aspirate 20 ml of Solution A1 to wash the loop. Take 5 ml of R37 mRNA diluted 5 times for sample loading. Set System B1 to 0% (i.e., 100% A1 + 0% B1) and run at a flow rate of 1.0 ml / min. However, start collecting the flow-through when the mAU rises to around 20. The operation is to click on "Manual", pull down to "Execute manual instruction", select "Fraction Collection", then pull down "Fractionation", set "Fraction Size" to 1.0 ml, and click "Execute" in the lower right corner to start the collection. After that, click on "Fraction Collection", pull down "Fractionation", click on "Stop Fraction", and then click "Execute" in the lower right corner to stop collecting the flow-through. After the collection is completed, maintain the proportion of Component B1 in the system at 0% (i.e., 100% A1 + 0% B1) and run at a flow rate of 1.0 ml / min to further remove unbound or loosely bound impurities.

[0191] 3. Elution: Set System B1 to 25% / 50% / 75% / 100% in sequence (i.e., the ratios of A1 + B1 are 75% A1 + 25% B1, 50% A1 + 50% B1, 25% A1 + 75% B1, and 0% A1 + 100% B1 corresponding to regions A, B, C, and D respectively). The system runs at a flow rate of 1.0 ml / min, and record the elution efficiency. Pause the system, place the B2 injection line in a bottle containing Reagent 3: 25 mM Cit - Buf + 3M NaCl pH6.6, set System B2 to 100% (i.e., 0% A1 + 100% B2), and run at a flow rate of 1.0 ml / min, corresponding to region E. Pause the system and directly place the B2 injection line into 0.5M NaOH, set System B2 to 100% (i.e., 0% A1 + 100% B2), and run at a flow rate of 1.0 ml / min until 140 min (corresponding to region F). Turn off the pH detection to protect the pH electrode of the instrument. When the entire pipeline is completely filled with NaOH, it enters the final regeneration stage (corresponding to region G).

[0192] 4. Water Washing: Place the A1 / B1 tubes in WFI, execute "Start A Wash Pump / Start B Wash Pump" for cleaning, set the proportion of System B1 to 50% (i.e., 50% A1 + 50% B1), and execute water washing at a flow rate of 1.0 ml / min.

[0193] The results are as Figure 3 shown in

[0194] Table 3

[0195] Different eluent combinations Elution peak area C 2.09％ F 36.12％ G 61.79％

[0196] Figure 3 The peak patterns that appear in the regions represented by A and A indicate the elution peaks when eluting with an eluent formed by mixing 75% of Reagent 1 and 25% of Reagent 2; the peak patterns that appear in the regions represented by B and B indicate the elution peaks when eluting with an eluent formed by mixing 50% of Reagent 1 and 50% of Reagent 2; the peak patterns that appear in the regions represented by C and C indicate the elution peaks when eluting with an eluent formed by mixing 25% of Reagent 1 and 75% of Reagent 2; the peak patterns that appear in the regions represented by D and D indicate the elution peaks when eluting with an eluent formed by mixing 0% of Reagent 1 and 100% of Reagent 2; the peak patterns that appear in the regions represented by E and E indicate the elution peaks when eluting with an eluent formed by 100% of Reagent 3; the peak patterns that appear in the regions represented by F and F indicate the elution peaks when directly introducing 0.5 M NaOH into Tube B2 filled with Reagent 3: 25 mM Cit-Buf + 3 M NaCl pH 6.6 for elution. The collected elution peaks are relatively stable for RNA because the overall pH of the eluent is slightly acidic to neutral; G represents the stage of column regeneration. If the elution and purification are complete, there will be no elution peaks in region G. Since 100% 0.5 M NaOH is used to elute the chromatography column during the regeneration stage, the corresponding elution peaks are strongly alkaline and often cause irreversible degradation of RNA; as Figure 3 It can be seen that when using eluents A, B, C, and D for elution, only one elution peak appears in region C under the UV line, and the peak area is 2.09%; when changing the eluent to 100% Reagent 3 (25 mM Cit-Buf + 3 M NaCl pH 6.6, corresponding to region E) and running, it is found that the UV is at the baseline level, indicating that the sample is not eluted, suggesting that under the combination of only Cit-Buf and NaCl, the increase in the concentration of NaCl cannot promote the elution of long-chain RNA at room temperature; when directly putting the B2 pipeline containing Reagent 3 into 0.5 M NaOH (corresponding to region F), it is found that the mAU starts to rise and presents an elution peak, and the peak area ratio is 36.12%. Finally, another UV peak appears in the regeneration stage (corresponding to region G), and the peak area ratio is 61.79%. If the elution is complete, there should be no obvious UV peak in the regeneration stage. The UV peak in the regeneration stage indicates that the target RNA was not completely eluted and collected in the previous elution stage, and a large amount of target RNA was not eluted, resulting in a significant reduction in the final RNA purification yield.

[0197] The overall results show that the elution buffer formed by mixing the buffer solution of 25 mM Cit-Buf + 3M NaCl pH6.6 and 0.5M NaOH buffer solution (there can be multiple mixing schemes. It can be, for example, directly placing the tube of 25 mM Cit-Buf + 3M NaCl pH6.6 buffer solution in the container of 0.5M NaOH buffer solution in this example to form a mixture at a certain flow rate. It can also be that the pipelines connected to the chromatography column are respectively filled with 25 mM Cit-Buf + 3M NaCl pH6.6 buffer solution and 0.5M NaOH buffer solution according to different instruments and equipment, and then mixed by setting the parameters of the equipment. It can also be directly mixed in vitro according to a certain ratio) can complete the elution of long-chain RNA at room temperature / normal temperature under the elution condition of a flow rate of 1.0 ml / min, indicating that the buffer solution formed by mixing the acidic buffer solution of pH6.6 containing 3M NaCl and 0.5M NaOH can be used for the elution of long-chain RNA at normal temperature, and the elution efficiency reaches 36.12%. The whole experimental results show that the addition of NaOH in the elution stage can significantly promote the elution of long-chain RNA at room temperature / normal temperature.

[0198] Example 2 The addition of urea is beneficial to further improve the mRNA elution efficiency

[0199] Reagent 1: 20 mM Cit-Buf pH4.0

[0200] Reagent 2: 20 mM Cit-Buf + 3M NaCl pH4.0

[0201] Reagent 3: 20 mM Cit-Buf + 3M NaCl + 8M urea pH4.0

[0202] Complete the equilibration and loading / washing according to the scheme of Example 1, and the loading sample is also R37 mRNA.

[0203] Elution: Set the proportion of system B1 to 25% / 50% / 75% / 100% in turn (that is, the ratio of A1 + B1 is 75%A1 + 25%B1, 50%A1 + 50%B1, 25%A1 + 75%B1, 0%A1 + 100%B1 in turn), and perform elution at a flow rate of 1.0 ml / min. Elute the corresponding regions as Figure 4A, B, C, and D shown represent the elution effects using the eluent components formed by mixing 75% reagent 1 + 25% reagent 2, 50% reagent 1 + 50% reagent 2, 25% reagent 1 + 75% reagent 2, and 0% reagent 1 + 100% reagent 2. Pause the system, place the B2 injection line into reagent 3, set the system B2 to 100% (i.e., 0% A1 + 100% B2), and run the elution at a flow rate of 1.0 ml / min, corresponding to the elution region E. After the previous run, the B2 injection line is filled with reagent 3. Place this B2 injection line directly into 0.5 M NaOH and run it at a flow rate of 1.0 ml / min (corresponding to region F), and the final collector collects the sample.

[0204] After the elution is completed, perform water washing according to step 4 of Example 1.

[0205] The results are as Figure 4 shown in

[0206] Table 4

[0207] A 0.35％ F 84.54％ G 15.11％

[0208] Figure 4 The peak patterns that appear in A, B, C, D, E, F, G and the regions they represent respectively indicate the elution peaks when using the eluents formed by mixing the buffer ratios corresponding to the above A, B, C, D, E, F, G regions for elution; the peak pattern that appears in the region represented by F indicates the elution peak when 0.5 M NaOH is directly added to the B2 tube filled with 20 mM Cit-Buf + 3 M NaCl + 8 M urea pH 4.0 buffer for elution; G represents the stage of column regeneration. If the elution and purification are complete, there will be no elution peak in region G. Since 100% 0.5 M NaOH is used to elute the chromatography column during the regeneration stage, the corresponding elution peak is alkaline and often causes irreversible degradation of RNA; as Figure 4 It can be seen that when the eluent components are A, B, C, D, and E in turn and elution is carried out at room temperature, it is found that there is only one elution peak in the AUV line in the region, accounting for about 0.35%, while the UV lines in regions B, C, D, and E are all at the baseline level, indicating that the sample has not been eluted. However, when 0.5 M NaOH is introduced (region F), the UV line starts to rise and forms an elution peak, and the peak area accounts for about 84.5%. Finally, another regeneration peak appears in the regeneration stage (region G), and the peak area accounts for about 15.1%. The experimental results show that the eluent formed by mixing the pH 4 acidic buffer of 20 mM Cit-Buffer pH4.0 + 3MNaCl + 8M urea and 0.5 M NaOH buffer can enable the specific elution recovery rate of long-chain RNA to reach 84.5% at room temperature, and the addition of urea in the pH 4 acidic buffer system helps to increase the elution efficiency of mRNA from 36% to 84.5%.

