An mRNA industrial separation and purification method and its application

Abstract

The present invention provides an industrial mRNA separation and purification method and its application. The method comprises the following steps: sequentially subjecting an mRNA IVT reaction solution to affinity chromatography and hydrophobic chromatography to obtain purified mRNA. The present invention utilizes a two-step purification process involving affinity chromatography and hydrophobic chromatography for the purification of mRNA products. This process is not only simple to operate and easily scalable, but also reduces the number of steps required, while also improving the overall recovery rate of purified mRNA and meeting pharmaceutical standards.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to an mRNA industrial separation and purification method and application thereof. Background Art

[0002] mRNA, with its inherent advantages, can theoretically express any protein, potentially enabling exploration into the treatment of virtually any protein-based disease. Currently, mRNA technology is demonstrating its unique advantages in the development of COVID-19 vaccines, particularly its robust protein expression capabilities and short development cycles. Furthermore, mRNA technology is now being progressively applied to protein replacement therapy, cell therapy, and other fields, promising a wide range of future industrial applications.

[0003] An ecosystem for mRNA-related drug development has essentially taken shape in China. As a relatively new technology, mRNA production technology is developing rapidly, and solutions for mRNA chromatography are constantly being optimized. In particular, when mRNA is used in therapeutic applications, purity control is crucial for efficacy. Commonly used mRNA purification methods in the industry include reversed-phase chromatography, ion exchange chromatography, and molecular sieve chromatography. Reverse-phase chromatography, the most widely used method, often requires the addition of ion pairs, has low binding capacity, and requires the use of flammable and toxic solvents. However, the widespread use of ion exchange chromatography and molecular sieve chromatography is limited by poor selectivity and low binding capacity.

[0004] Therefore, how to provide an industrial separation and purification method for mRNA with excellent purification effect, high loading capacity, strong repeatability and simple operation has become an urgent problem to be solved. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide an industrial mRNA separation and purification method and its application. The industrial mRNA separation and purification method provided by the present invention has excellent purification effect, high loading capacity, strong repeatability and simple operation.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an industrial separation and purification method for mRNA, comprising the following steps: subjecting an mRNA IVT reaction solution to affinity chromatography and hydrophobic chromatography in sequence to obtain purified mRNA.

[0008] The present invention utilizes a two-step purification process involving affinity chromatography and hydrophobic chromatography for the purification of mRNA products. This process is not only simple and easy to scale up, but also reduces the number of steps required. It also improves the overall recovery rate of purified mRNA and ensures compliance with pharmaceutical standards. The purification method provided by the present invention offers excellent purification efficacy, strong reproducibility, and simple operation, meeting the requirements of industrial purification production and significantly reducing production costs.

[0009] In the present invention, the affinity chromatography filler used in the affinity chromatography includes NanoGel dT20.

[0010] This method utilizes NanoGel dT20 affinity chromatography filler, which boasts high loading capacity, tolerance to high salt, high pH, ​​and high temperatures, and ease of use, eliminating the need for flammable or toxic reagents. This affinity chromatography removes process-related impurities, such as linearized DNA template and RNA polymerase, as well as product-related impurities, such as polyAd-free mRNA.

[0011] Preferably, the hydrophobic chromatography filler used in the hydrophobic chromatography includes UniHR Butyl 30L.

[0012] The present invention adopts UniHR Butyl 30L hydrophobic chromatography filler to remove dsRNA and uncapped RNA and remove RNA secondary structure impurities.

[0013] In the present invention, the mRNA industrial separation and purification method specifically comprises the following steps:

[0014] (1) Loading the mRNA IVT reaction solution onto the equilibrated affinity chromatography column, then washing with a washing buffer, eluting with an elution buffer, and collecting the first eluate;

[0015] (2) The first eluate obtained in step (1) is loaded onto the equilibrated hydrophobic chromatography column, and then washed with a washing buffer, eluted with an elution buffer, and the second eluate is collected.

[0016] In the present invention, in step (1), the affinity chromatography column is equilibrated with 4-6 column volumes (eg, 4 column volumes, 5 column volumes, 6 column volumes, etc.) of equilibration buffer.

