A multi-mode separation medium for one-step purification of mRNA and its preparation method and application

Abstract

The application discloses a kind of multi-mode separation medium for realizing mRNA one-step purification and its preparation method and application, it is related to biological macromolecule separation and purification technical field.The multi-mode separation medium includes matrix material and the imidazole derivative ligand with hydrogen bond bonding ability fixed on the surface of matrix material, utilizes the mixed mode specificity adsorption of hydrogen bond / electrostatic interaction in vitro transcription system mRNA, can remove oligonucleotide, plasmid DNA, T7 polymerase and double-stranded byproduct dsRNA and other multiple key impurities under the condition of no organic solvent, efficiently obtain the mRNA meeting clinical use standard from in vitro transcription system, overcome the problems that dsRNA is difficult to remove in current mRNA in vitro transcription production, purification step is complex, purification process involves organic solvent and the like, effectively improve mRNA purity and yield, easy to scale up, show great industrial production application potential.

Description

Technical Field

[0001] This invention relates to the field of biomolecule separation and purification technology, specifically to a multi-mode separation medium for one-step purification of mRNA, its preparation method, and its application. Background Technology

[0002] Nucleic acids, as one of the most important substances in living organisms, play a crucial role in vital biological activities such as protein synthesis and the transmission of genetic information. Nucleic acid-based therapies have become an important means of disease treatment. Among them, messenger RNA (mRNA) shows great potential in vaccine application due to its unique advantages such as high safety, rapid synthesis, and ease of production. mRNA can be extracted from matrices such as cells and tissues or synthesized using in vitro transcription (IVT) methods, including chemical and enzymatic methods. IVT is currently the main technical route for industrial production. However, the IVT reaction system contains a variety of reaction starting materials, such as RNA polymerase, template DNA, oligonucleotides, and transcription byproducts such as double-stranded RNA (dsRNA) and short transcripts, which seriously affect the safety of mRNA drugs. Therefore, multiple separation and purification processes must be used to achieve the clinical purity standards of mRNA.

[0003] Chromatography has become the most widely used platform for mRNA separation and purification due to its advantages such as high separation efficiency and fast separation speed. Commonly used chromatographic methods include ion exchange, Oligo dT affinity chromatography, and size exclusion chromatography. Although these chromatographic methods can remove most impurities in IVT (such as RNA polymerase, template DNA, oligonucleotides, etc.), they cannot effectively remove dsRNA, which has highly similar biophysical properties to mRNA. Studies have shown that excessive dsRNA content can activate innate cellular immune pathways and induce the secretion of various cytokines, thereby inhibiting the mRNA translation process. Therefore, it is generally considered that the dsRNA content in clinically usable mRNA molecules is less than 0.5%, and the removal of dsRNA has become a major challenge in mRNA production processes.

[0004] Currently, methods for specific removal of dsRNA include reverse ion-pair chromatography and cellulose chromatography. Reverse ion-pair chromatography is time-consuming, has low throughput, requires large amounts of organic solvents and downstream liquid exchange processes, severely limiting its use in large-scale industrial production. Cellulose chromatography, while primarily used for specific removal of dsRNA, has limited effectiveness in removing other impurities from in vitro transcription systems and cannot achieve high-purity mRNA in a single step, thus failing to meet the demands of high-efficiency production. Summary of the Invention

[0005] To address the aforementioned shortcomings in existing technologies, this invention provides a multi-mode separation medium for one-step mRNA purification, its preparation method, and its application. This invention features a highly specific, simple, and mild multi-mode separation medium that can effectively remove dsRNA, thus effectively solving the problem that current mRNA IVT purification processes cannot meet the demands of high-efficiency production.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: A multi-mode separation medium for one-step mRNA purification is provided, comprising a matrix material and an imidazole derivative multi-mode ligand with hydrogen bonding capability immobilized on the surface of the matrix material; the matrix material is one of natural polymers, synthetic polymers, inorganic materials, and composite materials; the structural formula of the imidazole derivative multi-mode ligand with hydrogen bonding capability is: , Wherein, X is a short-chain alkane with an amino, carboxyl, or hydroxyl functional group and a carbon chain length of less than 2; R1 and R2 are hydrogen or short-chain alkane with 1-4 carbons; and R3 is one of hydrogen, short-chain alkane with 1-4 carbons, or short-chain alkylamine with 1-4 carbons.

[0007] Furthermore, the imidazole derivative ligands with hydrogen bonding ability are one of 2-aminoimidazole, 2-methylaminoimidazole, and 2-amino-1-methylimidazole.

[0008] Furthermore, the coupling site between the imidazole derivative ligand with hydrogen bonding capability and the matrix material is the 3rd or 5th position of the imidazole ring.

[0009] Furthermore, the natural polymer is one or two of agarose, dextran, cellulose, chitosan and their derivatives.