[0209] Combining Example 1 and Example 2 shows that: in the solution of the present invention, introducing NaOH (the ratio of Cit-Buf acidic buffer to NaOH buffer) into the eluent composition can achieve the elution of long-chain RNA samples at normal temperature / room temperature; while only the urea component cannot complete the elution of long-chain RNA at normal temperature / room temperature. Introducing both NaOH and urea (the ratio of acidic buffer with pH 4 containing urea to NaOH buffer) into the eluent composition can significantly improve the elution efficiency of long-chain RNA samples at normal temperature / room temperature.

[0210] Example 3 Influence of the addition sequence of urea on the anion exchange chromatography purification of mRNA

[0211] Reagent 1: 20 mM Cit-Buffer + 8 M Urea pH 4.0

[0212] Reagent 2: 0.5 M NaOH

[0213] Reagent 3: 20 mM Cit-Buffer + 3 M NaCl pH 4.0

[0214] 1. Anion exchange chromatography purification

[0215] ·Equiliaration

[0216] Put the A1 injection tube into Reagent 1, put the B1 injection tube into Reagent 2, set the proportion of system B1 to 0% (i.e., 100% A1 + 0% B1), and equilibrate the chromatography column at a flow rate of 1 mL / min until the conductivity curve is balanced for a period of time, then pause and load the sample.

[0217] ·Sample loading

[0218] Use a syringe to aspirate 20 ml of deionized water to wash the 5 ml loop, and then aspirate 20 ml of Reagent 1 to wash the loop again. Take 2.5 ml of diluted 5-fold R37 mRNA and load it onto the loop, set the proportion of system B1 to 0% (i.e., 100% A1 + 0% B1), and run at a flow rate of 1 mL / min.

[0219] ·Columnwash

[0220] Set the proportion of system B1 to 0% (i.e., 100% A1 + 0% B1), and run at a flow rate of 1 mL / min. When the mAU rises to about 20, start collecting the flow-through. After the collection is completed, maintain the proportion of system B1 at 0% (i.e., 100% A1 + 0% B1) and run at a flow rate of 1 mL / min.

[0221] ·Elution

[0222] Place the A2 injection tube in the bottle corresponding to Reagent 2 (0.5M NaOH solution). First, elute at a flow rate of 1.0 ml / min with Region A: System B1 accounting for 100% (i.e., 0% A2 + 100% B1). As Figure 5 shown, a UV peak with an approximate proportion of 2.35% was found, and the sample was not eluted. Then, with Region B: System B1 accounting for 40% (i.e., 60% A2 + 40% B1), elute at a flow rate of 1 ml / min. A UV peak with a proportion of 97.65% was found, and finally, the eluate was collected.

[0223] ·Sanitization

[0224] Place the A2 injection tube in Reagent 2 (0.5M NaOH solution), set the proportion of System B1 to 0% (i.e., 100% A2 + 0% B1), and run at a flow rate of 1 mL / min until the mAU rises to about 50, then start collecting the NaOH eluate to regenerate the chromatography column.

[0225] The experimental results are as follows:

[0226] Table 5

[0227] Elution peak Peak area A 2.35％ B 97.65％

[0228] The results are as Figure 5 shown in Table 5. It can be seen that when eluting with 20 mM Cit - Buffer + 3M NaCl pH4.0 (corresponding to Region A), there is a UV peak with an approximate proportion of 2.35%. When using the buffer formed by mixing 40% Reagent 3 (20 mM Cit - Buffer + 3M NaCl pH4.0) and 60% Reagent 2 (0.5M NaOH) as the eluent (corresponding to Region B), the proportion of the UV peak is 97.65%, and there is no elution peak in the regeneration stage (corresponding to Region C). This shows that when urea is pre - added to the equilibration solution, and the entire chromatography column is treated with the buffer containing urea components (which can be equilibration, sample loading, and washing treatments), in the elution step, the acidic buffer part in the eluent ratio components can achieve efficient elution of long - chain RNA samples at room temperature without the presence of urea when mixed with 0.5M NaOH solution. It is shown that in the solution of the present invention, the addition of urea is not limited to the eluent. As long as urea exists in the chromatography column system environment, that is, urea can be introduced into any system solution in at least one of the equilibration, sample loading, washing, and elution stages. For example, urea can be added to the equilibration solution, sample dilution buffer, etc. Specifically, urea can be added to the buffer in this solution to form: 20 mM Cit - Buffer + 8M Urea pH4.0 buffer.

[0229] 2. RNA Stability Detection

[0230] Detect the purity of RNA by 1% agarose gel electrophoresis: Weigh 1.5 g of agarose and dissolve it in 1x TBE by heating. After cooling, add 10000x GelRed to prepare a 1% agarose gel. Mix 5 μl of the above elution product with 5 μl of 6x loading buffer and load the sample. Electrophorese at 130 V for 30 min for detection.

[0231] As Figure 6 shown in A, the RNA in the elution peak obtained by the above purification method has good integrity and the size is consistent with the expectation. Since there is urea in the eluate, the RNA peak will become wider during electrophoresis.

[0232] 3. RNA Activity Detection

[0233] Label the above chromatographic purification elution products as A7 and A8 respectively, and use a silica gel column to change the solution to remove the residual urea and sodium chloride: Take 500 μl of the product purified and collected by the chromatographic column, add 250 μl of Binding buffer to it, and mix gently. Then add 2812.5 μl of absolute ethanol to the above mixture. Transfer it to the binding column in fractions of 750 μl each time and centrifuge at 12000 RPM for 1 min to remove the liquid in the collection tube. Add 500 μl of WashBuffer to the binding column and wash it twice, centrifuge at 12000 RPM for 1 min. Remove the liquid in the collection tube and centrifuge at 12000 RPM for 5 min. Open the lid and place it in the fume hood for 5 min, then add 40 μl of EloutionBuffer to elute to obtain the RNA after changing the solution.

[0234] Verify the activity of the RNA after replacing the buffer by electrotransfecting 293T cells: Preheat the DMEM + 10% FBS complete medium, SF Cell Line Solution and Supplement I in advance. After the electrotransfection solution SF Cell Line Solution and Supplement I are equilibrated at room temperature, prepare 80 μl of electrotransfection working solution according to 82% + 18%. Resuspend 4 x 10 6 293T cells with 80 μl of electrotransfection solution, and aliquot the resuspended cell solution into 4 RNase-free EP tubes, 20 μl per tube. The experimental groups are A7 and A8 groups, and the positive control group is the R37 group. Add 2 μg of the corresponding RNA to each group and mix well.

[0235] Slowly add the cell sample mixture into the electroporation cuvette for electroporation. After electroporation, transfer the cells to a 6-well plate respectively, and culture them at 37°C with 5% CO2. The culture system is 2 ml. At the same time, for the non-electroporated control group, take 20 μl of the cell suspension into 2 ml of the culture medium in a 6-well plate for culture. After 16 h, collect the 293T cell precipitate, wash it once with PBS, centrifuge it at 1000 RPM for 5 min, resuspend it in 100 μl of PBS, and detect the RNA expression by flow cytometry.

[0236] As Figure 6 As shown in B, there is no significant difference in the activity of the RNA obtained by the above purification after changing the solution and electroporating 293T cells compared with the control R37, which proves that the anion exchange chromatography elution buffer containing NaOH will not damage the functional activity of RNA.

[0237] Example 4 Effects of the concentration ranges of NaCl, urea, and NaOH on the elution efficiency

[0238] 4A: Effects of the ratio range of 0.5 M NaOH to acidic buffer on the elution efficiency

[0239] Reagent 1: 20 mM Cit-Buffer pH 4.0

[0240] Reagent 2: 0.5 M NaOH

[0241] Reagent 3: 20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0

[0242] Anion exchange chromatography purification

[0243] ·Equiliaration

[0244] Put the A1 / B1 injection tubes into the bottles corresponding to the solution of Reagent 1 (20 mM Cit-Buffer pH 4.0), set the proportion of system B1 to 50% (that is, 50% A1 + 50% B1, the purpose is to pre-fill the B1 pipeline with the 20 mM Cit-Buffer pH 4.0 Reagent 1 solution), balance the chromatography column at a flow rate of 1.5 mL / min until the conductivity curve is balanced for a period of time, then pause and load the sample.