[0017] Preferably, the equilibration buffer comprises 8-12 mM (for example, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, etc.) Tris-HCl, 0.3-0.7 M (for example, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, etc.) NaCl and 0.8-1.2 mM (for example, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, etc.) EDTA on a molar basis, with a pH of 7.2-7.7 (for example, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, etc.), and the solvent is water.

[0018] In the present invention, in step (1), the washing buffer comprises, by molar concentration, 8-12 mM (for example, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, etc.) Tris-HCl, 0.3-0.7 M (for example, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, etc.) NaCl and 0.8-1.2 mM (for example, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, etc.) EDTA, with a pH of 7.2-7.7 (for example, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, etc.), and the solvent is water.

[0019] Preferably, in step (1), 4-6 column volumes (eg, 4 column volumes, 5 column volumes, 6 column volumes, etc.) of washing buffer are used for washing.

[0020] Preferably, in step (1), the elution buffer comprises deionized water.

[0021] Preferably, in step (1), elution is performed using 4-6 column volumes (eg, 4 column volumes, 5 column volumes, 6 column volumes, etc.) of elution buffer.

[0022] In the present invention, in step (2), the hydrophobic chromatography column is equilibrated with 4-6 column volumes (for example, 4 column volumes, 5 column volumes, 6 column volumes, etc.) of equilibration buffer.

[0023] Preferably, the equilibration buffer comprises 1.2-1.8 M (for example, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, etc.) ammonium sulfate and 45-55 mM (for example, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, etc.) Tris-HCl on a molar basis, with a pH of 7-7.4 (for example, 7, 7.1, 7.2, 7.3, 7.4, etc.), and the solvent is water.

[0024] In the present invention, in step (2), the washing buffer comprises, by molar concentration, 1.2-1.8 M (for example, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, etc.) ammonium sulfate and 45-55 mM (for example, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, etc.) Tris-HCl, a pH of 7-7.4 (for example, 7, 7.1, 7.2, 7.3, 7.4, etc.), and the solvent is water.

[0025] Preferably, in step (2), 4-6 column volumes (eg, 4 column volumes, 5 column volumes, 6 column volumes, etc.) of washing buffer are used for washing.

[0026] In the present invention, in step (2), the elution buffer comprises a first elution buffer and a second elution buffer.

[0027] Preferably, the first elution buffer comprises 1.2-1.8 M (for example, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, etc.) ammonium sulfate and 45-55 mM (for example, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, etc.) Tris-HCl on a molar basis, with a pH of 7-7.4 (for example, 7, 7.1, 7.2, 7.3, 7.4, etc.), and the solvent is water.

[0028] Preferably, the second elution buffer comprises 48-52 mM (for example, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, etc.) Tris-HCl in terms of molar concentration, a pH of 7-7.4 (for example, 7, 7.1, 7.2, 7.3, 7.4, etc.), and the solvent is water.

[0029] Preferably, in step (2), the elution includes a first elution and a second elution performed sequentially.

[0030] Preferably, the first elution is a linear gradient elution, and the linear gradient elution uses a first elution buffer and / or a second elution buffer for elution. During the linear gradient elution process, the volume fraction ratio of the second elution buffer to the first elution buffer is 0%:100%→100%:0%.

[0031] Preferably, the first elution is performed using 12-17 column volumes (for example, 12 column volumes, 13 column volumes, 14 column volumes, 15 column volumes, 16 column volumes, 17 column volumes, etc.) of elution buffer.

[0032] Preferably, the second elution is performed using a second elution buffer.

[0033] Preferably, the second elution is performed using 3-7 column volumes (eg, 3 column volumes, 4 column volumes, 5 column volumes, 6 column volumes, 7 column volumes, etc.) of elution buffer.

