A method for preparing a chromatographic packing for purifying mRNA

Abstract

This invention relates to the field of mRNA purification technology, specifically to a method for preparing a chromatographic packing material for purifying mRNA, comprising the following steps: coupling styrene resin with a first coupling agent to obtain a first intermediate; coupling the first intermediate with a second coupling agent and a third coupling agent to obtain a second intermediate; self-crosslinking the second intermediate to obtain a third intermediate; coupling the third intermediate with a fourth coupling agent to obtain a fourth intermediate, wherein the fourth coupling agent is an epoxy compound containing double bonds; brominating the fourth intermediate, coupling it with a 12-amine modified primer, and capping the ends to obtain the chromatographic packing material for purifying mRNA, wherein the primer is oligothymine. The chromatographic packing material provided by this invention not only improves the binding strength between the primer and styrene but also increases the mRNA recovery rate.

Description

Technical Field

[0001] This invention relates to the field of mRNA purification technology, and more specifically to a method for preparing chromatographic packing material for purifying mRNA. Background Technology

[0002] In recent years, with the increasing variety of viruses infecting humans, vaccine research has become crucial. mRNA vaccines, with their high protection rates and rapid development efficiency, have garnered widespread attention and research. Compared to other vaccines (such as inactivated and attenuated vaccines), mRNA vaccines not only respond well to viral antigens but also elicit a stronger immune response, facilitating rapid recognition and elimination of invading viruses by antibodies in the body. Furthermore, mRNA vaccines offer advantages such as predictable protein expression, low immunogenicity, and avoidance of host genome integration risks. In addition, the mRNA vaccine process is easily standardized. Therefore, mRNA vaccines can be used not only as preventative vaccines but also for the treatment of many diseases, such as infectious diseases caused by influenza and HIV, as well as cancers like non-small cell lung cancer and glioblastoma.

[0003] Downstream bioseparation and purification processes typically involve two stages: capture and purification. For the complex process of mRNA in vitro synthesis (mRNA IVT), due to the characteristic 3' terminal polyadenine ribonucleotide (poly(A)) tail structure of mRNA, affinity chromatography with high selectivity, utilizing the base complementarity pairing principle, is the most suitable capture method. Currently, the two main ligands used in mRNA affinity chromatography separation are oligodeoxythymidine (Oligo dT) and polyuridine (poly(U)). Studies have shown that affinity chromatography media with poly(U) ligands have strong binding affinity, making them difficult to elute and requiring the addition of substances to reduce non-specific adsorption. In contrast, affinity chromatography media with Oligo dT ligands ensure hydrogen bonding between AT molecules through high-salt loading, and rely on Coulomb repulsion for mRNA elution through low-salt or salt-free elution. Currently, Oligo dT affinity chromatography media include magnetic beads, cellulose, polystyrene microspheres, and agarose microspheres bonded with Oligo dT primers. However, these products result in low mRNA purification yields, are difficult to purify, and have low efficiency.

[0004] Existing technologies use polystyrene microspheres with rigid structures for large-scale purification of mRNA, but they also have obvious drawbacks. On the one hand, polystyrene microspheres have low mRNA loading capacity, and on the other hand, the surface of polystyrene microspheres is highly hydrophobic, which makes them difficult to elute during mRNA purification and also reduces the mRNA yield. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects in the purification mRNA loading capacity of the prior art, thereby providing a method for preparing chromatographic packing material for purifying mRNA.

[0006] Another technical problem to be solved by the present invention is to overcome the defect of low mRNA yield due to difficulty in elution during the purification of mRNA in the prior art, thereby providing a method for preparing chromatographic packing material for purifying mRNA.

[0007] Therefore, the present invention provides a method for preparing chromatographic packing material for purifying mRNA, comprising the following steps:

[0008] Styrene resin is coupled with a first coupling agent to obtain a first intermediate, wherein the first coupling agent is a compound having the chemical formula shown in formula (I) or (II).