[0245] ·Sample loading

[0246] Use a syringe to aspirate 20 ml of deionized water to wash the 5 ml loop, and then aspirate another 20 ml of 20 mM Cit-Buffer pH 4.0 solution to wash the loop. Take 5 ml of R37 mRNA diluted 5-fold with 20 mM Cit-Buffer pH 4.0 solution and load it onto the loop. Set the proportion of system B1 to 0% (i.e., 100% A1 + 0% B1), and run it at a flow rate of 1.5 mL / min.

[0247] ·Column wash

[0248] Maintain the proportion of 100% A1 + 0% B1 and run it at a flow rate of 1.5 mL / min for 3 CV. Then set the proportion of component B1 in the system to 100% (i.e., 0% A1 + 100% B1), and run it at a flow rate of 1.5 mL / min.

[0249] ·Elution

[0250] Place the A2 injection tube in reagent 2: 0.5 M NaOH, and then place the B1 injection tube in the bottle of reagent 3 (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0 solution). Set the proportion of system B1 to 100% (i.e., 0% A2 + 100% B1, (corresponding to region A)) and run for 4 CV for elution. As Figure 7 shown, a UV peak with an approximate proportion of 5.55% is found, and the elution efficiency is extremely low; reset the proportion of system B1 to 90% (i.e., 10% A2 + 90% B1 (corresponding to region B)) and elute at a flow rate of 1.5 ml / min; set the proportion of system B1 to 60% (i.e., 40% A2 + 60% B1 (corresponding to region E)) and elute at a flow rate of 1.5 ml / min.

[0251] ·Sanitization

[0252] Set up the instrument and use 0.5 M NaOH to clean the injection tubes A2 and B1 at a flow rate of 1.5 mL / min for column cleaning (corresponding to region F), and no elution peak is found.

[0253] The experimental results are as follows:

[0254] Table 6

[0255] Elution peak Peak area A 5.55％ E 94.45％

[0256] As Figure 7As can be seen from Table 6, when using 20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0 (Region A), there is a UV peak with a proportion of approximately 5.55%. When using the buffer solution formed by mixing 90% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 10% (0.5 M NaOH) as the eluent, the corresponding region is B. When using the buffer solution formed by mixing 80% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 20% (0.5 M NaOH) as the eluent, the corresponding region is C. When using the buffer solution formed by mixing 70% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 30% (0.5 M NaOH) as the eluent, the corresponding region is D, and in all these cases, the RNA could not be eluted. When using the buffer solution formed by mixing 60% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 40% (0.5 M NaOH) as the eluent, the corresponding region is E, that is, the RNA can be eluted only when 40% of 0.5 M NaOH is present, and there is no elution peak in the regeneration stage (the corresponding region F). This shows that when 0.5 M NaOH is used as one of the buffer solutions for the eluent ratio and its proportion is less than 30%, the RNA cannot be effectively eluted. That is, the optimal proportion of NaOH in the mixed solution is 40%. Combining Example 2 and Example 3, when the NaOH solution is used as one of the ratio solutions for the eluent, if it is a 0.5 M NaOH solution, the proportion range of 0.5 M NaOH is 40% - 60%. Based on this, those skilled in the art can infer that when the concentration of the NaOH solution can be any concentration in the range of 0.2 - 4 M, such as 0.2 M, 0.8 M, 1.0 M, 1.5 M, 1.8 M, 2.0 M, 2.5 M, 2.8 M, 3 M, 3.5 M, 4 M, etc.

[0257] 4A2: Influence of the Ratio Range of 1.0 M NaOH and Acidic Buffer on the Elution Efficiency

[0258] Reagent 1: 20 mM Cit-Buffer pH 4.0

[0259] Reagent 2: 1.0 M NaOH

[0260] Reagent 3: 20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0

[0261] Using the same experimental method as in Example 4A, experiments were carried out on the proportions of 10%, 20%, 50%, 70%, and 80% of 1.0 M NaOH, and the chromatogram results are as follows Figure 34 , Figure 35 , Figure 36 ,Figure 37 , similarly, 1% agarose gel electrophoresis was used to perform integrity analysis on the elution products of the eluents formed by mixing 20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0 solution and 1.0 M NaOH in different proportions. The results are as follows Figure 38 .

[0262] The experimental results are as follows:

[0263] As Figure 34 shown, when the buffer formed by mixing 90% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 10% (1.0 M NaOH) is used as the eluent, for the corresponding region B1, and when the buffer formed by mixing 40% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 60% (1.0 M NaOH) is used as the eluent, for the corresponding region B2, 85.91% of the RNA can be eluted and there is no elution peak in the regeneration stage (corresponding region C).

[0264] As Figure 35 shown, when the buffer formed by mixing 80% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 20% (1.0 M NaOH) is used as the eluent, for the corresponding region B, 94.64% of the RNA can be eluted, and there is almost no RNA residue in the regeneration stage (corresponding region C).

[0265] As Figure 36 shown, when the buffer formed by mixing 50% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 50% (1.0 M NaOH) is used as the eluent, for the corresponding region B, 84.69% of the RNA can be eluted, and there is almost no RNA residue in the regeneration stage (corresponding region C) (only an elution peak with a proportion of 2.28%).

[0266] As Figure 37 shown, when the buffer formed by mixing 30% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 70% (1.0 M NaOH) is used as the eluent, for the corresponding region B, 77.74% of the RNA can be eluted, and there is only a very small amount of RNA residue in the regeneration stage (corresponding region C).

[0267] When the buffer solution formed by mixing 20% (20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0) with 80% (1.0M NaOH) is used as the eluent, RNA can be eluted. However, the pH rises relatively fast in the second half of the elution process. 68.60% of the RNA is eluted in the first stage of the elution.

[0268] Such as Figure 38 It can be seen that the integrity and size of the elution products of the eluents formed by mixing 80% 20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 with 20% 1.0M NaOH, 50% 20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 with 50% 1.0M NaOH, 40% 20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 with 60% 1.0M NaOH, and 30% 20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 with 70% 1.0M NaOH are consistent with the expectations. For the eluent formed by mixing 20% 20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 with 80% 1.0M NaOH, the integrity and size of the elution products in the first half (corresponding to lane A6 in the 80% group of Figure 38) are consistent with the expectations. However, as the elution progresses and the pH rises, the elution products (corresponding to lane A8 in the 80% group of Figure 38) start to degrade.

[0269] Table 7 The elution ability of long - chain RNA at room temperature with 1.0M NaOH as the eluent formulation component and different volume ratios

[0270] NaOH volume ratio 10％ 20％ 50％ 70％ 80％ Elution percentage 85.91％ 94.64％ 84.69％ 77.74％ 68.60％

[0271] In summary, when the proportion of 1.0M NaOH is less than 10%, effective elution cannot be achieved. When the proportion of 1.0M NaOH is higher than 80%, it will promote the degradation of the elution products. At 80% proportion, elution can be achieved, but some products will degrade. In conclusion: When 1.0M NaOH is used as the buffer formulation concentration, as one of the formulation solutions of the eluent, 1.0M NaOH can be used for RNA chromatography purification when the proportion is not less than 10% and not more than 80%, that is, 10% ≤ 1.0M NaOH ≤ 80% when formulating the elution solution. It should be noted that there may be differences in elution efficiency between experimental batches, resulting in differences in elution efficiency, but the overall elution percentage is higher than 50%, indicating that the overall elution scheme is successful and can be used for the elution of long - chain RNA at room temperature.

[0272] 4A3: Influence of the ratio range of 2.0M NaOH and acidic buffer on elution efficiency

[0273] Reagent 1: 20 mM Cit-Buffer pH 4.0

[0274] Reagent 2: 2.0 M NaOH

[0275] Reagent 3: 20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0

[0276] The same experimental method as in Example 4A was used to conduct experimental verification on the proportions of 20%, 30%, 40%, 50%, and 60% of 2.0 M NaOH. The integrity of the elution products of the eluent formed by mixing 20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0 solution and 2.0 M NaOH in different proportions was also analyzed by 1% agarose gel electrophoresis. The results are as Figure 41 .

[0277] The experimental results are as follows:

[0278] As Figure 39 It can be seen that when the buffer formed by mixing 90% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 10% (2.0 M NaOH) is used as the eluent, 76.8% of the RNA can be eluted (corresponding to regions B1 and B2). The elution peak shows a relatively wide area, indicating that the elution strength is insufficient, and there is an elution peak with a proportion of 20.48% in the regeneration stage (corresponding to region C).