[0034] As a preferred technical solution of the present invention, the mRNA industrial separation and purification method comprises the following steps:

[0035] (1) Loading the mRNA IVT reaction solution onto the equilibrated affinity chromatography column, then washing with a washing buffer, eluting with an elution buffer, and collecting the first eluate;

[0036] The affinity chromatography column is balanced with 4-6 column volumes of an equilibration buffer; the equilibration buffer comprises 8-12 mM Tris-HCl, 0.3-0.7 M NaCl and 0.8-1.2 mM EDTA on a molar basis, with a pH of 7.2-7.7, and the solvent is water; the wash buffer comprises 8-12 mM Tris-HCl, 0.3-0.7 M NaCl and 0.8-1.2 mM EDTA on a molar basis, with a pH of 7.2-7.7, and the solvent is water; the elution buffer comprises deionized water;

[0037] (2) loading the first eluate obtained in step (1) onto the equilibrated hydrophobic chromatography column, then washing with a washing buffer, performing a first elution with the first elution buffer and / or the second elution buffer, performing a second elution with the second elution buffer, and collecting the second eluate;

[0038] The hydrophobic chromatography column is balanced with 4-6 column volumes of an equilibration buffer; the equilibration buffer comprises 1.2-1.8 M ammonium sulfate and 45-55 mM Tris-HCl by molar concentration, the pH is 7-7.4, and the solvent is water; the wash buffer comprises 1.2-1.8 M ammonium sulfate and 45-55 mM Tris-HCl by molar concentration, the pH is 7-7.4, and the solvent is water;

[0039] The first elution buffer comprises 1.2-1.8M ammonium sulfate and 45-55mM Tris-HCl by molar concentration, pH 7-7.4, and the solvent is water; the second elution buffer comprises 48-52mM Tris-HCl by molar concentration, pH 7-7.4, and the solvent is water;

[0040] The first elution is linear gradient elution, and the volume fraction ratio of the second elution buffer to the first elution buffer during the linear gradient elution is 0%:100%→100%:0%.

[0041] In a second aspect, the present invention provides an application of the mRNA industrial separation and purification method according to the first aspect in the preparation of mRNA drugs.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention uses a two-step purification process of affinity chromatography and hydrophobic chromatography for the purification of mRNA products. This process is not only simple and easy to scale up, but also reduces the number of steps required. It also improves the total recovery rate of purified mRNA and meets pharmaceutical standards. The initial purity of the mRNA IVT reaction solution is 69%. After the first step of affinity chromatography purification, the recovery rate reaches 87.1-88.3%, and the purity reaches 89.9-93%. The second step of hydrophobic purification of UniHR Phenyl-30L reaches a recovery rate of 89.5-92%, and the purity reaches 94.3-96.5%. This shows that the purification method provided by the present invention has good purification effect, strong repeatability, simple operation, can meet the requirements of industrial purification production, and greatly reduces production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the chromatogram of the affinity chromatography filler NanoGel dT20 used in Example 1.

[0045] Figure 2 This is the SEC spectrum of the first eluate in Example 1.

[0046] Figure 3 This is the chromatogram of the hydrophobic chromatography filler UniHR Butyl 30L used in Example 1.

[0047] Figure 4 This is the SEC spectrum of the second eluate in Example 1. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0049] In the following examples, the affinity chromatography filler is NanoGel dT20, manufactured by Nanovitamin CMS, with a product number of 17030-050150-1001; the hydrophobic chromatography filler is UniHR Butyl 30L, manufactured by Nanovitamin CMS, with a product number of 06132-030100.

[0050] Example 1

[0051] This embodiment provides an mRNA industrial separation and purification method, which includes the following steps:

[0052] (1) Load the mRNA IVT reaction solution onto an equilibrated affinity chromatography column (filled with NanoGel dT20), then wash with 5 column volumes of wash buffer and elute with 5 column volumes of elution buffer, collecting the first eluate;

[0053] The affinity chromatography column is balanced with 5 column volumes of an equilibrium buffer; the equilibrium buffer comprises, by molar concentration, 10 mM Tris-HCl, 0.5 M NaCl, and 1 mM EDTA, with a pH of 7.4, and the solvent is water;

[0054] The washing buffer comprises 10 mM Tris-HCl, 0.5 M NaCl and 1 mM EDTA in terms of molar concentration, with a pH of 7.4 and the solvent being water;

[0055] The elution buffer comprises deionized water;