[0010] Furthermore, the synthesized polymer is one or two of polystyrene-divinylbenzene, polymethacrylate, polyacrylate, polyethersulfone, polyvinylidene fluoride, polyamide, and polysulfone.

[0011] Furthermore, the inorganic material is one or two of silica gel, hydroxyapatite, zirconium dioxide, and alumina.

[0012] Furthermore, the composite material is at least one of a core-shell structure, an organic-inorganic hybrid material, or a magnetic composite material.

[0013] Furthermore, the matrix material is in the form of at least one of porous microspheres, porous membranes, magnetic microspheres, and monolithic columns.

[0014] The method for preparing the above-mentioned multi-mode separation medium is characterized by comprising the following steps: S1. Activate the matrix material to give its surface reactive groups; S2. Imidazole derivative multimode ligands with hydrogen bonding ability are coupled to the matrix material through reactive groups to obtain multimode separation media.

[0015] Furthermore, if step S1 is required, activation is performed using a silane coupling agent or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0016] Furthermore, the silane coupling agents include 3-chloropropyltrimethoxysilane and 3-glycidyl etheroxypropyltrimethoxysilane.

[0017] Furthermore, step SI can be omitted, and the reactive groups on the surface of the matrix material itself can be used directly.

[0018] The above-mentioned mRNA purification method using multimode separation media includes the following steps: washing the multimode separation media, then adding the mRNA IVT mixture to be separated, then adding elution buffer A and elution buffer B to remove impurities, adding elution buffer, and collecting the target mRNA component.

[0019] Further, washing was performed using a binding buffer.

[0020] Furthermore, the binding buffer is a buffer solution with 0-0.1 M inorganic salt added.

[0021] Furthermore, the pH of the binding buffer is 8.0-9.5.

[0022] Furthermore, the elution buffer A is a buffer solution containing 0.1-0.5 M inorganic salt.

[0023] Furthermore, the pH value of the rinsing buffer A is 8.0-9.5.

[0024] Furthermore, the elution buffer B is a buffer solution containing 0.5-1.0 M inorganic salt.

[0025] Furthermore, the pH value of the rinsing buffer B is 8.0-9.5.

[0026] The beneficial effect of taking the above-mentioned further measures is that it effectively removes a variety of impurities.

[0027] Furthermore, the elution buffer is a buffer solution containing 1.0 M inorganic salt.

[0028] Furthermore, the pH of the elution buffer is 9.5-10.5; Furthermore, the inorganic salt is one or two of sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, magnesium chloride, magnesium sulfate, and ammonium sulfate.

[0029] Furthermore, the buffer solution is one or two of the following: citrate buffer, acetate buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer, carbonate buffer, and glycine buffer.

[0030] The beneficial effect of adopting the above-mentioned further measures is: efficient recovery of mRNA target components.

[0031] Furthermore, the mRNA can be derived from any form of in vitro transcription synthesis.

[0032] Furthermore, the in vitro transcription system includes a mixture of post-transcriptional tailing and / or capping.

[0033] Furthermore, the in vitro transcription system includes a DNA template, T7 polymerase, NTP, dsRNA, enzyme inhibitor, and salt.

[0034] Application of multi-mode separation media in the purification of mRNA in vitro transcription systems or the preparation of mRNA purification kits.

[0035] In summary, the present invention has the following beneficial effects: 1. The multi-mode separation medium described in this invention has a simple preparation process and low cost.

[0036] 2. The multi-mode separation medium described in this invention can remove key impurities such as oligonucleotides, plasmid DNA, T7 polymerase, and dsRNA in one step, yielding mRNA products that meet clinical purity standards, with an mRNA yield >65.70%. Currently used Oligo-dT microspheres cannot effectively remove dsRNA (removal rate is only 45.91%, Comparative Example 4), requiring downstream purification steps (such as cellulose chromatography), resulting in an actual overall mRNA production yield of only 55.57% (Comparative Example 6), significantly lower than the one-step purification yield of this invention. Furthermore, this invention replaces a multi-step tandem process with a single step, effectively simplifying the process flow, reducing production costs, and meeting the demands of high-efficiency production.

[0037] 3. The imidazole derivative ligand in the multi-mode separation medium of the present invention significantly improves the difference in binding energy between mRNA and dsRNA through the hydrogen-bonding functional group of the side chain at position 2. By regulating electrostatic and hydrogen bonding forces, the separation of mRNA from its highly similar byproduct dsRNA can be effectively achieved. Although existing technologies use 2-aminoimidazole for protein purification, most of them are based on hydrophobic or electrostatic interactions and have never guided or implied that the difference in hydrogen bonding ability caused by the single and double strand structures of mRNA and dsRNA can be used to design separation materials. Furthermore, they have not pointed out that only the hydrogen bond donor of the imidazole ring at position 2 can produce high selectivity. The results of Comparative Examples 1-3 and Experimental Examples 1-2 have demonstrated that the ΔΔE and dsRNA removal rates of the same functional groups at positions 1 and 4 are significantly lower than those of the present invention.