[0279] As Figure 40 It can be seen that when the buffer formed by mixing 70% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 30% (2.0 M NaOH) is used as the eluent, 78.82% of the RNA can be eluted (corresponding to region B), and there is only a small amount of RNA residue (about 1.26% in region C) in the regeneration stage (corresponding to region C).

[0280] When the buffer formed by mixing 80% (20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0) with 20% (2.0M NaOH) is used as the eluent, 89.98% of the RNA can be eluted, and only a small amount of RNA (10%) remains during the regeneration stage. When the buffer formed by mixing 60% (20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0) with 40% (2.0M NaOH) is used as the eluent, 92.55% of the RNA can be eluted, and only a very small amount of RNA (7%) remains during the regeneration stage. When the buffer formed by mixing 50% (20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0) with 50% (2.0M NaOH) is used as the eluent, 94.99% of the RNA can be eluted, and only a very small amount of RNA (5%) remains during the regeneration stage. When the buffer formed by mixing 40% (20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0) with 60% (2.0M NaOH) is used as the eluent, 68.65% of the RNA can be eluted, but the pH starts to rise in the second half of the elution.

[0281] As Figure 41 It can be seen that the integrity and size of the elution products of the eluents formed by mixing 80% 20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 with 20% 2.0M NaOH, 70% 20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 with 30% 2.0M NaOH, 60% 20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 with 40% 2.0M NaOH, 50% 20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 with 50% 2.0M NaOH are consistent with the expectations. However, for the eluent formed by mixing 40% 20 mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 with 60% 2.0M NaOH, the integrity and size of the elution products in the first half of the elution (corresponding to lane A7 in the 60% group of Figure 41) are consistent with the expectations, but the elution products (corresponding to lane B1 in the 60% group of Figure 41) start to degrade as the pH rises during the elution.

[0282] Table 8 Elution ability of long - chain RNA at room temperature with different volume ratios of 2.0M NaOH as the eluent components:

[0283] NaOH volume ratio 10％ 30％ 40％ 50％ 60％ Elution percentage 76.80％ 78.82％ 92.55％ 94.99％ 68.65％

[0284] In summary, when the proportion of 2.0M NaOH is less than 10%, effective elution cannot be achieved. When the proportion of 2.0M NaOH is higher than 60%, it will promote the degradation of elution products. At a ratio of 60%, elution can be achieved, but partial product degradation will occur. To sum up: When 2.0M NaOH is used as the buffer concentration ratio, the 2.0M NaOH can be used as one of the formula solutions of the eluent at a ratio of not less than 10% and not more than 60% for RNA chromatography purification. When preparing the elution solution, 10% ≤ 1.0M NaOH ≤ 60%. It should be noted that there may be differences in elution efficiency between experimental batches, resulting in differences in elution efficiency, but the overall elution percentage is higher than 50%, indicating that the overall elution scheme is successful and can be used for the elution of long-chain RNA at room temperature.

[0285] 4A4: Influence of the ratio range of 3.0M NaOH and acidic buffer on elution efficiency

[0286] Reagent 1: 20mM Cit-Buffer pH4.0

[0287] Reagent 2: 3.0M NaOH

[0288] Reagent 3: 20mM Cit-Buffer + 3M NaCl + 8M Urea pH4.0

[0289] The same experimental method as in Example 4A was used to verify the ratios of 10%, 20% and 50% of 3.0M NaOH. The integrity of the elution products of the eluents formed by different ratios of 20mM Cit-Buffer + 3M NaCl + 8M Urea pH4.0 solution and 3.0M NaOH was also analyzed by 1% agarose gel electrophoresis. The results are as Figure 42 .

[0290] The experimental results are as follows:

[0291] When the buffer formed by mixing 90% (20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0) with 10% (3.0 M NaOH) is used as the eluent, 88.85% of the RNA can be eluted, and only a very small amount of RNA remains during the regeneration stage (corresponding to region C). When the buffer formed by mixing 80% (20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0) with 20% (3.0 M NaOH) is used as the eluent, 87.44% of the RNA can be eluted, and only a very small amount of RNA remains during the regeneration stage. When the buffer formed by mixing 50% (20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0) with 50% (3.0 M NaOH) is used as the eluent, 93.45% of the RNA can be eluted, and only a very small amount of RNA remains during the regeneration stage.

[0292] As Figure 45 shown, when the buffer formed by mixing 90% (20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0) with 10% (3.0 M NaOH) is used as the eluent, 90.66% of the RNA can be eluted (corresponding to region B). In addition, we also verified the elution of RNA at room temperature using the eluents formed by mixing 80% 20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0 with 20% 3.0 M NaOH and 50% 20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0 with 50% 3.0 M NaOH. The elution efficiency of all is above 80%; and the integrity and size of the eluted RNA were detected:

[0293] As Figure 42 shown, the integrity and size of the elution products using the eluents formed by mixing 90% 20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0 with 10% 3.0 M NaOH, 80% 20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0 with 20% 3.0 M NaOH, and 50% 20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0 with 50% 3.0 M NaOH are consistent with the expectations.

[0294] Table 93.0 M NaOH as the eluent ratio components, long - chain RNA elution ability at different volume ratios at room temperature

[0295] NaOH volume ratio 10％ Elution percentage 90.66％

[0296] In summary, when 3.0M NaOH is used as the buffer concentration ratio, the eluents formed by the ratios of 90% 20mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 and 10% 3.0M NaOH, 80% 20mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 and 20% 3.0M NaOH, 70% 20mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 and 30% 3.0M NaOH can be used for RNA chromatography purification. 3.0M NaOH, as one of the formulation solutions of the eluent, can be used for RNA chromatography purification, where 10% ≤ 3.0M NaOH ≤ 50%. It should be noted that there may be differences in the elution efficiency between experimental batches, resulting in different elution efficiencies, but the overall elution percentage is higher than 50%, indicating that the overall elution scheme is successful and can be used for the elution of long - chain RNA at room temperature.

[0297] 4A5: Influence of the ratio range of 4.0M NaOH and acidic buffer on the elution efficiency

[0298] Reagent 1: 20mM Cit - Buffer pH4.0

[0299] Reagent 2: 4.0M NaOH

[0300] Reagent 3: 20mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0

[0301] The different proportions of 4.0M NaOH were experimentally verified using the same experimental method as in Example 4A. The chromatogram results are as Figure 43 , and the integrity of the elution products of the eluents formed by different proportions of 20mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0 solution and 4.0M NaOH was also analyzed using 1% agarose gel electrophoresis. The results are as Figure 44 .

[0302] The experimental results are as follows:

[0303] As Figure 43As can be seen, when the buffer formed by mixing 95% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 5% (4.0 M NaOH) is used as the eluent, RNA cannot be eluted (corresponding to region B1). When the buffer formed by mixing 90% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 10% (4.0 M NaOH) is used as the eluent, RNA can be eluted (corresponding to region B2) by 94.14%, and there is no RNA residue in the regeneration stage (corresponding to region C). As Figure 43 As can be seen: When the buffer formed by mixing 50% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 50% (4.0 M NaOH) is used as the eluent, 80.86% of the RNA can be eluted, and there is only a small amount of RNA residue in the regeneration stage (corresponding to region C).

[0304] When the buffer formed by mixing 60% (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0) and 40% (4.0 M NaOH) is used as the eluent, 61.53% of the RNA can be eluted, but the pH starts to rise in the second half of the elution.

[0305] As Figure 44 As can be seen, the integrity and size of the elution product of the eluent formed by mixing 90% 20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0 and 10% 4.0 M NaOH are consistent with the expectation.

[0306] Referring to the method for detecting the RNA activity in Example 3, the RNA activities of the RNAs eluted with the eluents prepared by mixing different NaOH concentrations in different ratios in Example 4 were detected. The results are as Figure 46 shown. The RNAs eluted with the highest mixing ratios of 1 M NaOH (80%), 2 M NaOH (60%), 3 M NaOH (50%), and 4 M NaOH (12.5%, 50%) can all be transcribed and expressed in the target cells, and there is no difference compared with the activity of R37 purified by the standard method. The RNA purified by the scheme described in the present invention has excellent activity, and it can be inferred that the RNA activities of the RNAs eluted under other lower mixing ratios are also excellent.