[0056] (2) loading the first eluate obtained in step (1) onto a equilibrated hydrophobic chromatography column (filled with UniHRButyl 30L), then washing with 5 column volumes of washing buffer, performing a first elution with 15 column volumes of the first elution buffer and / or the second elution buffer, performing a second elution with 5 column volumes of the second elution buffer, and collecting the second eluate;

[0057] The hydrophobic chromatography column is balanced with 5 column volumes of equilibration buffer; the equilibration buffer comprises 1.5 M ammonium sulfate and 50 mM Tris-HCl on a molar basis, with a pH of 7.2, and the solvent is water; the wash buffer comprises 1.5 M ammonium sulfate and 50 mM Tris-HCl on a molar basis, with a pH of 7.2, and the solvent is water;

[0058] The first elution buffer comprises 1.5M ammonium sulfate and 50mM Tris-HCl at a molar concentration of 7.2, and the solvent is water; the second elution buffer comprises 50mM Tris-HCl at a molar concentration of 7.2, and the solvent is water;

[0059] The first elution is linear gradient elution, and the volume fraction ratio of the second elution buffer to the first elution buffer during the linear gradient elution is 0%:100%→100%:0%.

[0060] The purity of the mRNA IVT reaction solution was calculated to be 69% by SEC analysis.

[0061] Figure 1 The chromatogram of the affinity chromatography filler NanoGel dT20 used in Example 1 shows that NanoGel dT20 can effectively separate mRNA; Figure 2 This is the SEC spectrum of the first eluate in Example 1. It can be calculated that the purity of the mRNA in the first eluate is 93%.

[0062] Figure 3 This is the chromatogram of the hydrophobic chromatography filler UniHR Butyl 30L in Example 1. It can be seen from the figure that as the conductivity decreases, the mRNA is gradually eluted; Figure 4 This is the SEC spectrum of the second eluate in Example 1. It can be calculated that the purity of the mRNA IVT reaction solution is 96.5%.

[0063] Example 2

[0064] This embodiment provides an mRNA industrial separation and purification method, which includes the following steps:

[0065] (1) Load the mRNA IVT reaction solution onto an equilibrated affinity chromatography column (filled with NanoGel dT20), then wash with 4 column volumes of wash buffer and elute with 4 column volumes of elution buffer, collecting the first eluate;

[0066] The affinity chromatography column is balanced with 4 column volumes of an equilibrium buffer; the equilibrium buffer comprises 8 mM Tris-HCl, 0.7 M NaCl, and 0.8 mM EDTA in terms of molar concentration, with a pH of 7.2, and the solvent is water;

[0067] The washing buffer comprises 8 mM Tris-HCl, 0.7 M NaCl and 0.8 mM EDTA in terms of molar concentration, with a pH of 7.2 and a solvent of water;

[0068] The elution buffer comprises deionized water;

[0069] (2) loading the first eluate obtained in step (1) onto a equilibrated hydrophobic chromatography column (filled with UniHRButyl 30L), then washing with 4 column volumes of washing buffer, performing a first elution with 12 column volumes of the first elution buffer and / or the second elution buffer, performing a second elution with 7 column volumes of the second elution buffer, and collecting the second eluate;

[0070] The hydrophobic chromatography column is balanced with 4 column volumes of equilibration buffer; the equilibration buffer comprises 1.2 M ammonium sulfate and 55 mM Tris-HCl on a molar basis, with a pH of 7.1, and the solvent is water; the wash buffer comprises 1.2 M ammonium sulfate and 55 mM Tris-HCl on a molar basis, with a pH of 7.1, and the solvent is water;

[0071] The first elution buffer comprises 1.2M ammonium sulfate and 55mM Tris-HCl by molar concentration, pH 7.1, and the solvent is water; the second elution buffer comprises 48mM Tris-HCl by molar concentration, pH 7.1, and the solvent is water;

[0072] The first elution is linear gradient elution, and the volume fraction ratio of the second elution buffer to the first elution buffer during the linear gradient elution is 0%:100%→100%:0%.