[0038] 4. The purification method of the multi-mode separation medium described in this invention is mild, requires no organic reagents, and operates at room temperature, effectively improving mRNA stability and showing great potential for industrial production applications.

[0039] In summary, this invention provides a highly specific, efficient, simple, low-cost, and easily scalable multi-mode mRNA separation medium and purification method, which has great application value for the efficient and low-cost production of mRNA drugs. Attached Figure Description

[0040] Figure 1 A comparison of dsRNA removal rates in post-transcriptional capped in vitro transcription systems using multi-mode microsphere separation media with 2-aminoimidazole ligands, microsphere media with 1-methylimidazole, commercial Oligo dT media, commercial microcrystalline cellulose media, and a two-step chromatography process combining Oligo-dT and microcrystalline cellulose. Figure 2 The graph shows a comparison of dsRNA content in mRNA samples purified by single-step chromatography using multi-mode microspheres with 2-aminoimidazolium ligand, 1-methylimidazolium microspheres, commercial Oligo dT media, commercial microcrystalline cellulose media, and a two-step chromatography process using Oligo-dT and microcrystalline cellulose. Figure 3 The graph shows a comparison of the recovery rates of mRNA samples purified by single-step chromatography using multi-mode microspheres with 2-aminoimidazolium ligand, microspheres with 1-methylimidazolium, commercial Oligo dT medium, commercial microcrystalline cellulose medium, and a two-step chromatography process using Oligo-dT and microcrystalline cellulose. Figure 4A comparison of the removal rates of other impurities (T7 polymerase, DNA template, NTPs) in a post-transcriptional capped in vitro transcription system using multi-mode microsphere separation media with 2-aminoimidazole ligand, microsphere media with 1-methylimidazole, commercial Oligo dT media, commercial microcrystalline cellulose media, and a two-step chromatography process using Oligo-dT and microcrystalline cellulose. Figure 5 This is a comparison of dsRNA removal rate and mRNA recovery rate after one-step purification of IVT samples using a multi-mode microsphere separation medium with 2-aminoimidazole ligand and two-step purification using Oligo-dT and microcrystalline cellulose. Detailed Implementation

[0041] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0042] Example 1 A method for preparing a multi-mode separation medium for one-step mRNA purification includes the following steps: S1. Take 15 g of polymethacrylate microspheres with epoxy groups on the surface, wash thoroughly with plenty of water to remove isopropanol, freeze-dry, resuspend the dried microspheres in 180 mL of toluene, and then transfer to a 250 mL three-necked flask. Add 15 mL of 3-chloropropyltrimethoxysilane, heat to reflux at 115 °C under mechanical stirring at 200 rpm, and react for 24 h. After the reaction mixture cools to room temperature, transfer it to a vacuum glass filter and filter with a 0.45 mm filter membrane. Wash successively with 100 mL of toluene, 200 mL of 50% ethanol aqueous solution, 500 mL of deionized water, and 100 mL of methanol to obtain activated microspheres crosslinked with 3-chloropropyltrimethoxysilane. S2. After drying the microspheres obtained in step S1 in a vacuum drying oven at 60°C for 8 h, resuspend them in 60 mL of toluene containing approximately 0.6 mmol of active groups. Add 0.5 g of 2-aminoimidazole and heat under reflux at 115°C for 24 h. After the reaction system cools to room temperature, transfer it to a vacuum glass filter and wash thoroughly with 350 mL of methanol, 300 mL of water, and 150 mL of methanol in sequence to obtain the microsphere separation medium modified with 2-aminoimidazole ligands. Freeze-dry the packing material for later use.

[0043] The purification effect of the multimodal microsphere separation medium synthesized in Example 1 on mRNA in the post-transcriptional capping in vitro synthesis system was evaluated. The required buffer solutions are as follows: Sample loading buffer: 0.0865 M sodium chloride, 50 mM glycine buffer, pH 9.1 Eluent A: 0.3 M sodium chloride, 50 mM glycine buffer, pH 9.1 Eluent B: 1.0 M sodium chloride, 50 mM glycine buffer, pH 9.1 Elution buffer: 1.0 M sodium chloride, 50 mM glycine buffer, pH 10.5 The specific steps are as follows: (1) The microsphere separation medium modified with 2-aminoimidazolium ligand obtained in Example 1 was loaded into a 1 mL empty C-type column tube with a column medium volume of 1.0 mL. The column was connected to a GE Akta Explorer 100 chromatography system and the signal was detected by ultraviolet light at a wavelength of 260 nm. (2) Pre-equilibrate with loading buffer at a flow rate of 0.5 mL / min for 10 CVs (column volume). Take 200 μL of green fluorescent protein mRNA in vitro transcription solution, dilute it to 1 mL with loading buffer, and then feed it. The mRNA content is 0.8 mg / mL. Collect the eluent corresponding to the breakthrough peak until the 260 nm UV absorption signal returns to the baseline; (3) Elute with elution buffer A at a flow rate of 0.5 mL / min and collect the elution fraction; (4) Elute with elution buffer B at a flow rate of 0.5 mL / min and collect the elution fraction; (5) Elute with elution buffer at a flow rate of 0.5 mL / min and collect the mRNA target component; (6) Adjust the pH of the mRNA target component to 7.0 with 3 M sodium acetate / acetic acid buffer (pH 5.0), and save the components collected at each stage for subsequent high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) detection.