[0009]

[0010]

[0011] In formula (Ⅰ), n takes the value of 0 to 6, and in formula (Ⅱ), R1 is independently selected from hydrogen atoms or C1-C6 alkyl groups;

[0012] The first intermediate, the second coupling agent, and the third coupling agent are coupled together to obtain the second intermediate, wherein the second and third coupling agents are independently selected from compounds having the chemical formula shown in formula (III).

[0013]

[0014] Wherein, R2 is independently selected from hydroxyl, halogen atom, At least one of them,

[0015] In the structural formula of either the second or third coupling agent, R2 is selected from a halogen atom;

[0016] The second intermediate is self-crosslinked to obtain the third intermediate;

[0017] The third intermediate is coupled with the fourth coupling agent to obtain the fourth intermediate, wherein the fourth coupling agent is an epoxy compound containing a double bond;

[0018] The fourth intermediate was bromine-coated with a 12-amine modified primer and capped to obtain a purified mRNA chromatography packing material, wherein the primer was oligothymine.

[0019] In some embodiments, n in formula (I) takes the value of 0, 2 or 6, and R1 in formula (II) is selected from methyl;

[0020] Preferably, the fourth coupling agent includes at least one selected from allyl glycidyl ether, glycidyl methacrylate, 1,2-epoxy-5-hexene, and 1,2-epoxy-7-butene.

[0021] Preferably, the first coupling agent comprises at least one of glycidyl methacrylate, 3,4-epoxy-1-butene, 1,2-epoxy-9-decene, 1,2-epoxy-5-hexene, and glycidyl acrylate.

[0022] Preferably, the second and / or third coupling agent comprises at least one of glycidyl ether, epichlorohydrin, epibromopropane, glycerol triglycidyl ether, and triglycidyl isocyanurate. Preferably, at least one of the second or third coupling agent contains a halogen. More preferably, one of the second or third coupling agent is at least one of epichlorohydrin and epibromopropane.

[0023] In some embodiments, before coupling with the first coupling agent, the polystyrene resin is extracted with propanol, stirred with hot water at 60-80°C for 2-5 hours, circulated 3-5 times, and then washed with acetone. After washing, the resin is washed with pure water until it is odorless. The mass ratio of the styrene resin to the hot water is 1:10-15.

[0024] In some embodiments, the formation of the first intermediate involves reacting styrene resin and a first coupling agent at a reaction temperature of 60-80°C for 10-15 hours in the presence of an initiator and an organic solvent. The ratio of the styrene resin, the first coupling agent, the first initiator, and the organic solvent is 1:0.7-1.0:0.01-0.05:5-10, in g:g:g:mL.

[0025] Preferably, the organic solvent includes at least one of amide compounds, aromatic hydrocarbons, cycloalkanes, halogenated hydrocarbons, or ethers; more preferably, the organic solvent includes at least one of N,N-dimethylformamide, toluene, dimethyl sulfoxide, nitrobenzene, benzene, cyclohexane, dichloroethane, dioxane, and xylene.

[0026] Preferably, the first initiator includes an azo initiator and benzoyl peroxide, wherein the azo initiator includes azobisisobutyronitrile and azobisisoheptanenitrile.

[0027] In some embodiments, when forming the second intermediate, a mixture of the second coupling agent and the third coupling agent is dripped into a swelling solution containing the second initiator and the first intermediate, and the reaction is carried out at 25-40°C for 5-10 hours at a dripping rate of 1.25-2 g / h. The mass ratio of the first intermediate to the swelling agent is 1:20-30.

[0028] Preferably, the swelling solution of the first intermediate is prepared by swelling the first intermediate and dichloromethane at 25-40°C for 5-10 hours.

[0029] Preferably, the mass ratio of the first intermediate, the second initiator, the second coupling agent, and the third coupling agent is 1:0.01-0.08:1-1.5:1-4.5, in g:mL:g:g.

[0030] Preferably, the second initiator comprises boron trifluoride ethyl ether.

[0031] In some embodiments, when forming the third intermediate, the second intermediate is stirred with an alkaline solution at 25-40°C for 1-3 hours, and then reacted at 50-70°C for 15-20 hours. The mass ratio of the second intermediate to the alkaline solution is 1:5-8, and the concentration of alkaline substances in the alkaline solution is 20-50 wt%.

