A method for purifying mRNA
By adjusting pH and concentration through ammonium sulfate precipitation, the problems of complexity and high cost of existing mRNA purification methods are solved, achieving high-purity and high-yield mRNA purification, which is suitable for industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INSTITUTE OF PROCESS ENGINEERING CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2022-12-21
- Publication Date
- 2026-07-07
AI Technical Summary
Existing mRNA purification methods are complex, costly, and pose biological hazards, making it difficult to achieve high purity and high yield of mRNA purification.
mRNA was purified by ammonium sulfate precipitation. The purification of mRNA was achieved by adjusting the pH of the precipitation solution, the concentration of ammonium sulfate, and the precipitation time.
It enables the simple and efficient acquisition of high-purity and high-yield mRNA, suitable for industrial-scale production, and has high safety.
Smart Images
Figure CN116179536B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of ribonucleic acid isolation and purification technology, and relates to a method for purifying mRNA. Background Technology
[0002] With the effective immunization against influenza virus, Zika virus, and rabies virus by mRNA vaccines, especially after the research on SARS-CoV-2 mRNA vaccines, mRNA has become a current research focus. Large-scale production of non-replicating messenger RNA mainly uses linearized plasmid DNA as a template and NTPs as raw materials. With the assistance of magnesium ions and spermidine, the target mRNA is synthesized through in vitro enzymatic transcription by RNA polymerase. However, mRNA is highly susceptible to degradation. To improve its stability and translation efficiency, the transcribed mRNA needs to be capped at the 5' end and tailed at the 3' end.
[0003] Currently, commonly used mRNA purification methods include precipitation, chromatography, and membrane filtration. Commonly used chromatographic methods include ion exchange, OligodT affinity chromatography, cellulose chromatography, and reversed-phase ion-pair chromatography. These methods offer high separation precision, but require differences in charge, size, etc., between the mRNA and other impurities for purification to be feasible. Furthermore, different chromatographic principles need to be explored for mRNAs of different sequences and lengths, often requiring combinations of multiple chromatographic modes, resulting in long operation times. In addition, the media are expensive, especially dT affinity media, which have limited loading capacity and high ligand costs, making them more suitable for subsequent purification steps. While membrane separation methods offer fast separation speeds, the intense shearing can easily destroy the product, limiting separation precision.
[0004] Compared to chromatography and membrane separation, precipitation separation offers advantages such as ease of operation, scale-up capability, and high yield. Furthermore, mRNA products are more stable in precipitation and can be stored as intermediates, making it advantageous for large-scale production processes. Currently, commonly used precipitants for mRNA precipitation in laboratories include lithium chloride, ammonium acetate / sodium acetate, and the chloroform-isopropanol method, which can concentrate mRNA while simultaneously purifying it. Lithium chloride precipitation is the most commonly used precipitation method in laboratory settings; however, it requires operation at -20°C, resulting in high energy consumption. Additionally, while lithium chloride is a low-toxicity reagent, it can irritate and corrode the eyes and mucous membranes, and residual lithium salts in the sample can harm the central nervous system, thus limiting its application. Ammonium acetate and sodium acetate precipitation methods also require low temperatures. The chloroform-isopropanol method uses large amounts of organic reagents, which also poses a biohazard to subsequent research.
[0005] CN115287282A discloses an industrial-scale method for the separation and purification of mRNA and its application. The method includes sequentially subjecting an mRNA IVT reaction solution to affinity chromatography and hydrophobic chromatography to obtain purified mRNA. This method utilizes a two-step purification process of affinity chromatography and hydrophobic chromatography for mRNA product purification, is easily scaled up, and improves the overall recovery rate of purified mRNA. However, this method suffers from problems such as high solvent consumption and high cost.