[0044] Example 2 The preparation method of the microsphere separation medium modified with 2-aminoimidazole ligand is the same as in Example 1.

[0045] The microsphere separation medium modified with 2-aminoimidazole ligand synthesized in Example 1 was used to evaluate the purification effect of mRNA in the co-transcriptional capping in vitro synthesis system, using the same method as in Example 1.

[0046] Example 3 A method for preparing a multi-mode separation medium for one-step mRNA purification includes the following steps: S1. Take 15 g of polymethacrylate microspheres with hydroxyl groups on the surface, wash thoroughly with plenty of water to remove isopropanol, freeze-dry, resuspend the dried microspheres in 180 mL of toluene, transfer to a 250 mL three-necked flask, add 20 mL of 3-chloropropyltrimethoxysilane, heat under reflux at 115 °C for 24 h with mechanical stirring at 170 rpm, after the reaction mixture cools to room temperature, transfer to a vacuum glass filter, filter with a 0.45 mm filter membrane, and wash successively with 100 mL of toluene, 200 mL of 50% ethanol aqueous solution, 500 mL of deionized water, and 100 mL of methanol to obtain activated microspheres crosslinked with 3-chloropropyltrimethoxysilane; S2. After drying the microspheres obtained in step S1 in a vacuum drying oven at 60°C for 8 h, resuspend them in 60 mL of toluene. Add 0.6 g of 2-aminomethylimidazole to the active group (approximately 0.6 mmol). Heat the mixture under reflux at 115°C for 24 h. After the reaction system cools to room temperature, transfer it to a vacuum glass filter and wash thoroughly with 350 mL of methanol, 300 mL of water, and 150 mL of methanol in sequence to obtain the microsphere separation medium modified with 2-amino-1-methylimidazole ligands. Freeze-dry the packing material for later use.

[0047] The microsphere separation medium modified with 2-amino-1-methylimidazolium ligand synthesized in Example 2 was used to evaluate the purification effect of mRNA in the post-transcriptional capping in vitro synthesis system, using the same method as in Example 1.

[0048] Example 4 A method for preparing a multi-mode separation medium for one-step mRNA purification includes the following steps: S1. Weigh 15 g of agarose microspheres with epoxy groups on the surface, wash them thoroughly with plenty of water, and then freeze-dry them. S2. The dried agarose microspheres were placed in a sodium phosphate-disodium hydrogen phosphate solution (pH 11-12), and 0.6 g of 2-aminomethylimidazole was added. The mixture was then reacted at 37°C for 24 h to complete ligand coupling. After the reaction system cooled to room temperature, it was transferred to a vacuum glass filter and washed sequentially with water and ethanol to obtain the microsphere separation medium modified with the 2-aminomethylimidazole ligand. The packing material was then freeze-dried for later use.

[0049] Example 5 A method for preparing a multi-mode separation medium for one-step mRNA purification includes the following steps: S1. Glycidyl methacrylate, ethylene glycol dimethacrylate, cyclohexanol, dodecanol, and sodium persulfate were mixed in a volume ratio of 20:20:50:9:1. After thorough dissolution, 10 mL of the mixture was poured into a designated mold and placed in an 80 °C water bath. After reacting for 24 h, the monolithic column was formed. The monolithic column was then trimmed into a 2 mL column and placed in a specific component. The column was thoroughly washed with ethanol to remove the pore-forming agent, resulting in a blank monolithic column sample. The monolithic column was treated with 20% sulfuric acid, and then 20 mL of 3-glycidyl etheroxypropyltrimethoxysilane was added. The column was treated at room temperature for 4 h and then washed with deionized water to obtain the activated monolithic column matrix material. S2. Dissolve 2.0 g of 2-aminoimidazole in carbonate buffer (pH 9-10), react overnight at 50-60℃, and then wash thoroughly with deionized water to prepare a monolithic column separation medium modified with 2-aminoimidazole ligand.