[0032] In some embodiments, when forming the fourth intermediate, the third intermediate and the reducing agent are mixed in the presence of an alkaline solution, and a fourth coupling agent is added thereto, and the mixture is reacted at 40-70°C for 13-18 hours. The mass ratio of the third intermediate, the reducing agent and the fourth coupling agent is 1:0.001-0.008:0.8-1.2.

[0033] Preferably, the reducing agent includes at least one of potassium borohydride, sodium borohydride, and sodium cyanoborohydride.

[0034] In some embodiments, when forming a third intermediate and / or a fourth intermediate, the alkaline solution comprises an inorganic base and an organic base, wherein the inorganic base comprises at least one of sodium hydroxide and potassium hydroxide, and the organic base comprises at least one of sodium alkoxides or potassium alkoxides.

[0035] In some embodiments, the step of bromine-adding the fourth intermediate specifically involves mixing the fourth intermediate with bromine in the presence of water for 1-3 hours, adding a bromine removal agent and reacting for 3-6 hours, wherein the bromine removal agent includes a sodium salt.

[0036] Preferably, the sodium salt includes at least one of sodium formate, sodium acetate, sodium bicarbonate, and sodium carbonate.

[0037] Preferably, the mass ratio of the fourth intermediate, bromine, and bromine removal agent is 1:0.1-0.5:0.5-1.0;

[0038] In some embodiments, the fourth intermediate of bromination is mixed with a buffer containing primers modified with 12amines and reacted at 20-30°C for 30-50 h in the presence of a neutral salt.

[0039] In some embodiments, the fourth intermediate of the brominated compound, containing a 12-amine-modified primer and a neutral salt, is present in a mass ratio of 1:0.005-0.01:0.1-0.5.

[0040] Preferably, the mass ratio of the fourth intermediate coupled with the 12-amine modified primer to the capping agent is 1:0.001-0.005.

[0041] Preferably, the oligomeric thymine in the primer has a length of 18-25 bp.

[0042] In some embodiments, the specific capping step involves coupling a fourth intermediate of a 12-amine modified primer with a capping agent in a buffer solution at a temperature of 40-60°C for 6-8 hours.

[0043] Preferably, the capping agent includes at least one selected from Tris base, ethanolamine, glutathione, serine, and threonine.

[0044] Preferably, the neutral salt includes at least one of sodium chloride, potassium sulfate, and sodium sulfate.

[0045] The technical solution of this invention has the following advantages:

[0046] This invention provides a chromatography packing material for purifying mRNA, comprising the following steps: coupling styrene resin with a first coupling agent to obtain a first intermediate, wherein the first coupling agent is a compound having the chemical formula described in formula (I) or (II).

[0047]

[0048] In formula (Ⅰ), n takes the value of 0 to 6, and in formula (Ⅱ), R1 is independently selected from hydrogen atoms or C1-C6 alkyl groups;

[0049] The first intermediate, the second coupling agent, and the third coupling agent are coupled together to obtain the second intermediate, wherein the second and third coupling agents are independently selected from compounds having the chemical formula described in formula (III).

[0050]

[0051] Wherein, R2 is independently selected from hydroxyl, halogen atom, At least one of the following, wherein R2 in the structural formula of at least one of the second or third coupling agents is selected from a halogen atom; the second intermediate is self-crosslinked to obtain a third intermediate; the third intermediate is coupled with a fourth coupling agent to obtain a fourth intermediate, wherein the fourth coupling agent is an epoxy compound containing a double bond; the fourth intermediate is brominated, coupled with a 12-amine modified primer, and capped to obtain a purified mRNA chromatography packing material, wherein the primer is oligothymine. This invention, by sequentially coupling styrene resin with a first, second, and third coupling agent, and then self-crosslinking the second and third coupling agents, forms a hydrophilic network on styrene microspheres. This not only greatly reduces the non-specific adsorption performance of the styrene microspheres but also enhances the crosslinking degree of the styrene microspheres. Furthermore, by activating the double bond and grafting the 12-amine modified primer with bromine via CN coupling, not only is the binding strength between the primer and styrene improved, but the mRNA recovery rate is also increased.