[0006] CN114907430A discloses a method for isolating and purifying mRNA, comprising: (1) injecting the mRNA raw material to be isolated and purified into a hydrophobic chromatography column, rinsing the hydrophobic chromatography column with equilibration buffer, collecting the breakthrough fraction, and discarding or reusing the adsorbed fraction; (2) injecting the breakthrough fraction into a hydrophobic chromatography column, rinsing the hydrophobic chromatography column with equilibration buffer, and then eluting with a mobile phase, collecting the mRNA fraction based on the ultraviolet absorption signal at 260 nm. This method yields mRNA with high recovery rate and high purity, can be operated at room temperature, and is easy to scale up to industrial production. However, this method has high requirements for the properties of mRNA, limited applicability, and is complex to operate.
[0007] In summary, current mRNA purification methods suffer from problems such as complex operation, high cost, and potential biohazards. Developing a safe, room-temperature-operable, and high-yield mRNA purification method has become one of the most pressing issues to be addressed in the field of ribonucleic acid (RNA) isolation and purification technology. Summary of the Invention
[0008] To address the shortcomings of existing technologies and practical needs, this invention provides an mRNA purification method that solves the problems of complex operation, low safety, and high cost of current mRNA purification methods. It enables the simple and efficient acquisition of high-purity and high-yield mRNA, which is easy to scale up for application.
[0009] To achieve this objective, the present invention adopts the following technical solution:
[0010] In a first aspect, the present invention provides an mRNA purification method, the mRNA purification method comprising:
[0011] The mRNA to be purified was mixed with ammonium sulfate, and the precipitate was collected to obtain purified mRNA.
[0012] This invention creatively utilizes ammonium sulfate precipitation to purify mRNA. The purification process has a wide range of conditions, is stable, and is easy to scale up to industrial production. It is also highly safe and yields a high amount of mRNA.
[0013] Preferably, the mRNA is derived from any one or a combination of at least two of the mRNAs from fungi, bacteria, plants, or animals.
[0014] Preferably, the mRNA is derived from an in vitro transcription mixture.
[0015] Preferably, the in vitro transcription system comprises a mixture of post-transcriptional tailing and / or capping.
[0016] Preferably, the in vitro transcription system further includes a DNA template, T7 polymerase, NTP, and salt.
[0017] Preferably, the mRNA purification method includes the following steps:
[0018] (1) Mix the precipitant solution containing ammonium sulfate with the mRNA to be purified and collect the precipitate;
[0019] (2) The precipitate was mixed with the reconstitution solution to obtain purified mRNA.
[0020] Preferably, the pH of the ammonium sulfate precipitant solution in step (1) is 3-10.
[0021] The specific point values in 3-10 above can be 3, 4, 5, 6, 7, 8, 9, 10, etc.
[0022] Preferably, the mixing temperature in step (1) is 0-40°C.
[0023] The specific point values in the range 0-40 can be selected from 0, 10, 15, 20, 25, 30, 35, 36, 37, 38, 39, 40, etc.
[0024] Preferably, the mixing time in step (1) is 0.5-10 hours.
[0025] The specific point values in the range of 0.5-10 can be selected from 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.
[0026] Preferably, the final concentration of ammonium sulfate in the precipitant solution containing ammonium sulfate is 0.5-4M.
[0027] The specific point values in the above 0.5-4 can be selected from 0.5, 1, 2, 2.5, 3, 3.5, 3.8, 4, etc.
[0028] Preferably, step (1) further includes washing the precipitate.
[0029] Preferably, the washing solution includes ethanol.
[0030] Preferably, the ethanol includes cold ethanol.
[0031] Preferably, the ethanol concentration is 70-80%.
[0032] Preferably, the ammonium sulfate precipitant solution further contains an inorganic salt buffer solution.
[0033] Preferably, the inorganic salt buffer solution comprises any one or a combination of at least two of Tris-HCl, citric acid, acetate, phosphate, or HEPES.
[0034] Preferably, the acetate includes any one or a combination of at least two of ammonium acetate, sodium acetate, or potassium acetate.
[0035] Preferably, the phosphate includes any one or a combination of at least two of sodium dihydrogen phosphate, disodium hydrogen phosphate, or potassium dihydrogen phosphate.
[0036] Preferably, the concentration of the inorganic salt buffer solution is 0.01-1M.