[0307] In summary, when the proportion of 4.0M NaOH is less than 10%, effective elution cannot be achieved. When the proportion of 4.0M NaOH is higher than 40%, it will promote the degradation of the eluted product. At a 40% ratio, elution can be achieved, but partial product degradation will occur. In conclusion: when 4.0M NaOH is used as the buffer concentration, as one of the formula solutions of the eluent, 4.0M NaOH can be used for RNA chromatography purification at a ratio of not less than 10% and not more than 40%. When preparing the elution solution, 10% ≤ 4.0M NaOH ≤ 40%. It should be noted that there may be differences in elution efficiency between experimental batches, but the overall elution percentage is higher than 50%, indicating that the overall elution scheme is successful and can be used for the elution of long-chain RNA at room temperature. In summary, as one of the formula solutions for preparing the eluent of long-chain RNA at room temperature, NaOH with a concentration of 0.5 - 4M can be used, and the proportion can be selected from 10% - 80%.

[0308] 4B: Influence of NaCl gradient on elution efficiency

[0309] Reagent 1: 20mM Cit-Buffer pH4.0

[0310] Reagent 2: 0.5M NaOH

[0311] Reagent 3: 20mM Cit-Buffer + 0M NaCl + 8M Urea pH4.0

[0312] 20mM Cit-Buffer + 1M NaCl + 8M Urea pH4.0

[0313] 20mM Cit-Buffer + 3M NaCl + 8M Urea pH4.0

[0314] 20mM Cit-Buffer + 3.5M NaCl + 8M Urea pH4.0

[0315] Complete the equilibration and sample loading / washing according to the scheme of Example 4. The sample loaded is also R37 mRNA.

[0316] Place the A2 pump in the bottle containing Reagent 2 (0.5M NaOH solution), and then place B1 in the bottle containing Reagent 3 (20mM Cit-Buffer + 8M Urea + 0M NaCl pH4.0 or 20mM Cit-Buffer + 8M Urea + 1M NaCl pH4.0 or 20mM Cit-Buffer + 8M Urea + 3M NaCl pH4.0 or 20mM Cit-Buffer + 8M Urea + 3.5M NaClin a bottle with pH 4.0, when the proportion of component B1 in system B is set to 60% (i.e., 40% A2 + 60% B1) (i.e., 40% (0.5M NaOH) is successively mixed with 60% (20mM Cit-Buffer + 0M NaCl + 8M Urea pH4.0), 60% (20mM Cit-Buffer + 1M NaCl + 8M Urea pH4.0), 60% (20mM Cit-Buffer + 3M NaCl + 8M Urea pH4.0), 60% (20mM Cit-Buffer + 3.5M NaCl + 8M Urea pH4.0) to form the eluent, respectively corresponding to Figure 8 / 9 / 10 / 11 area B), the system is eluted at a flow rate of 1.5 ml / min.

[0317] · Sanitization regeneration

[0318] Keep the A2 pump in the bottle of reagent 2 (0.5M NaOH), set the proportion of component B1 in system B to 0% (i.e., 100% A2 + 0% B1), and perform column cleaning at a flow rate of 1.5 mL / min.

[0319] The experimental results are as follows:

[0320] Table 10

[0321] Elution peak Peak area A 0.64％ B 99.36％

[0322] Table 11

[0323] Elution peak Peak area A 2.12％ B 96.55％ C 1.33％

[0324] Table 12

[0325] Elution peak Peak area A 1.13％ B 98.87％

[0326] Table 13

[0327] Elution peak Peak area A 4.35％ B 95.65％

[0328] As Figure 8 - 11 shown in Tables 10 - 13, it can be seen that when using the eluent formed by mixing 40% (0.5M NaOH) with 60% (20mM Cit-Buffer + 0M NaCl + 8M Urea pH4.0), 60% (20mM Cit-Buffer + 1M NaCl + 8M Urea pH4.0), 60% (20mM Cit-Buffer + 3M NaCl + 8M Urea pH4.0), 60% (20mM Cit-Buffer + 3.5M NaCl + 8M Urea pH4.0) respectively for elution, it corresponds toFigure 8 , 9 , areas 10 and 11, region B. The specific elution areas are shown in Tables 9 - 12 respectively. The results show that in the solution of the present invention, when the final concentration of the eluent contains NaCl greater than 0 M, RNA can be completely eluted at room temperature, and the elution peak areas are all greater than 95%. However, Figure 8 As shown, when the UV line of the elution peak in region B rises, the pH also rises linearly, indicating that the elution collection solution in this region is alkaline, suggesting that 20 mM Cit - Buffer + 0 M NaCl + 8 M Urea pH 4.0 as one of the buffer solutions for the eluent ratio may damage the stability of RNA; but Figure 9 - 11 As shown, after the UV line of the elution peak in region B drops to the baseline level, the pH curve in the figure begins to rise linearly to alkaline, that is, 20 mM Cit - Buffer + 1 M NaCl + 8 M Urea pH 4.0, 20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0, and 20 mM Cit - Buffer + 3.5 M NaCl + 8 M Urea pH 4.0 can all be used as one of the buffer solutions for the eluent ratio to complete the elution of long - chain RNA at room temperature and maintain the stability of RNA. Based on this, to achieve the purification of long - chain RNA at room temperature, the acidic buffer solution in the ratio should contain an NaCl component. Combining Example 3 and Example 4, the ratio of the acidic buffer solution can be 40% - 60%, and the NaCl component in the acidic buffer solution can be 1 M - 3.5 M.

[0329] 4C: Influence of 20 mM Cit - Buffer pH value gradient on elution efficiency

[0330] Reagent 1: 20 mM Cit - Buffer pH 4.0

[0331] Reagent 2: 0.5 M NaOH

[0332] Reagent 3: 20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 3.0

[0333] 20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0

[0334] 20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 5.0

[0335] 20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 6.2

[0336] Complete equilibration and sample loading / washing according to the protocol of Example 4. The sample loaded is also R37 mRNA.

[0337] Place the A2 injection tube in a bottle containing Reagent 2 (0.5 M NaOH solution), and place B1 in a bottle containing Reagent 3 (20 mM Cit-Buffer + 8 M Urea + 3 M NaCl pH 3.0 or 20 mM Cit-Buffer + 8 M Urea + 3 M NaCl pH 4.0 or 20 mM Cit-Buffer + 8 M Urea + 3 M NaCl pH 5.0 or 20 mM Cit-Buffer + 8 M Urea + 3 M NaCl pH 6.2). Set the proportion of component B1 in the system to 60% (i.e., 40% A2 + 60% B1). When the buffer formed by mixing 40% (0.5 M NaOH) with 60% (20 mM Cit-Buffer + 8 M Urea + 3 M NaCl pH 3.0 or 20 mM Cit-Buffer + 8 M Urea + 3 M NaCl pH 4.0 or 20 mM Cit-Buffer + 8 M Urea + 3 M NaCl pH 5.0 or 20 mM Cit-Buffer + 8 M Urea + 3 M NaCl pH 6.2) in sequence is used as the eluent, it corresponds to Figure 12 、 13 、14, 15 Region B), and the specific elution areas are shown in Tables 14 - 17. Elute at a flow rate of 1.5 ml / min.

[0338] Table 14

[0339] Elution peak Peak area A 1.13％ B 98.87％

[0340] Table 15

[0341] Elution peak Peak area A 4.86％ B 95.14％

[0342] Table 16

[0343] Elution peak Peak area A 1.21％ B 98.79％

[0344] Table 17

[0345] Elution peak Peak area A 9.1％ B 90.9％

[0346] ·Sanitization

[0347] Place the A2 injection tube in a 0.5 M NaOH bottle, set the proportion of component B1 in the system to 0% (i.e., 100% A2 + 0% B1), and perform column cleaning at a flow rate of 1.5 mL / min.

[0348] The experimental results are as follows:

[0349] As shown Figure 12 - 15 in Tables 14 to 17, it can be seen that when using a buffer formed by mixing 40% (0.5M NaOH) with 60% (20mM Cit-Buffer + 8M Urea + 3M NaCl at pH 3.0 or 20mM Cit-Buffer + 8M Urea + 3M NaCl at pH 4.0 or 20mM Cit-Buffer + 8M Urea + 3M NaCl at pH 5.0 or 20mM Cit-Buffer + 8M Urea + 3M NaCl at pH 6.2) in sequence as the eluent, respectively corresponding to Figure 12 、 13 、regions 14 and 15 (B), and performing elution at a flow rate of 1.5 ml / min, UV peaks were all found, indicating that the RNA was eluted, suggesting that 20mM Cit-Buffer is helpful for protecting RNA elution within the range of pH 3.0 - pH 6.2. That is, the optimal range of 20mM Cit-Buffer is pH 3.0 - pH 6.2.