[0052] The present invention provides a chromatography packing material for purifying mRNA, which exhibits good resistance to pressure flow rate and acid and alkali, and has a high dynamic loading capacity during mRNA purification. Attached Figure Description

[0053] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0054] Figure 1 This is a schematic diagram of the chromatographic packing material specifically binding to the PolyA end of mRNA obtained in an embodiment of the present invention;

[0055] Figure 2 This is a graph showing the pressure resistance flow rate of the chromatographic packing material prepared in Example 1 of Experimental Example 1 of this invention;

[0056] Figure 3 This is a graph showing the pressure resistance and flow rate of the commercially available medium chromatography packing material, Thermo Fisher Scientific POROS Oligo(dT) 25 from the USA.

[0057] Figure 4 This is the dynamic loading test result of the chromatography packing material prepared in Example 1 of Experimental Example 3 of the present invention;

[0058] Figure 5 The results are the dynamic loading test results of the commercially available chromatography packing material POROS Oligo(dT)25 in Experimental Example 3 of this invention;

[0059] Figure 6The elution curves are those of the chromatography packing material prepared in Example 1 of Experimental Example 3 of this invention and the commercially available chromatography packing material POROS Oligo(dT)25. Figure 6 In this context, A represents the elution effect curve of the chromatography packing material prepared in Example 1. Figure 6 In this context, B represents the elution curve of the commercially available chromatography packing material POROS Oligo(dT)25. Detailed Implementation

[0060] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0061] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0062] The 12amine-modified primers used in the embodiments and comparative examples of this invention were purchased from Genscript Biotech.

[0063] A schematic diagram of the specific binding of the chromatographic packing material to the polyA terminus of mRNA obtained in this embodiment of the invention is shown below. Figure 1 , Figure 1 In this context, LINKER represents the coupling structure between the hydrophilic spacer arm of the base sphere and the dodecylamine primer CN.

[0064] Example 1

[0065] This embodiment provides a method for preparing a chromatography packing material for purifying mRNA. The specific steps and parameters are as follows:

[0066] (1) Pretreatment of polystyrene microspheres: The polystyrene microspheres after sieving were extracted with acetone (brand: macroporous ion exchange resin; particle size: 40-60μm; pore size: 150nm; source: Xi'an Lanxiao Technology New Material Co., Ltd.). 50g of the extracted polystyrene microspheres were added to 50g of water at 60℃ and stirred for 2h. After stirring, the mixture was dried. One cycle was defined as adding hot water until the mixture was dried. The cycle was repeated 3 times. The polystyrene microspheres were washed with acetone and pure water in turn until they were odorless. They were then dried for later use.

[0067] (2) Coupling with glycidyl methacrylate: Mix 30g of the resin dried in step (1), 21g of glycidyl methacrylate, 0.3g of azobisisobutyronitrile and 150mL of N,N-dimethylformamide, stir at 25℃ for 2h, react at 60℃ for 10h, wash 3 times with N,N-dimethylformamide, wash 3 times with acetone, wash 3 times with pure water until odorless, dry in an oven at 50℃ until the product moisture content is less than 1%, and pack for later use.

[0068] (3) Coupling glycidyl ether and epichlorohydrin: Take 10g of the product from step (2) and 100g of dichloromethane and swell at 25°C for 5h. Add 0.1mL of boron trifluoride ether as an initiator and stir for 0.5h. Use a peristaltic pump to slowly add a mixture of glycidyl ether and epichlorohydrin. The mixture consists of 10g of glycidyl ether and 10g of epichlorohydrin. Add the mixture at a rate of 2g / h. After adding the mixture, react at 25°C for 5h. Wash the product after the reaction is complete with ethanol 3 times and pure water 3 times until the product is odorless. Dry the product for later use.

[0069] (4) Self-crosslinking: Mix 30g of the product from step (3) with 150g of 30wt% sodium hydroxide solution at 30℃ and stir for 1h. Increase the temperature to 50℃ and react for 15h. Wash the product with pure water and dry it for later use.