[0037] The specific point values in the range 0.01-1 above can be 0.01, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.
[0038] Preferably, the reconstitution solution comprises sterile, enzyme-free water and / or a buffer solution.
[0039] Preferably, the buffer solution comprises any one or a combination of at least two of Tris-HCl, citric acid, acetate, phosphate, or HEPES.
[0040] Preferably, the acetate includes any one or a combination of at least two of sodium acetate, potassium acetate, or ammonium acetate.
[0041] Preferably, the phosphate includes any one or a combination of at least two of sodium dihydrogen phosphate, disodium hydrogen phosphate, or potassium dihydrogen phosphate.
[0042] Compared with the prior art, the present invention has the following beneficial effects:
[0043] This invention creatively adjusts the pH, ammonium sulfate concentration, precipitation time, and precipitation temperature of the precipitation solution to purify mRNA using the ammonium sulfate precipitation method. The operation is simple, the purification conditions are wide-ranging, the purification process is stable, and it is easy to scale up to industrial production. At the same time, it is highly safe and yields a high amount of mRNA. Attached Figure Description
[0044] Figure 1 HPLC chromatograms of mRNA purified from in vitro transcription products by lithium chloride precipitation (Comparative Example 1) and ammonium sulfate precipitation (Example 1);
[0045] Figure 2Agarose gel images of mRNA purified from in vitro transcription products by lithium chloride precipitation (Comparative Example 1) and ammonium sulfate precipitation (Example 1). Detailed Implementation
[0046] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.
[0047] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0048] Preparation Example 1
[0049] This preparation example provides an mRNA transcription system (reaction volume 100 μL), the preparation method of which is as follows:
[0050] (1) Prepare 10× Transcription Buffer: 400mM Tris-HCl pH 8.0, 60mM KCl, 10mM MgCl2, 20mM MTT (dithiothreitol);
[0051] (2) Place the required T7 RNA polymerase, RNase inhibitor, and pyrophosphatase on ice. Mix ATP, CTP, GTP, and UTP and place on ice. Keep sterile enzyme-free water, DNA template, and 10× transcription buffer at room temperature for later use.
[0052] (3) Add 10 μL of 10×Transcription Buffer, 41.5 μL of RNase Free Water, 8 μL of NTP (nucleoside triphosphate), 30 μL of DNA template, 5 μL of T7, 4 μL of RNase inhibitor and 1.5 μL of pyrophosphatase to a sterile, enzyme-free centrifuge tube, gently mix the components and incubate at 37°C for 3 h.
[0053] (4) Add 5 μL of DNase I to each centrifuge tube and incubate at 37°C for 15 min to digest the transcribed DNA template.
[0054] Preparation Example 2
[0055] This preparation example provides an mRNA capping system, the preparation method of which is as follows:
[0056] (1) The formulation of 10×CappingBuffer is as follows: 500mM Tris-HCl (pH 8.0 at 25℃), 60mM MgCl2, 100mM MDTT, 100mM MKCl, 20mM Spermidine.
[0057] (2) Capping reaction procedure (reaction system 100 μL)
[0058] Take 50 μg of mRNA sample and denature it by heating at 65 °C for 10 min. After denaturation, place it on ice for 5 min. Then, add 10 μL of 10× Capping Buffer, 10 μL of GTP, 2.5 μL of SAM, 2.5 μL of RNA inhibitor, 4 μL of mRNA Cap2′-O-methyltransferase, and 4 μL of vaccinia virus capping enzyme to the sample in sequence. Add sterile enzyme-free water to make up to 100 μL. Mix each group thoroughly and incubate at 37 °C for 30 min.