[0073] Example 3

[0074] This embodiment provides an mRNA industrial separation and purification method, which includes the following steps:

[0075] (1) Load the mRNA IVT reaction solution onto an equilibrated affinity chromatography column (filled with NanoGel dT20), then wash with 6 column volumes of wash buffer and elute with 6 column volumes of elution buffer, collecting the first eluate;

[0076] The affinity chromatography column is balanced with 6 column volumes of an equilibrium buffer; the equilibrium buffer comprises, by molar concentration, 12 mM Tris-HCl, 0.3 M NaCl, and 1.2 mM EDTA, with a pH of 7.6, and the solvent is water;

[0077] The washing buffer comprises 12 mM Tris-HCl, 0.3 M NaCl and 1.2 mM EDTA in terms of molar concentration, with a pH of 7.6 and the solvent being water;

[0078] The elution buffer comprises deionized water;

[0079] (2) loading the first eluate obtained in step (1) onto a equilibrated hydrophobic chromatography column (filled with UniHRButyl 30L), then washing with 6 column volumes of washing buffer, performing a first elution with 17 column volumes of the first elution buffer and / or the second elution buffer, performing a second elution with 3 column volumes of the second elution buffer, and collecting the second eluate;

[0080] The hydrophobic chromatography column is balanced with 6 column volumes of equilibration buffer; the equilibration buffer comprises 1.8 M ammonium sulfate and 45 mM Tris-HCl on a molar basis, with a pH of 7.3, and the solvent is water; the wash buffer comprises 1.8 M ammonium sulfate and 45 mM Tris-HCl on a molar basis, with a pH of 7.3, and the solvent is water;

[0081] The first elution buffer comprises 1.8 M ammonium sulfate and 45 mM Tris-HCl at a molar concentration of 7.3, and the solvent is water; the second elution buffer comprises 55 mM Tris-HCl at a molar concentration of 7.3, and the solvent is water;

[0082] The first elution is linear gradient elution, and the volume fraction ratio of the second elution buffer to the first elution buffer during the linear gradient elution is 0%:100%→100%:0%.

[0083] Example 4

[0084] This embodiment provides an industrial separation and purification method for mRNA. The only difference from Example 1 is that in step (1), the filler used in the affinity chromatography column is replaced with Monomix dT20 (manufacturer: Saifen Technology, product number 283030950). The other steps are the same as in Example 1.

[0085] Example 5

[0086] This embodiment provides an industrial separation and purification method for mRNA. The only difference from Example 1 is that in step (2), the filler used in the hydrophobic chromatography column is replaced with Capto Butyl (manufacturer GE, product number 17545901). The other steps are the same as in Example 1.

[0087] Example 6

[0088] This embodiment provides an industrial separation and purification method for mRNA, which differs from Example 1 only in that, in step (2), the first elution is a linear gradient elution, and the volume fraction ratio of the second elution buffer to the first elution buffer during the linear gradient elution process is 30%:70%→100%:0%. The other steps are the same as in Example 1.

[0089] Example 7

[0090] This embodiment provides an industrial separation and purification method for mRNA, which differs from Example 1 only in that, in step (2), the first elution is a linear gradient elution, and the volume fraction ratio of the second elution buffer to the first elution buffer during the linear gradient elution process is 70%:30%→100%:0%. The other steps are the same as in Example 1.

[0091] Comparative Example 1

[0092] This comparative example provides an industrial separation and purification method for mRNA, which differs from Example 1 only in that hydrophobic chromatography is performed first and then affinity chromatography, and the other steps are the same as Example 1.

[0093] Comparative Example 2

[0094] This comparative example provides an industrial separation and purification method for mRNA, which differs from Example 1 only in that the affinity chromatography in step (1) is replaced by ion chromatography, and the ion chromatography includes the following steps:

[0095] Load the mRNA IVT reaction solution onto an equilibrated ion chromatography column (UniGel-30DEAE, manufactured by Nanotech, catalog number 04083-030100), then wash with 5 column volumes of wash buffer and elute with 5 column volumes of elution buffer, collecting the elution buffer.