[0070] (5) Coupling allyl glycidyl ether: Take 30g of the product from step (4), 9g of water, 60g of 45% sodium hydroxide solution, and 0.03g of potassium borohydride. Stir the above materials at 30°C for 2 hours. Add 24g of allyl glycidyl ether to the stirred mixture and react at 70°C for 13 hours. Wash the product after reaction with ethanol 3 times and then with pure water 3 times until it is odorless. Dry it for later use.

[0071] (6) Bromine addition: Take 30g of the product from step (5) and add 30g of water. Stir at 25°C for 0.5h, add 3g of bromine, and continue stirring at 25°C for 1h. After stirring, add 5g of sodium formate and continue stirring at 25°C for 3h. Wash with pure water, requiring a conductivity of less than or equal to 10μs / cm, and dry for later use.

[0072] (7) Coupling with 12-amine modified Oligo(dT)20 primers: Add 0.15g of 12-amine modified Oligo(dT)20 primers to 60mL of 0.2M carbonate buffer (pH=10) to form a mixture. Add 30g of the product from step (6) to the mixture, and then add 3g of sodium sulfate. React at 20℃ for 30h. Wash the product with pure water, requiring a conductivity of less than or equal to 10μs / cm. Dry the product for later use.

[0073] (8) End-capping: Add 0.03g of Tris base to 30mL of 0.2M carbonate buffer (pH=10) to form a mixture (pH=10). Mix 30g of the product from step (7) with the mixture and stir. React at 40℃ for 6h. Wash the product with pure water. The conductivity should be less than or equal to 10μs / cm. Dry the product and store it in 20vol% ethanol solution.

[0074] Example 2

[0075] This embodiment provides a method for preparing a chromatography packing material for purifying mRNA. The specific steps and parameters are as follows:

[0076] (1) Pretreatment of polystyrene microspheres: The polystyrene microspheres after sieving were extracted with acetone (brand: macroporous ion exchange resin; particle size: 40-60μm; pore size: 150nm; source: Xi'an Lanxiao Technology New Material Co., Ltd.). 50g of the extracted polystyrene microspheres were added to 50g of water at 60℃ and stirred for 5h. After stirring, the mixture was dried. One cycle was formed by adding hot water until the mixture was dried. The cycle was repeated 3 times. The polystyrene microspheres were washed with acetone and pure water in turn until the microspheres were odorless. The mixture was then dried for later use.

[0077] (2) Coupling of 3,4-epoxy-1-butene: Mix 30g of the dried resin from step (1), 30g of 3,4-epoxy-1-butene, 1.5g of azobisisobutyronitrile and 300mL of toluene, stir at 25℃ for 2h, react at 80℃ for 15h, wash 3 times with toluene, wash 3 times with acetone, wash 3 times with pure water until odorless, dry in an oven at 50℃ until the product moisture content is less than 1%, and pack for later use.

[0078] (3) Coupling glycidyl ether and epibromopropane: Take 10g of the product from step (2) and 300g of dichloromethane and swell at 40℃ for 5h. Add 0.8mL of boron trifluoride ether as an initiator and stir for 2h. Use a peristaltic pump to slowly add a mixture of glycidyl ether and epibromopropane. The mixture consists of 15g of glycidyl ether and 15g of epibromopropane. Add the mixture at a rate of 1.25g / h. After adding the mixture, react at 40℃ for 10h. Wash the product after the reaction is complete with ethanol 3 times and pure water 3 times until the product is odorless. Dry the product for later use.

[0079] (4) Self-crosslinking: Mix 30g of the product in step (3) with 240g of 20wt% potassium hydroxide solution at 40℃ and stir for 3h. Increase the temperature to 50℃ and react for 20h. Wash the product with pure water and dry it for later use.

[0080] (5) Coupling with glycidyl methacrylate: Take 30g of the product from step (4), 9g of water, 60g of 45% potassium hydroxide solution, and 0.24g of sodium borohydride. Stir the above materials at 30°C for 5h. Add 36g of glycidyl methacrylate to the stirred mixture and react at 40°C for 18h. Wash the product after reaction with ethanol 3 times and then with pure water 3 times until it is odorless. Dry it for later use.