[0059] Example 1
[0060] This embodiment provides a method for precipitating and purifying mRNA, the specific steps of which are as follows:
[0061] Take 50 μL of green fluorescent protein mRNA transcription mixture, add 36 μL of buffer B1 (20 mM Tris-HCl, pH 7.0), add 114 μL of buffer B2 (20 mM Tris-HCl, 3.5 M ammonium sulfate, pH 7.0) (final ammonium sulfate concentration is 2 M), mix the sample thoroughly, place the mixture in a 25℃ water bath and incubate for 1 h, then centrifuge the mixture at 20℃ and 12000 rpm for 10 min to separate the supernatant and precipitate, add 200 μL of sterile enzyme-free water to dissolve the precipitate;
[0062] Precipitation rate calculation method: (1 - (mRNA content in supernatant / mRNA content in transcription mixture)) * 100%;
[0063] Precipitation yield calculation method: mRNA content of ammonium sulfate precipitate resolvate / mRNA content in transcription mixture * 100%.
[0064] Example 2
[0065] This embodiment provides a method for precipitating and purifying mRNA, the specific steps of which are as follows:
[0066] Take 50 μL of the green fluorescent protein mRNA transcription mixture, add 36 μL of buffer B1 (20 mM Tris-HCl, pH 5.0), and add 114 μL of buffer B2 (20 mM Tris-HCl, 3.5 M ammonium sulfate, pH 5.0) (final ammonium sulfate concentration is 2 M). Mix the sample thoroughly and incubate in a 20°C water bath for 1 h. Then, centrifuge the mixture at 12000 rpm for 10 min at 20°C to separate the supernatant and precipitate. Dissolve the precipitate in 200 μL of sterile, enzyme-free water.
[0067] Example 3
[0068] This embodiment provides a method for precipitating and purifying mRNA, the specific steps of which are as follows:
[0069] Take 50 μL of green fluorescent protein mRNA transcription mixture, add 64 μL of buffer B1 (20 mM Tris-HCl, pH 7.0) and 86 μL of buffer B2 (20 mM Tris-HCl, 3.5 M ammonium sulfate, pH 7.0) (final ammonium sulfate concentration is 1.5 M), mix the sample thoroughly, place the mixture in a 20℃ water bath and incubate for 1 h, then centrifuge the mixture at 12000 rpm for 10 min at 20℃ to separate the supernatant and precipitate, and dissolve the precipitate in 200 μL of sterile enzyme-free water.
[0070] Example 4
[0071] This embodiment provides a method for precipitating and purifying mRNA, the specific steps of which are as follows:
[0072] Take 50 μL of green fluorescent protein mRNA transcription mixture, add 36 μL of buffer B1 (20 mM Tris-HCl, pH 7.0), and add 114 μL of buffer B2 (20 mM Tris-HCl, 3.5 M ammonium sulfate, pH 7.0) (final ammonium sulfate concentration is 2 M). Mix the sample thoroughly and place the mixture in a 20℃ water bath for 1 h. Then, centrifuge the mixture at 25℃ and 12000 rpm for 10 min to separate the supernatant and precipitate. Dissolve the precipitate in 200 μL of sterile enzyme-free water.
[0073] Example 5
[0074] This embodiment provides a method for precipitating and purifying mRNA, the specific steps of which are as follows:
[0075] Take 50 μL of green fluorescent protein mRNA transcription mixture, add 36 μL of buffer B1 (20 mM Tris-HCl, 3.5 M ammonium sulfate, pH 7.0), add 114 μL of buffer B2 (20 mM Tris-HCl, 3.5 M ammonium sulfate, pH 7.0) (final ammonium sulfate concentration is 2 M), mix the sample thoroughly, place the mixture in a 20℃ water bath and incubate for 2 h, then centrifuge the mixture at 25℃ and 12000 rpm for 10 min to separate the supernatant and precipitate, add 200 μL of sterile enzyme-free water to dissolve the precipitate.
[0076] Example 6
[0077] This embodiment provides a method for precipitating and purifying mRNA, the specific steps of which are as follows:
[0078] Take 50 μL of the capped green fluorescent protein mRNA transcription mixture, add 36 μL of buffer B1 (20 mM Tris-HCl, pH 7.0) and 114 μL of buffer B2 (20 mM Tris-HCl, 3.5 M ammonium sulfate, pH 7.0) (final ammonium sulfate concentration is 2 M), mix the sample thoroughly, and incubate the mixture in a 20°C water bath for 2 h. Then, centrifuge the mixture at 12000 rpm for 10 min at 20°C to separate the supernatant and precipitate. Dissolve the precipitate in 200 μL of sterile enzyme-free water.