[0350] 4D: Influence of Cit-Buffer concentration gradient on elution efficiency

[0351] Reagent 1: 0mM Cit-Buffer

[0352] 20mM Cit-Buffer pH4.0

[0353] 50mM Cit-Buffer pH4.0

[0354] 100mM Cit-Buffer pH4.0

[0355] Reagent 2: 0.5M NaOH

[0356] Reagent 3: 0mM Cit-Buffer + 3M NaCl + 8M Urea

[0357] 20mM Cit-Buffer + 3M NaCl + 8M Urea pH4.0

[0358] 50mM Cit-Buffer + 3M NaCl + 8M Urea pH4.0

[0359] 100mM Cit-Buffer + 3M NaCl + 8M Urea pH4.0

[0360] Complete equilibration and loading / washing according to the scheme of Example 4, and the loading sample is also R37 mRNA.

[0361] Place the A2 injection tube in a bottle containing Reagent 2 (0.5M NaOH solution), and place B1 in a bottle containing Reagent 3 (0mM Cit-Buffer + 8M Urea + 3M NaCl or 20mM Cit-Buffer + 8M Urea + 3M NaCl pH4.0 or 50mM Cit-Buffer + 8M Urea + 3M NaCl pH4.0 or 100mM Cit-Buffer + 8M Urea + 3M NaCl pH4.0). Set the proportion of component B1 in the system to 60% (i.e., 40% A2 + 60% B1), and perform elution at a flow rate of 1.5 ml / min. The results are as Figure 16 、 17 、shown in 18, 19.

[0362] ·Sanitization

[0363] Maintain the A2 injection tube in the 0.5M NaOH bottle, set the proportion of component B1 in the system to 0% (i.e., 100% A2 + 0% B1), and perform column cleaning at a flow rate of 1.5 mL / min.

[0364] The experimental results are as follows:

[0365] Table 18

[0366] Elution peak Peak area A 0.85％ B 99.15％

[0367] Table 19

[0368] Elution peak Peak area A 4.86％ B 95.14％

[0369] Table 20

[0370] Elution peak Peak area A 3.68％ B 96.32％

[0371] Table 21

[0372] Elution peak Peak area A 1.67％ B 97.33％

[0373] As Figure 16 - 19As shown in Tables 18 to 21, it can be seen that the above elution protocols can all promote the elution of RNA, indicating that acidic buffers containing 0M - 100mM Cit - Buffer, 8M Urea + 3M NaCl and a buffer formed by mixing 0.5M NaOH in a ratio of 60% / 40% can all be used as eluents for RNA elution. However, when the buffer formed by mixing an acidic buffer containing 0M Cit - Buffer, 8M Urea, 3M NaCl and 0.5M NaOH in a ratio of 60% / 40% is used as the eluent, it shows a slightly alkaline pH before elution, while when the buffer formed by mixing an acidic buffer containing 20 - 100mM Cit - Buffer, 8M Urea, 3M NaCl and 0.5M NaOH in a ratio of 60% / 40% is used as the eluent, it is in a stable pH state during the process before RNA is eluted. In addition, the Cit - Buffer not only stabilizes the pH of the system but also has the function of a metal chelator, which helps to improve the stability of the RNA purification process. That is, the optimal introduction concentration range of Cit - Buffer is 20 - 100mM. Combining Examples 3, 4, and 5, the ratio of the acidic buffer can be 40% - 60%, so the final concentration of Cit - Buffer can also be 8 - 60mM.

[0374] 4E: Influence of Urea Concentration Gradient on Elution Efficiency

[0375] Reagent 1: 20mM Cit - Buffer pH4.0

[0376] Reagent 2: 0.5M NaOH

[0377] Reagent 3: 20mM Cit - Buffer + 3M NaCl + 0M Urea pH4.0

[0378] 20mM Cit - Buffer + 3M NaCl + 2M Urea pH4.0

[0379] 20mM Cit - Buffer + 3M NaCl + 4M Urea pH4.0

[0380] 20mM Cit - Buffer + 3M NaCl + 8M Urea pH4.0

[0381] Complete equilibration and loading / washing according to the protocol of Example 4. The loaded sample is also R37 mRNA.

[0382] Place the A2 injection tube in the bottle of Reagent 2 (0.5M NaOH solution), and place B1 in the bottle containing Reagent 3 (20mM Cit-Buffer + 0M Urea + 3M NaCl pH4.0 or 20mM Cit-Buffer + 2M Urea + 3M NaCl pH4.0 or 20mM Cit-Buffer + 4M Urea + 3M NaCl pH4.0 or 20mM Cit-Buffer + 8M Urea + 3M NaCl pH4.0). Set the proportion of component B1 in the system to 60% (i.e., 40% A2 + 60% B1), and elute at a flow rate of 1.5 ml / min.

[0383] ·Sanitization

[0384] Keep the A2 pump in the 0.5M NaOH bottle, set the proportion of B1 in the system to 0% (i.e., 100% A2 + 0% B1), and perform column cleaning at a flow rate of 1.5 mL / min.

[0385] The experimental results are as follows:

[0386] Table 22

[0387] A 2.53％ B 97.47％

[0388] Table 23

[0389] Elution peak Peak area A 2.71％ B 95.27％ C 2.02％

[0390] Table 24

[0391] Elution peak Peak area A 1.22％ B 98.78％

[0392] Table 25

[0393] Elution peak Peak area A 4.86％ B 95.14％

[0394] As Figure 20 - 23 and Tables 22 - 25 show, when using the buffer formed by mixing the acidic buffer containing 3M NaCl, 20mM Cit-Buffer with 0M Urea or 2M Urea or 4M Urea or 8M Urea and 0.5M NaOH in a 60% / 40% ratio as the eluent and eluting at a flow rate of 1.5 ml / min, UV peaks are all found (corresponding respectively to Figure 20 / 21 / 22 / 23 Region B), it is indicated that the RNA is eluted, indicating that the addition of 0M - 8M urea can be used for RNA elution. 20 mM Cit - Buffer + 0M Urea + 3M NaCl pH4.0 or 20 mM Cit - Buffer + 2M Urea + 3M NaCl pH4.0 or 20 mM Cit - Buffer + 4M Urea + 3M NaCl pH4.0 or 20 mM Cit - Buffer + 8M Urea + 3M NaCl pH4.0 can all be used as one of the formulation solutions for the eluent. This eluent can be used for the purification of long - chain RNA at room temperature. Comparing the trend of the pH curve in Region B during the elution stage as it decreases with the UV line (indicating the elution of RNA), Figure 21 - 23 the rising amplitude of the pH curve in Region B is less than Figure 20 as shown, indicating that the presence of urea helps to maintain the pH stability of the RNA elution environment. Therefore, the introduction of urea in the formulated buffer is more preferred, and the concentration of urea is preferably 2 - 8M.

[0395] Example 5 Influence of NaCl Component Substitution on Elution Efficiency

[0396] Reagent 1: 20 mM Cit - Buffer pH4.0

[0397] Reagent 2: 0.5M NaOH

[0398] Reagent 3: 20 mM Cit - Buffer + 3M MgCl2 + 8M Urea pH4.0

[0399] 20 mM Cit - Buffer + 3M KCl + 8M Urea pH4.0

[0400] 20 mM Cit - Buffer + 3M (NH4)2SO4 + 8M Urea pH4.0

[0401] Complete the equilibration and loading / washing according to the scheme of Example 4. The loaded sample is also R37 mRNA.

[0402] Place the A2 injection tube in the bottle containing Reagent 2 (0.5M NaOH solution), and place B1 in the bottle containing Reagent 3 (20mM Cit-Buffer + 3M MgCl2 + 8M Urea pH4.0 or 20mM Cit-Buffer + 3M KCl + 8M Urea pH4.0 or 20mM Cit-Buffer + 3M (NH4)2SO4 + 8M Urea pH4.0). Set the proportion of component B1 in the system to 60% (i.e., 40% A2 + 60% B1), and perform elution at a flow rate of 1.5 ml / min. It is found that only when KCl replaces NaCl, there is a UV peak in elution region B, indicating that RNA is eluted, while when using (NH4)2SO4 and MgCl2, there is no UV peak shown in region B.

[0403] ·Sanitization

[0404] Place the A2 injection tube in the bottle containing Reagent 2 (0.5M NaOH), set the proportion of component B1 in the system to 0% (i.e., 100% A2 + 0% B1), and perform column cleaning at a flow rate of 1.5 mL / min.

[0405] The experimental results are as Figure 24 - 26 shown in Tables 26 - 28.