[0050] Example 6 A method for preparing a multi-mode separation medium for one-step mRNA purification includes the following steps: S1. Place the polyamide nanofiltration membrane with carboxyl groups on its surface in an aqueous solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and hydroxybenzotriazole (HOBt) (pH 9.0) and activate it at room temperature for 0.5 h. S2. Add the aqueous solution containing 5-(4-aminophenyl)-2-aminoimidazole and N-methylmorpholine (NMM) (pH 9.0) to the above reaction, shake the reaction at room temperature for 24 h, remove the membrane and rinse it thoroughly with ultrapure water for 2 min, then soak it alone in ultrapure water at 4°C for at least 24 h to obtain the membrane separation medium modified with 2-aminoimidazole ligand.

[0051] Example 7 A method for preparing a multi-mode separation medium for one-step mRNA purification includes the following steps: S1. Weigh 15 g of magnetic composite microspheres with epoxy groups on the surface, wash them thoroughly with a large amount of water, and then freeze-dry them. S2. Take the dried magnetic composite microspheres and put them into a sodium phosphate-disodium hydrogen phosphate solution (pH 11-12). Add 0.5 g of 2-aminoimidazole and place them at 37℃ for 24 h to complete the ligand coupling. After the reaction system cools to room temperature, transfer it to a vacuum glass filter and wash it with water and ethanol in sequence to obtain the magnetic composite microsphere separation medium modified with 2-aminoimidazole ligand. Freeze-dry the packing material for later use.

[0052] Comparative Example 1 The purification effect of multimodal microsphere separation media with 1-methylimidazole as the ligand was evaluated in a post-transcriptional capped in vitro synthesis system for mRNA purification.

[0053] The preparation process of this microsphere separation material is as follows: (1) Weigh 15 g (wet weight) of polymethyl methacrylate microspheres (with epoxy groups on the surface), wash thoroughly with a large amount of water to remove isopropanol, and then freeze dry.

[0054] (2) The dried microspheres were resuspended in 180 mL of toluene and then transferred to a 250 mL three-necked flask. 15 mL of 3-chloropropyltrimethoxysilane was added, and the mixture was heated to reflux at 115 °C under mechanical stirring at 200 rpm for 24 h. After the reaction mixture cooled to room temperature, it was transferred to a vacuum glass filter and filtered through a 0.45 mm filter membrane. The mixture was washed successively with toluene (100 mL), 50% ethanol aqueous solution (200 mL), deionized water (500 mL), and methanol (100 mL) to obtain activated microspheres crosslinked with 3-chloropropyltrimethoxysilane.

[0055] (3) After drying the microspheres obtained in step (2) in a vacuum drying oven at 60°C for 8 h, resuspend them in 60 mL of toluene (approximately 0.6 mmol of active group), add 0.5 g of 1-methylimidazole, and heat under reflux at 115°C for 24 h. After the reaction system cools to room temperature, transfer it to a vacuum glass filter and wash thoroughly with methanol (350 mL), water (300 mL), and methanol (150 mL) in sequence to obtain microsphere separation material modified with 1-methylimidazole ligand. Freeze-dry the packing material for later use.

[0056] The synthesized multimodal microsphere separation medium was used to evaluate the purification effect of mRNA in the post-transcriptional capping in vitro synthesis system. The required buffer is as follows: Sample loading buffer: 0.0865 M sodium chloride, 50 mM glycine buffer, pH 9.1 Eluent A: 0.3 M sodium chloride, 50 mM glycine buffer, pH 9.1 Eluent B: 1.0 M sodium chloride, 50 mM glycine buffer, pH 9.1 Elution buffer: 1.0 M sodium chloride, 50 mM glycine buffer, pH 10.5 The specific steps are as follows: (1) The prepared multi-mode microsphere separation material was loaded into a 1 mL C-type empty column tube with a column medium volume of 1.0 mL. The column was connected to a GE Akta Explorer 100 chromatography system and the ultraviolet light was set to detect the signal at a wavelength of 260 nm. (2) Pre-equilibrate with loading buffer at a flow rate of 0.5 mL / min for 10 CVs (column volume). Take 200 μL of green fluorescent protein mRNA in vitro transcription solution, dilute it to 1 mL with loading buffer, and then feed it. The mRNA content is 0.8 mg / mL. Collect the eluent corresponding to the breakthrough peak until the 260 nm UV absorption signal returns to the baseline; (3) Elute with elution buffer A at a flow rate of 0.5 mL / min and collect the elution fraction; (4) Elute with elution buffer B at a flow rate of 0.5 mL / min and collect the elution fraction; (5) Elute with elution buffer at a flow rate of 0.5 mL / min and collect the mRNA target component; (6) Adjust the pH of the mRNA target component to 7.0 with 3 M sodium acetate / acetic acid buffer (pH 5.0), and save the components collected at each stage for subsequent high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) detection.

[0057] Comparative Example 2 The purification effect of multimodal microsphere separation media with 1-(3-aminopropyl)imidazole as a ligand in a post-transcriptional capping in vitro synthesis system was evaluated. The amount of 1-(3-aminopropyl)imidazole added was 0.75 g, and the preparation method was the same as that of Comparative Example 1.