[0079] Example 7
[0080] This embodiment provides a method for precipitating and purifying mRNA, the specific steps of which are as follows:
[0081] Take 50 μL of the green fluorescent protein mRNA transcription mixture treated with LDNaseI, add 36 μL of buffer B1 (20 mM Tris-HCl, pH 7.0) and 114 μL of buffer B2 (20 mM Tris-HCl, 3.5 M ammonium sulfate, pH 7.0) (final ammonium sulfate concentration is 2 M), mix the sample thoroughly, and incubate the mixture in a 20℃ water bath for 2 h. Then, centrifuge the mixture at 12000 rpm for 10 min at 20℃ to separate the supernatant and precipitate. Dissolve the precipitate in 200 μL of sterile enzyme-free water.
[0082] Comparative Example 1
[0083] This comparative example uses lithium chloride precipitation to purify mRNA in the transcription system. The difference between this example and Example 1 is that the precipitant is different, and the precipitation buffer is 7.5M LiCl buffer.
[0084] Take 50 μL of green fluorescent protein mRNA transcription mixture with an mRNA concentration of 1000 μg / μL, add 83.3 μL of sterile enzyme-free water and 66.7 μL of 7.5 M LiCl buffer to it, with a final LiCl concentration of 2.5 M. Incubate the mixture at -20℃ for 1 h. Then, centrifuge the mixture at 12000 rpm for 10 min at 4℃ to separate the supernatant and precipitate. Wash the precipitate three times with 70% ethanol, and centrifuge at 12000 rpm for 5 min at 4℃ to separate the supernatant and precipitate. Dissolve the precipitate in 200 μL of sterile enzyme-free water.
[0085] Test Example 1
[0086] T7 enzyme residue was determined by SDS-PAGE, DNA template residue was quantitatively detected by HPLC, and mRNA content and purity in the samples were determined by agarose gel electrophoresis and high performance liquid chromatography. The yield of mRNA and impurity removal rate obtained by the methods of each example and comparative example were calculated, and the results are listed in Table 1.
[0087] DNA removal rate: (DNA in 1-mRNA sample / total DNA) * 100%
[0088] T7 enzyme removal rate: (T7 enzyme in 1-mRNA sample / total T7 enzyme) * 100%
[0089] Table 1
[0090]
[0091]
[0092] The results show that the ammonium sulfate precipitation method of this invention can achieve a high mRNA precipitation rate and yield, and the removal of impurities such as DNA, T7 polymerase, and NTP is very effective. In addition, the comparative examples show that the ammonium sulfate precipitation method of this invention can significantly improve the mRNA yield compared with the traditional lithium chloride precipitation method. This indicates that the ammonium sulfate precipitation method of this invention has a wide range of purification conditions and significantly improves the mRNA yield.
[0093] Test Example 2
[0094] HPLC tests were performed on Example 1 and Comparative Example 1. The LiCl precipitate and (NH4)2SO4 precipitate were both diluted 4-fold. The test method was as follows: HPLC analysis of each sample was performed using a SEC-1000 (Sepax, 300×7.8mm) analytical column through a WatersArc HPLC series system. UV detection wavelengths were 260 nm and 280 nm. In each operation, 100 μL of sample was injected and eluted with a buffer containing 50 mM PB + 100 mM Na2SO4 (pH 7.0) at a flow rate of 0.6 mL / min for 30 min. The results are as follows. Figure 1 As shown.
[0095] The results showed that ammonium sulfate precipitation effectively removed impurities such as DNA template, NTPs, and transcriptase, resulting in high-purity mRNA with a higher yield than that of lithium chloride precipitation.