[0406] Table 26

[0407] Elution peak Peak area C 100％

[0408] Table 27

[0409] Elution peak Peak area A 5.06％ B 94.94％

[0410] Table 28

[0411] Elution peak Peak area A + B 25.76％ C 74.24％

[0412] The results show that when using the buffer formed by mixing 20mM Cit-Buffer + 3M MgCl2 + 8M Urea pH4.0 or 20mM Cit-Buffer + 3M KCl + 8M Urea pH4.0 or 20mM Cit-Buffer + 3M (NH4)2SO4 + 8M Urea pH4.0 with 0.5M NaOH in a 60% / 40% ratio as the eluent, they respectively correspond to Figure 24 、 25, elute the 26 regions B). Only when NaCl is replaced by KCl, there is a UV peak in the elution region B, indicating that RNA is eluted. However, when using (NH4)2SO4 and MgCl2, no UV peak is shown in region B. In summary, NaCl can be replaced by KCl, but cannot be replaced by (NH4)2SO4 and MgCl2. This shows that in the solution of the present invention, NaCl can be replaced by a monovalent Cl ion salt, such as KCl. Combining with Example 4, to achieve the purification of long-chain RNA at room temperature / normal temperature, one of the eluent ratio solutions, the acidic buffer, should contain a monovalent Cl ion salt, and the preferred monovalent Cl ion salts are NaCl and KCl.

[0413] Example 6 Effect of Component Substitution of 20 mM Cit-Buffer pH 4.0 on Elution Efficiency

[0414] Reagent 1: 20 mM acetic acid-sodium acetate

[0415] 20 mM glycine-hydrochloric acid

[0416] Reagent 2: 0.5 M NaOH

[0417] Reagent 3: 20 mM acetic acid-sodium acetate + 3 M NaCl + 8 M Urea pH 4.0

[0418] 20 mM glycine-hydrochloric acid + 3 M NaCl + 8 M Urea pH 4.0

[0419] Put the A1 / B1 injection tubes into the bottle containing Reagent 1 (20 mM acetic acid-sodium acetate pH 4.0 or 20 mM glycine-hydrochloric acid pH 4.0 solution), set the proportion of component B1 to be 50% (i.e., 50% A1 + 50% B1), and balance the chromatography column at a flow rate of 1.5 mL / min until the conductivity curve is balanced for a period of time, then pause and load the sample.

[0420] Use a syringe to aspirate 20 ml of deionized water to wash the 5 ml loop, and then aspirate 20 ml of 20 mM acetic acid-sodium acetate pH 4.0 or 20 mM glycine-hydrochloric acid pH 4.0 to wash the loop. Take a total of 5 ml of R37 mRNA diluted 5 times with 20 mM acetic acid-sodium acetate pH 4.0 or 20 mM glycine-hydrochloric acid pH 4.0 solution and load it onto the column through the loop. Set the proportion of component B1 in the system to be 0% (i.e., 100% A1 + 0% B1), and run at a flow rate of 1.5 mL / min. Complete the washing according to the scheme of Example 4.

[0421] ·Elution

[0422] Place the A2 injection tube in a bottle containing Reagent 2 (0.5 M NaOH solution), and place B1 in 20 mM acetic acid - sodium acetate + 3 M NaCl + 8 M Urea pH 4.0 or 20 mM glycine - hydrochloric acid + 3 M NaCl + 8 M Urea pH 4.0. Set the proportion of component B1 in the system to 60% (i.e., 40% A2 + 60% B1), and perform elution at a flow rate of 1.5 ml / min. It is found that there are UV peaks in elution region B when acetic acid - sodium acetate or glycine - hydrochloric acid replaces Cit - Buffer, indicating that RNA is eluted.

[0423] Perform column cleaning according to the scheme of Example 4.

[0424] The experimental results are as Figure 27 - 28 shown in Table 29 - 30.

[0425] Table 29

[0426]

[0427]

[0428] Table 30

[0429] Elution peak Peak area Elution peak Peak area A 0.6％ B 99.01％ C 0.39％

[0430] As Figure 27 - 28 shown in Table 29 - 30, when using the buffer formed by mixing 20 mM acetic acid - sodium acetate + 3 M NaCl + 8 M Urea pH 4.0 or 20 mM glycine - hydrochloric acid + 3 M NaCl + 8 M Urea pH 4.0 with 0.5 M NaOH in a 60% / 40% ratio as the eluent, corresponding to Figure 27 、 28 region B) for elution, it is found that there are UV peaks in elution region B when acetic acid - sodium acetate or glycine - hydrochloric acid replaces Cit - Buffer, and the proportion of the peak area > 97%. In summary, 20 mM acetic acid - sodium acetate pH 4.0 and 20 mM glycine - hydrochloric acid pH 4.0 solutions can both replace 20 mM Cit - Buffer pH 4.0. Therefore, in the scheme described in the present invention, the buffer Buffer is not limited to a specific buffer pair salt, and any buffer solution / buffer system with a pH of 4.0 and a concentration in the range of 20 mM - 100 mM can be applied to this scheme, for example: citric acid - sodium citrate system, sodium dihydrogen phosphate - citric acid system, citric acid - sodium hydroxide - hydrochloric acid system, acetic acid - sodium acetate system or glycine - hydrochloric acid system.

[0431] Example 7 Influence of NaOH Component Substitution on Elution Efficiency

[0432] Reagent 1: 20 mM Cit-Buffer pH 4.0

[0433] Reagent 2: 0.5 M Disodium Hydrogen Phosphate

[0434] 0.5 M Tris

[0435] 0.5 M KOH

[0436] Reagent 3: 20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0

[0437] Complete equilibration and sample loading / washing according to the protocol of Example 4. The sample loaded is also R37 mRNA.

[0438] Place the A2 injection tube in the bottle containing Reagent 2 (0.5 M Disodium Hydrogen Phosphate or 0.5 M Tris or 0.5 M KOH solution), and place B1 in the bottle containing Reagent 3 (20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0). Set the proportion of component B1 in the system to 60% (i.e., 40% A2 + 60% B1), and perform elution at a flow rate of 1.5 ml / min. It is found that only when KOH replaces NaOH, there is a UV peak in elution region B, indicating that RNA is eluted, while no UV peak is shown in region B when using Disodium Hydrogen Phosphate and Tris.

[0439] ·Sanitization

[0440] Keep the A2 pump placed in the 0.5 M NaOH bottle, set the proportion of component B1 in the system to 0% (i.e., 100% A2 + 0% B1), and perform column cleaning at a flow rate of 1.5 mL / min.

[0441] The experimental results are as follows:

[0442] Table 31

[0443] Elution peak Peak area A 7.64％ C 92.36％

[0444] Table 32

[0445] Elution peak Peak area C 10.28％ D 89.72％

[0446] Table 33

[0447] Elution peak Peak area A 2.69％ B 95.22％ C 2.1％

[0448] As Figure 29 - 31As shown in Tables 31 - 33, when using the buffer formed by mixing 20 mM Cit - Buffer + 3 M NaCl + 8 M Urea pH 4.0 with 0.5 M disodium hydrogen phosphate or 0.5 M Tris or 0.5 M KOH in a 60% / 40% ratio as the eluent, respectively corresponding to Figure 29 , 30 , and Region B of 31), it was found that only when KOH replaced NaOH was there a UV peak in Elution Region B, indicating that the RNA was eluted, while when using disodium hydrogen phosphate and Tris, no UV peak was shown in Region B. In summary, KOH can replace NaOH, while disodium hydrogen phosphate and Tris cannot replace NaOH. It shows that in the scheme of the present invention, NaOH can be replaced by a salt containing OH - , such as KOH.

[0449] Example 8 Effect of guanidine hydrochloride replacing urea on elution efficiency

[0450] Reagent 1: 20 mM Cit - Buffer pH 4.0

[0451] Reagent 2: 0.5 M NaOH

[0452] Reagent 3: 20 mM Cit - Buffer + 3 M NaCl + 3.4 M guanidine hydrochloride pH 4.0

[0453] Complete the equilibration and loading / washing according to the scheme of Example 4, and the loading sample is also R37 mRNA.

[0454] Place the A2 injection tube in the bottle containing Reagent 2 (0.5 M NaOH solution), and then place B1 in the bottle containing Reagent 3 (20 mM Cit - Buffer + 3 M NaCl + 3.4 M guanidine hydrochloride pH 4.0). Set the proportion of component B1 in the system to 60% (i.e., 40% A2 + 60% B1, corresponding to Figure 32 Region B), and perform elution at a flow rate of 1.5 ml / min. It was found that after guanidine hydrochloride replaced urea, there were two UV peaks in Elution Region B, accounting for 77.75% and 21.44% respectively.

[0455] ·Sanitization

[0456] Keep the A2 pump placed in the 0.5 M NaOH bottle, set the proportion of component B1 in the system to 0% (i.e., 100% A2 + 0% B1), and perform column cleaning at a flow rate of 1.5 mL / min.