[0096] The ion chromatography column is balanced with 5 column volumes of a balancing buffer solution; the balancing buffer solution comprises 50 mM Tris-HCl and 10 mM EDTA in terms of molar concentration, with a pH of 7.2, and the solvent is water;

[0097] The washing buffer comprises 50 mM Tris-HCl and 10 mM EDTA in terms of molar concentration, with a pH of 7.2, and the solvent is water;

[0098] The elution buffer comprises, by molar concentration, 50 mM Tris-HCl, 0.5 M NaCl, and 10 mM EDTA, with a pH of 7.2;

[0099] Other steps are the same as in Example 1.

[0100] Comparative Example 3

[0101] This comparative example provides an industrial separation and purification method for mRNA, which differs from Example 1 only in that the hydrophobic chromatography in step (2) is replaced by gel filtration chromatography, and the gel filtration chromatography comprises the following steps:

[0102] The elution buffer obtained in step (1) was loaded onto the equilibrated gel filtration chromatography column (filler: NW Rose 6FF, manufacturer: Nanotech, catalog number: 60012-014900), and then eluted with 5 column volumes of equilibration buffer, and the elution buffer was collected;

[0103] The NW Rose 6FF chromatography column was equilibrated with 5 column volumes of an equilibration buffer solution comprising, by molar concentration, 2.1 M (NH4)2SO4, 10 mM EDTA, and 100 mM Tris-HCl, with a pH of 8.0, and the solvent was water.

[0104] The elution buffer comprises, by molar concentration, 2.1 M (NH 4 ) 2 SO 4 , 10 mM EDTA and 100 mM Tris-HCl, with a pH of 8.0;

[0105] Other steps are the same as in Example 1.

[0106] Comparative Example 4

[0107] This comparative example provides an industrial separation and purification method for mRNA, which differs from Example 1 only in that two affinity chromatography steps are used consecutively for purification, and the steps of the first affinity chromatography and the second affinity chromatography are the same as step (1) in Example 1.

[0108] Comparative Example 5

[0109] This comparative example provides an industrial separation and purification method for mRNA, which differs from Example 1 only in that two hydrophobic chromatography steps are used continuously for purification, and the steps of the first hydrophobic chromatography and the second hydrophobic chromatography are the same as step (2) in Example 1.

[0110] Test Case

[0111] Recovery and purity testing

[0112] The initial purity of the mRNA IVT reaction was 69%.

[0113] Test samples: the first eluate and the second eluate provided in Examples 1-9 and Comparative Examples 1-5.

[0114] Test method: SEC gel permeation chromatography was used to analyze the purity of mRNA in the eluate. The formula for calculating the mRNA recovery rate is as follows:

[0115] mRNA recovery rate (%) = [(C 上样液 V 上样液 ) / (C 洗脱液 V 洗脱液 )】×100%.

[0116] The test results are shown in Table 1 below:

[0117] Table 1

[0118]

[0119]

[0120] As shown in Table 1, as the preferred technical solution of the present invention (Examples 1-3), the first step of affinity chromatography NanoGel dT20 purification achieved a recovery rate of 87.1-88.3% and a purity of 89.9-93%. The second step of hydrophobic purification using UniHR Phenyl-30L achieved a recovery rate of 89.5-92% and a purity of 94.3-96.5%. This demonstrates that the two-step mRNA purification process can efficiently produce high-quality mRNA samples.

[0121] A comparison between Example 1 and Examples 4-5 shows that changing the filler used in affinity chromatography or hydrophobic chromatography will lead to a worse mRNA purification effect.

[0122] A comparison between Example 1 and Examples 6-7 shows that changing the gradient of the elution buffer during the hydrophobic chromatography process is not conducive to the purification of mRNA.

[0123] By comparing Example 1 and Comparative Examples 1-4, it can be seen that changing the order of affinity chromatography and hydrophobic chromatography, replacing affinity chromatography or hydrophobic chromatography with other types of chromatography, and using affinity chromatography twice or hydrophobic chromatography twice in succession will all lead to a decrease in the mRNA purification recovery rate and mRNA purity in the final eluate.

[0124] The mRNA separation and purification method provided by the present invention only requires two steps of chromatography separation and purification of mRNA, which not only has high purity and stable yield, but also is simple and convenient to operate, has a relatively short purification cycle, and greatly reduces costs.