[0096] Test Example 3
[0097] Agarose gel electrophoresis was performed on Example 1 and Comparative Example 1. The test method was as follows: a 1.5% agarose gel was prepared. 5 μL of the transcribed sample was mixed with 5 μL of sterile, enzyme-free water and 2 μL of 6× RNA loading buffer. 10 μL of the mixture was then loaded for agarose gel electrophoresis. Wherein, 1 represents lithium chloride precipitation and 2 represents ammonium sulfate precipitation. The results are as follows. Figure 2 As shown.
[0098] The results showed that the purity of the mRNA obtained after ammonium sulfate precipitation was not significantly different from that of the LiCl precipitated sample, which fully demonstrates that ammonium sulfate precipitation can obtain high-purity mRNA.
[0099] In summary, this invention creatively adjusts the pH, ammonium sulfate concentration, precipitation time, and precipitation temperature of the precipitation solution to purify mRNA using the ammonium sulfate precipitation method. The purification conditions are wide-ranging, the purification process is stable, and it is easy to scale up to industrial production. At the same time, it is highly safe and yields a high amount of mRNA.
[0100] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A method for purifying mRNA, characterized in that, The mRNA purification method includes the following steps: (1) Mix the mRNA to be purified with a precipitant solution composed of ammonium sulfate and inorganic salt buffer, and collect the precipitate; wherein the mixing temperature is 20-25℃, the mixing time is 0.5-10 h, the pH of the precipitant solution composed of ammonium sulfate and inorganic salt buffer is 5-7, and the final concentration of ammonium sulfate in the precipitant solution composed of ammonium sulfate and inorganic salt buffer is 1.5-2M; (2) The precipitate is mixed with the reconstitution solution to obtain purified mRNA.
2. The mRNA purification method according to claim 1, characterized in that, The mRNA is derived from any one or a combination of at least two of the mRNAs from fungi, bacteria, plants, or animals.
3. The mRNA purification method according to claim 1, characterized in that, The mRNA was derived from an in vitro transcription mixture.
4. The mRNA purification method according to claim 3, characterized in that, The in vitro transcription system comprises a mixture of post-transcriptional tailing and / or capping.
5. The mRNA purification method according to claim 4, characterized in that, The in vitro transcription system also includes a DNA template, T7 polymerase, NTPs, and salt.
6. The mRNA purification method according to claim 1, characterized in that, Step (1) also includes the step of washing the precipitate.
7. The mRNA purification method according to claim 6, characterized in that, The washing solution includes ethanol.
8. The mRNA purification method according to claim 7, characterized in that, The ethanol includes cold ethanol.
9. The mRNA purification method according to claim 7, characterized in that, The ethanol concentration is 70-80%.
10. The mRNA purification method according to claim 1, characterized in that, The inorganic salt buffer solution includes any one or a combination of at least two of Tris-HCl, citric acid, acetate, phosphate, or HEPES.
11. The mRNA purification method according to claim 10, characterized in that, The acetate includes any one or a combination of at least two of sodium acetate, ammonium acetate, or potassium acetate.
12. The mRNA purification method according to claim 10, characterized in that, The phosphate includes any one or a combination of at least two of sodium dihydrogen phosphate, disodium hydrogen phosphate, or potassium dihydrogen phosphate.
13. The mRNA purification method according to claim 10, characterized in that, The concentration of the inorganic salt buffer solution is 0.01-1 M.
14. The mRNA purification method according to claim 1, characterized in that, The reconstitution solution includes sterile, enzyme-free water and / or a buffer solution.
15. The mRNA purification method according to claim 14, characterized in that, The buffer solution comprises any one or a combination of at least two of Tris-HCl, citric acid, acetate, phosphate, or HEPES.
16. The mRNA purification method according to claim 15, characterized in that, The acetate includes any one or a combination of at least two of sodium acetate, ammonium acetate, or potassium acetate.
17. The mRNA purification method according to claim 15, characterized in that, The phosphate includes any one or a combination of at least two of sodium dihydrogen phosphate, disodium hydrogen phosphate, or potassium dihydrogen phosphate.