[0058] The synthesized multimodal microsphere separation medium was used to evaluate the purification effect of mRNA in the post-transcriptional capping in vitro synthesis system. The evaluation method was the same as that of Comparative Example 1.

[0059] Comparative Example 3 The purification effect of multimodal microspheres with 4-aminoimidazole as the ligand was evaluated in a post-transcriptional capping in vitro synthesis system for mRNA. The amount of 4-aminoimidazole added was 0.5 g, and the preparation method was the same as in Comparative Example 1.

[0060] The synthesized multimodal microsphere separation medium was used to evaluate the purification effect of mRNA in the post-transcriptional capping in vitro synthesis system. The evaluation method was the same as that of Comparative Example 1.

[0061] Comparative Example 4 The purification efficiency of commercially available Oligo dT affinity chromatography packing material (Thermo) in post-transcriptional capping in vitro synthesis systems was evaluated. The required buffers are as follows: Sample loading buffer: 0.5 M sodium chloride, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4 Eluent: 0.1 M sodium chloride, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4 Elution buffer: 10 mM Tris-HCl, pH 7.4 The specific steps are as follows: (1) Load 1 mL of Oligo dT chromatography packing material into a 1 mL empty C-type column tube, connect it to the GE Akta Explorer 100 chromatography system, and set the ultraviolet absorption wavelength to 260 nm.

[0062] (2) Pre-equilibrate with loading buffer at a flow rate of 0.5 mL / min for 3-5 CVs (column volumes). Take 200 μL of green fluorescent protein mRNA transcription solution, dilute it to 1 mL with loading buffer, and then feed it. The mRNA content is 0.8 mg / mL. Collect the eluent corresponding to the breakthrough peak until the UV absorption signal at 260 nm returns to the baseline; (3) Elute with elution buffer at a flow rate of 0.5 mL / min for 2-3 CVs until the conductivity signal stabilizes, and collect the eluted fraction.

[0063] (4) Elute with elution buffer at a flow rate of 0.5 mL / min for 3-5 CVs and collect the mRNA target component.

[0064] (5) The components eluted at each stage were subjected to the same quantitative detection as in Example 1.

[0065] Comparative Example 5 The purification efficiency of commercially available microcrystalline cellulose chromatography packing material (Sigma-Aldrich) in "mRNA flow-through" mode for post-transcriptional capping in vitro synthesis systems was evaluated. The required buffer solutions are as follows: Sample loading buffer: 125 mM sodium chloride, 10 mM HEPES, 0.1 mM EDTA, pH 7.2, 16% (v / v) ethanol The specific steps are as follows: (1) Load 1 mL of microcrystalline cellulose chromatography packing material into a 1 mL C-type empty column tube, connect it to the GE Akta Explorer100 chromatography system, and set the ultraviolet absorption wavelength to 260 nm.

[0066] (2) Pre-equilibrate with loading buffer at a flow rate of 0.5 mL / min for 3-5 CVs (column volumes). Take 200 μL of green fluorescent protein mRNA transcription solution, dilute it to 1 mL with loading buffer, and then feed it. The mRNA concentration is 0.8 mg / mL. Collect the eluent (mRNA target component) corresponding to the breakthrough peak until the UV absorption signal at 260 nm returns to the baseline; (3) The components eluted at each stage were subjected to the same quantitative detection as in Example 1.

[0067] Comparative Example 6 The purification efficiency of a two-step chromatography process using Oligo-dT coupled with microcrystalline cellulose in a co-transcriptional capping in vitro synthesis system was evaluated.

[0068] The buffer required for Oligo-dT chromatography purification is as follows: Sample loading buffer 1: 0.5 M sodium chloride, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4 Eluent: 0.1 M sodium chloride, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4 Elution buffer: 10 mM Tris-HCl, pH 7.4 The buffer solution required for microcrystalline cellulose chromatography purification is as follows: Loading buffer 2: 125 mM sodium chloride, 10 mM HEPES, 0.1 mM EDTA, pH 7.2, 16% (v / v) ethanol The specific steps are as follows: (1) Load 1 mL of Oligo dT chromatography packing material into a 1 mL empty C-type column tube, connect it to the GE Akta Explorer 100 chromatography system, and set the ultraviolet absorption wavelength to 260 nm.

[0069] (2) Pre-equilibrate with loading buffer 1 at a flow rate of 0.5 mL / min for 3-5 CVs (column volumes). Take 200 μL of green fluorescent protein mRNA transcription solution, dilute it to 1 mL with loading buffer, and then feed it. The mRNA content is 0.8 mg / mL. Collect the eluent corresponding to the breakthrough peak until the UV absorption signal at 260 nm returns to the baseline; (3) Elute with elution buffer at a flow rate of 0.5 mL / min for 2-3 CVs until the conductivity signal stabilizes, and collect the eluted fraction.