[0125] The applicant states that while the above-described embodiments illustrate the process of the present invention, the present invention is not limited to the above-described process steps, nor does it imply that the present invention must rely on the above-described process steps for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for the raw materials used, additions of auxiliary components, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for industrial separation and purification of mRNA, characterized in that: The industrial separation and purification method of mRNA comprises the following steps: subjecting the mRNA IVT reaction solution to affinity chromatography and hydrophobic chromatography in sequence to obtain purified mRNA; The affinity chromatography filler used in the affinity chromatography is NanoGel dT20; The hydrophobic chromatography filler used in the hydrophobic chromatography is UniHR Butyl 30L; The mRNA industrial separation and purification method specifically comprises the following steps: (1) Loading the mRNA IVT reaction solution onto the equilibrated affinity chromatography column, then washing with a washing buffer, eluting with an elution buffer, and collecting the first eluate; (2) loading the first eluate obtained in step (1) onto the equilibrated hydrophobic chromatography column, washing with a washing buffer, eluting with an elution buffer, and collecting a second eluate; In step (1), the elution buffer comprises deionized water; In step (2), the elution buffer comprises a first elution buffer and a second elution buffer; The first elution buffer comprises 1.2-1.8 M ammonium sulfate and 45-55 mM Tris-HCl in molar concentration, has a pH of 7-7.4, and the solvent is water; The second elution buffer comprises 48-52 mM Tris-HCl in molar concentration, pH 7-7.4, and the solvent is water; In step (2), the elution includes a first elution and a second elution performed sequentially; The first elution is a linear gradient elution, wherein the linear gradient elution is performed using the first elution buffer and / or the second elution buffer, and the volume fraction ratio of the second elution buffer to the first elution buffer during the linear gradient elution is 0%:100%→100%:0%; The second elution is performed using a second elution buffer.

2. The mRNA industrial separation and purification method according to claim 1, wherein In step (1), the affinity chromatography column is equilibrated with 4-6 column volumes of equilibration buffer.

3. The mRNA industrial separation and purification method according to claim 2, wherein The equilibration buffer comprises 8-12 mM Tris-HCl, 0.3-0.7 M NaCl and 0.8-1.2 mM EDTA in terms of molar concentration, has a pH of 7.2-7.7, and the solvent is water.

4. The mRNA industrial separation and purification method according to claim 1, wherein In step (1), the washing buffer comprises 8-12 mM Tris-HCl, 0.3-0.7 M NaCl and 0.8-1.2 mM EDTA in terms of molar concentration, has a pH of 7.2-7.7, and the solvent is water.

5. The mRNA industrial separation and purification method according to claim 1, wherein In step (1), the affinity chromatography column is washed with 4-6 column volumes of washing buffer.

6. The mRNA industrial separation and purification method according to claim 1, characterized in that In step (1), the affinity chromatography column is eluted using 4-6 column volumes of elution buffer.

7. The mRNA industrial separation and purification method according to claim 1, characterized in that In step (2), the hydrophobic chromatography column is equilibrated with 4-6 column volumes of equilibration buffer.

8. The mRNA industrial separation and purification method according to claim 7, characterized in that: The equilibration buffer comprises 1.2-1.8 M ammonium sulfate and 45-55 mM Tris-HCl in terms of molar concentration, has a pH of 7-7.4, and the solvent is water.

9. The mRNA industrial separation and purification method according to claim 1, wherein In step (2), the washing buffer comprises 1.2-1.8 M ammonium sulfate and 45-55 mM Tris-HCl in terms of molar concentration, has a pH of 7-7.4, and the solvent is water.

10. The mRNA industrial separation and purification method according to claim 1, characterized in that: In step (2), the hydrophobic chromatography column is washed with 4-6 column volumes of washing buffer.

11. The mRNA industrial separation and purification method according to claim 1, characterized in that: The first elution is performed using 12-17 column volumes of elution buffer.

12. The mRNA industrial separation and purification method according to claim 1, characterized in that: The second elution is performed using 3-7 column volumes of elution buffer.

13. Use of the mRNA industrial separation and purification method according to any one of claims 1 to 12 in the preparation of mRNA drugs.

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