[0457] The experimental results are shown in Figure 32 and Table 34.

[0458] Table 34

[0459] Elution peak Peak area A 0.81％ B1 77.75％ B2 21.44％

[0460] As Figure 32 can be seen, when using the buffer formed by mixing 20 mM Cit-Buffer + 3 M NaCl + 3.4 M guanidine hydrochloride at pH 4.0 and 0.5 M NaOH in a ratio of 60% / 40% as the eluent, corresponding to Figure 32 Region B) for elution, it was found that after replacing urea with guanidine hydrochloride, there were 2 UV peaks in elution region B, accounting for 77.75% and 21.44% respectively, and the second peak coincided with the increase in pH. That is, the area of peak B1 was the recovery rate of 77.75%, indicating that guanidine hydrochloride can replace urea. Based on the common knowledge of R & D personnel in this field, it can be inferred that the urea component in the acidic buffer for forming the elution buffer can be replaced by other chaotropic agents, including but not limited to: lithium perchlorate or other perchlorates, guanidine chloride or other guanidine salts, urea, butanol, ethanol, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, or one or several of them.

[0461] Example 9 Effect of Column Replacement on RNA Elution Efficiency

[0462] Reagent 1: 20 mM Cit-Buffer pH 4.0

[0463] Reagent 2: 0.5 M NaOH

[0464] Reagent 3: 20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0

[0465] Complete equilibration and loading / washing according to the protocol of Example 4, and the loaded sample was also R37 mRNA.

[0466] The A2 injection tube was placed in a 0.5 M NaOH solution, and then B1 was placed in a bottle containing Reagent 3 (20 mM Cit-Buffer + 8 M Urea + 3 M NaCl pH 4.0). Set the proportion of component B1 in the system to 60% (i.e., 40% A2 + 60% B1, corresponding to Figure 33 Region B), and elute at a flow rate of 1.5 ml / min. It was found that there was a UV peak with an area proportion of 92.96%, indicating that RNA was eluted, suggesting that the buffer and purification method of the present invention are also applicable to the 50HQ column.

[0467] ·Sanitization

[0468] Keep the A2 injection tube placed in the 0.5 M NaOH bottle, set the proportion of component B1 in the system to 0% (i.e., 100% A2 + 0% B1), and perform column cleaning at a flow rate of 1.5 mL / min.

[0469] The experimental results are as follows:

[0470] Table 35

[0471] Elution peak Peak area A 7.24％ B 92.96％

[0472] As Figure 33 can be seen from Table 35, when using the buffer formed by mixing 20 mM Cit-Buffer + 3 M NaCl + 8 M Urea pH 4.0 and 0.5 M NaOH in a ratio of 60% / 40% as the eluent for elution in the corresponding Figure 33 region B), a UV peak was found with an area ratio of 92.96%, indicating that the RNA was eluted, suggesting that the method of this study is also applicable to the 50HQ column. Further, it is shown that the solution described in the present invention uses all ion exchange column solutions, including but not limited to: weak anion chromatography columns such as UniGel-30DEAE, DEAE Chromstar FF, MaXtar DEAE, UniDEAE-50XS, UniDEAE-30S, or strong anion chromatography columns such as 50HQ column, MaXtar Q, MaXtar Q HR, Q Chromstar HP, POROS TM GoPure TM HQ, POROS TM XQ, NanoGel TM -50Q HC, etc. as columns filled with the above-mentioned fillers or columns with commercial fillers described in Table 1.

[0473] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Eluent, characterized in that, It includes Solution 1 and Solution 2. Among them, Solution 1 is an acidic buffer solution with a pH of 3.0 - 6.6, and Solution 2 is a 0.2 - 4M monobasic strong base solution.

2. The eluent according to claim 1, characterized in that, Solution 1 also contains a 0.6 - 3.5M monovalent salt of Cl ions.

3. The eluent according to claim 2, characterized in that, In Solution 1, the monovalent salt of Cl ions is NaCl and / or KCl.

4. The eluent according to claim 1, wherein In Solution 1, the acidic buffer solution is selected from the citric acid - sodium citrate system, sodium dihydrogen phosphate - citric acid system, citric acid - sodium hydroxide - hydrochloric acid system, acetic acid - sodium acetate system, or glycine - hydrochloric acid system with a concentration of 20mM - 100mM.

5. The buffer solution according to any one of claims 1 to 5, characterized in that, Solution 1 also includes a 2 - 8M chaotropic agent, and the chaotropic agent is one or more of lithium perchlorate or other perchlorates, guanidine chloride or other guanidine salts, urea, butanol, ethanol, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea.

6. The eluent according to claim 1, wherein, In Solution 2, the monobasic strong base is NaOH and / or KOH.

7. The eluent according to claim 1, wherein Solution 1 is a 20mM - 100mM Cit - Buffer buffer solution with a pH of 3.0 - 6.6 containing a 0.6 - 3.5M monovalent salt of Cl ions and a 2 - 8M chaotropic agent; Solution 2 is a 0.5 - 4M monobasic strong base solution.

8. The eluent according to claim 7, wherein The monovalent salt of Cl ions is NaCl or KCl; The monobasic strong base is NaOH or KOH; The chaotropic agent is urea or guanidine hydrochloride.

9. The eluent according to any one of claims 1 to 8, characterized in that, The eluent is prepared by mixing Solution 1 and Solution 2, and the proportion of Solution 2 in the preparation accounts for 10% - 80%.

10. Solution combination, characterized in that, It includes a equilibration buffer, a washing solution, a sample dilution solution, and the eluent according to any one of claims 1 - 9; At least one of the equilibration buffer, the sample dilution solution, the washing solution, and the eluent contains a 2 - 8M chaotropic agent; The chaotropic agent is one or more of lithium perchlorate or other perchlorates, guanidine chloride or other guanidine salts, urea, butanol, ethanol, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea.

11. The solution combination according to claim 10, characterized in that, The equilibration buffer, the sample dilution solution, and the washing solution are selected from any one of the citric acid - sodium citrate system, sodium dihydrogen phosphate - citric acid system, citric acid - sodium hydroxide - hydrochloric acid system, acetic acid - sodium acetate system, or glycine - hydrochloric acid system with a pH of 3.0 - 6.

6.

12. Use of the eluent according to any one of claims 1 - 9 and the solution combination according to any one of claims 10 - 11 in purifying long - chain mRNA at room temperature.

13. The application according to claim 12, characterized in that, The long - chain mRNA > 300nt.

14. A method for purifying long-chain RNA at normal temperature / room temperature, characterized in that, Dilute the sample containing long - chain mRNA with the sample dilution solution, load it onto an anion exchange chromatography column after equilibration, wash it, elute it with the eluent according to any one of claims 1 - 9, and collect the elution peak; At least one of the equilibration buffer for equilibration, the sample dilution solution, the washing solution for washing, and the eluent for elution contains 2 - 8M urea.

15. The method according to claim 14, wherein The anion exchange chromatography column includes a weak anion exchange chromatography column and a strong ion exchange chromatography column.

16. According to the method of claim 10, wherein The sample diluent, equilibration buffer, and wash buffer are acidic buffers with a pH of 3.0 - 6.2, which may or may not contain 2 - 8 M urea; the acidic buffer is selected from any one or more of the citric acid - sodium citrate system, sodium dihydrogen phosphate - citric acid system, citric acid - sodium hydroxide - hydrochloric acid system, acetic acid - sodium acetate system, or glycine - hydrochloric acid system; When the eluent does not contain 2 - 8 M urea, at least one of the sample diluent, equilibration buffer, and wash buffer contains 2 - 8 M urea.

17. The method according to claim 14, wherein The elution specifically includes: before the elution, filling one pipeline of the purification device with solution 1 of the eluent according to any one of claims 1 - 9, placing the other pipeline in solution 2 of the eluent of the present invention, and setting any ratio of the volume ratio of the pipeline placed in solution 2 between 10% and 80%.

18. The method according to claim 14, wherein Before the elution, mix solution 1 and solution 2 of the eluent according to any ratio of the volume percentage of solution 2 in the eluent between 10% and 80%, and use the obtained eluent for elution purification to collect the elution peak.

19. The method according to claim 14, wherein The elution method is isocratic elution or gradient elution; specifically, for isocratic elution: when the elution speed is 1.0 - 1.5 ml / min, set the volume of solution 2 to be constant at any ratio between 10% and 80%, and elute for at least 3 CVs; specifically, for gradient elution: when the elution speed is 1.0 - 1.5 ml / min, set the volume of solution 2 to increase from 0% to 80% within 25 minutes.

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