[0070] (4) Elute with elution buffer at a flow rate of 0.5 mL / min for 3-5 CVs and collect the mRNA target component.

[0071] (5) Load 1 mL of microcrystalline cellulose chromatography packing material into a 1 mL C-type empty column tube, connect it to the GE Akta Explorer100 chromatography system, and set the ultraviolet absorption wavelength to 260 nm.

[0072] (6) Pre-equilibrate with loading buffer 2 at a flow rate of 0.5 mL / min for 3-5 CVs (column volumes). Dilute the mRNA target component collected in step (4) with loading buffer and then feed it. Collect the eluent corresponding to the breakthrough peak until the UV absorption signal at 260 nm returns to the baseline; (7) The components eluted at each stage were subjected to the same quantitative detection as in Example 1.

[0073] Experimental Example 1 The effects of the position (positions 1, 2, and 4 of the imidazole ring) and type of functional groups in several imidazole derivative ligands with hydrogen bonding capability on the binding energies of mRNA and dsRNA were compared using the molecular docking software AutoDock vina 1.2.

[0074] The mRNA was homology-modeled using the Alphafold 3 server to obtain a three-dimensional PDB structure. The dsRNA was obtained from the Protein Data Bank database (PDB sequence number 1QC0).

[0075] The specific steps are as follows: (1) Use AutodockTools to prepare the acceptor input file, including removing water molecules and ligand molecules, synthesizing nonpolar hydrogen atoms, adding some charges, and adding atom types.

[0076] (2) The grid center coordinates of mRNA and dsRNA on the x, y and z axes were set to 187.259 × 57.318 × -96.532 and 12.932 × 25.537 × 36.125, respectively. The grid box sizes of mRNA and dsRNA were 350 Å × 250 Å × 250 Å and 37.41 Å × 40.29 Å × 107.91 Å, respectively.

[0077] (3) Molecular docking was performed based on the settings to obtain the binding energy ΔE (kcal / mol), and the results are shown in Table 1.

[0078] Table 1. Comparison of molecular docking results for different ligand sites and types.

[0079] As shown in Table 1, the binding energy difference between the ligand and mRNA and dsRNA is significantly increased only when the functional group with hydrogen bonding ability is located on the side chain at position 2 of the imidazole ring, which is conducive to the separation of the two.

[0080] Experimental Example 2 The yield of purified mRNA and the impurity removal rate in each example and comparative example were quantitatively analyzed by methods such as high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA).

[0081] The purified components in Examples 1-3 and Comparative Examples 1-6 were analyzed by high-performance liquid chromatography (HPLC) to quantitatively compare the removal rates of key impurities NTPs and DNA, and the recovery rates of target mRNA under different chromatographic processes. The specific HPLC procedure was as follows: an SRT SEC-2000 (300×7.8 mm, Sepax Technologies, USA) column was used, and analysis was performed using a WatersArc HPLC series. The UV detection wavelength was 260 nm, the sample loading volume was 200 ng, and elution was performed 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.

[0082] The purified components in each example and comparative example were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit to quantitatively compare the removal rate of the key impurity dsRNA under different chromatographic processes. The specific procedure was as follows: Purified mRNA samples were added to 96-well plates pre-immobilized with dsRNA antibodies and incubated at 37°C for 1 hour; after washing, detection antibodies were added and incubation continued at 37°C for 1 hour; after washing, horseradish peroxidase (HRP)-labeled detection antibodies were added and incubated at 37°C for 15 minutes. After the colorimetric reaction was complete, the signal was recorded at 450 nm using a microplate reader (Thermo Scientific, USA). The dsRNA content was calculated based on the concentration-absorbance standard curve of the dsRNA standard.

[0083] The purified components in each example and comparative example were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit to quantitatively compare the removal rate of the key impurity T7 polymerase under different chromatographic processes. The specific procedure was as follows: Purified mRNA samples were added to 96-well plates pre-immobilized with T7 polymerase antibody and incubated at 37°C for 1 hour; after washing, detection antibody was added and incubation continued at 37°C for 1 hour; after washing, horseradish peroxidase (HRP)-labeled detection antibody was added and incubated at 37°C for 15 minutes. After the colorimetric reaction was complete, the signal was recorded at 450 nm using a microplate reader (Thermo Scientific, USA). The T7 polymerase removal rate for each example and comparative example was calculated based on the concentration-absorbance standard curve of the T7 polymerase standard.

[0084] The results are as follows Figure 1-5 As shown in Table 2. Figure 1 dsRNA removal rate Figure 2 For comparison of dsRNA content, Figure 3 For comparison of mRNA sample recovery rates, Figure 4 For the comparison of removal rates, Figure 5 Comparison of dsRNA removal rate and mRNA recovery rate.

[0085] Table 2 Comparison of Key Impurity Removal Rate and mRNA Yield Results

[0086] The results above show that, compared with separation media where the ligand is located at other sites (Comparative Examples 1, 2, and 3), the multi-mode separation media (Examples 1, 2, and 3) composed of imidazole derivative ligands with hydrogen bonding functional groups at position 2 have slightly higher DNA template and NTP removal rates, and significantly improved dsRNA removal and mRNA yield, demonstrating a significant advantage. Compared with commercial Oligo-dT chromatographic packing material (Comparative Example 4), the multi-mode separation media provided by this invention have comparable DNA template and NTP removal rates, and a higher dsRNA removal rate, but the mRNA yield is slightly lower than that of commercial Oligo-dT affinity chromatographic packing material. However, due to the limited dsRNA removal effect of commercial Oligo-dT media, the purified dsRNA content is 4.60%, which does not meet the requirements for clinical use. It needs to be used in conjunction with a dsRNA-specific purification process, such as the classic cellulose chromatography process (Comparative Example 6), whose actual total mRNA production yield is reduced to 55.57%, significantly lower than the one-step purification yield of this invention.

[0087] In summary, this invention designs and synthesizes a multi-mode separation medium that can separate mRNA from other key impurities (template DNA, NTPs, dsRNA) in an in vitro transcription system in one step without the use of organic solvents. This allows for the efficient preparation of mRNA target products that meet clinical purity standards. The purification process is simple and the conditions are mild, which greatly reduces the economic and time costs of removing dsRNA and makes it easy to scale up to industrial production.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-mode separation medium for one-step purification of mRNA, characterized in that, The invention comprises a matrix material and a multimodal ligand of imidazole derivatives with hydrogen bonding capability immobilized on the surface of the matrix material; the matrix material is one of natural polymers, synthetic polymers, inorganic materials, and composite materials; the structural formula of the multimodal ligand of imidazole derivatives with hydrogen bonding capability is: Wherein, X is a short-chain alkane with an amino, carboxyl, or hydroxyl functional group and a carbon chain length of less than 2; R1 and R2 are hydrogen or short-chain alkane with 1-4 carbons; and R3 is one of hydrogen, short-chain alkane with 1-4 carbons, or short-chain alkylamine with 1-4 carbons.

2. The multi-mode separation medium as described in claim 1, characterized in that, The imidazole derivative ligand with hydrogen bonding capability is one of 2-aminoimidazole, 2-methylaminoimidazole and 2-amino-1-methylimidazole, and the coupling site between the imidazole derivative ligand with hydrogen bonding capability and the matrix material is the 3rd or 5th position of the imidazole ring.

3. The multi-mode separation medium as described in claim 1, characterized in that, The natural polymer is one or two of agarose, dextran, cellulose, chitosan and their derivatives; the synthetic polymer is one or two of polystyrene-divinylbenzene, polymethacrylate, polyacrylate, polyethersulfone, polyvinylidene fluoride, polyamide and polysulfone; the inorganic material is one or two of silica gel, hydroxyapatite, zirconium dioxide and alumina; the composite material is at least one of core-shell structure, organic-inorganic hybrid material or magnetic composite material.

4. The multi-mode separation medium as described in claim 1, characterized in that, The matrix material is at least one of porous microspheres, porous membranes, magnetic microspheres, and monolithic columns.

5. The method for preparing the multi-mode separation medium according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Activate the matrix material to give its surface reactive groups; S2. Imidazole derivative multimode ligands with hydrogen bonding ability are coupled to the matrix material through reactive groups to obtain multimode separation media.

6. The preparation method according to claim 5, characterized in that, Step S1 involves activation using a silane coupling agent or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

7. The method for purifying mRNA using the multi-mode separation medium according to any one of claims 1-4, characterized in that, Includes the following steps: The multi-mode separation medium was washed, and then the mRNA IVT mixture to be separated was added. Elution buffer A and elution buffer B were added, followed by elution buffer, and the mRNA target component was collected.

8. The method as described in claim 7, characterized in that, Washing was performed using a binding buffer solution, which was a buffer solution containing 0-0.1 M inorganic salts with a pH of 8.0-9.

5. The elution buffer A is a buffer solution containing 0.1-0.5 M inorganic salts, with a pH of 8.0-9.

5. The elution buffer B is a buffer solution containing 0.5-1.0 M inorganic salt, and the pH value of the elution buffer is 8.0-9.5; The elution buffer is a buffer solution containing 1.0 M inorganic salt with a pH of 9.5-10.5; The inorganic salt is one or two of sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, magnesium chloride, magnesium sulfate, and ammonium sulfate; the buffer solution is one or two of citrate buffer, acetate buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer, carbonate buffer, and glycine buffer.

9. The method as described in claim 7, characterized in that, The mRNA is derived from any one of the in vitro transcriptional synthesis methods.

10. The use of the multi-mode separation medium according to any one of claims 1-4 in the purification of mRNA in vitro transcription systems or in the preparation of mRNA purification kits.

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