[0081] (6) Bromine addition: Take 30g of the product from step (5) and add 30g of water. Stir at 25°C for 3 hours. Add 3g of bromine and continue stirring at 25°C for 4 hours. After stirring, add 30g of sodium acetate and continue stirring at 25°C for 6 hours. Wash with pure water, ensuring the conductivity is less than or equal to 10μs / cm. Dry and set aside.

[0082] (7) Coupling with 12-amine modified Oligo dT(18) primers: Add 0.3g of 12-amine modified Oligo dT(18) primers to 60mL of 0.2M carbonate buffer (pH=10) to form a mixture. Add 30g of the product from step (6) to the mixture, and then add 5g of sodium chloride. React at 30℃ for 50h. Wash the product with pure water, requiring a conductivity of less than or equal to 10μs / cm. Dry the product for later use.

[0083] (8) End-capping: Add 0.05g of ethanolamine to 30mL of 0.2M carbonate buffer (pH=10) to form a mixture (pH=10). Mix 30g of the product from step (7) with the mixture and stir. React at 60℃ for 6h. Wash the product with pure water. The conductivity should be less than or equal to 10μs / cm. Dry the product and store it in 20vol% ethanol solution.

[0084] Example 3

[0085] This embodiment provides a method for preparing a chromatography packing material for purifying mRNA. The specific steps and parameters are as follows:

[0086] (1) Pretreatment of polystyrene microspheres: The polystyrene microspheres after sieving were extracted with acetone (brand: macroporous ion exchange resin; particle size: 40-60μm; pore size: 150nm; source: Xi'an Lanxiao Technology New Material Co., Ltd.). 50g of the extracted polystyrene microspheres were added to 50g of water at 60℃ and stirred for 2h. After stirring, the mixture was dried. One cycle was defined as adding hot water until the mixture was dried. The cycle was repeated 3 times. The polystyrene microspheres were washed with acetone and pure water in turn until they were odorless. They were then dried for later use.

[0087] (2) Coupling 1,2-epoxy-9-decene: Mix 30g of the dried resin from step (1), 21g of 1,2-epoxy-9-decene, 0.3g of benzoyl peroxide and 150mL of dichloroethane, stir at 25℃ for 2h, react at 60℃ for 10h, wash 3 times with dichloroethane, wash 3 times with acetone, wash 3 times with pure water until odorless, dry in an oven at 50℃ until the product moisture content is less than 1%, and pack for later use.

[0088] (3) Coupling of glycerol triglycidyl ether and epichlorohydrin: Take 10g of the product from step (2) and 100g of dichloromethane and swell at 25°C for 5h. Add 0.1mL of boron trifluoride ether as an initiator and stir for 0.5h. Use a peristaltic pump to slowly add a mixture of glycerol triglycidyl ether and epichlorohydrin. The mixture consists of 10g of glycerol triglycidyl ether and 10g of epichlorohydrin. Add the mixture at a rate of 2g / h. After adding the mixture, react at 25°C for 5h. Wash the product after the reaction is complete with ethanol 3 times and pure water 3 times until the product is odorless. Dry the product for later use.

[0089] (4) Self-crosslinking: Mix 30g of the product from step (3) with 150g of 50wt% sodium methoxide solution at 30℃ and stir for 1h. Increase the temperature to 50℃ and react for 15h. Wash the product with pure water and dry it for later use.

[0090] (5) Coupling with 1,2-epoxy-5-hexene: Take 30g of the product from step (4), 9g of water, 60g of 45% potassium ethoxide solution, and 0.03g of sodium cyanoborohydride. Stir the above materials at 30°C for 2h. Add 24g of 1,2-epoxy-5-hexene to the stirred mixture and react at 70°C for 13h. Wash the product after reaction with ethanol 3 times and then with pure water 3 times until it is odorless. Dry it for later use.

[0091] (6) Bromine addition: Take 30g of the product from step (5) and add 30g of water. Stir at 25°C for 0.5h. Add 3g of bromine and continue stirring at 25°C for 1h. After stirring, add 5g of sodium bicarbonate and continue stirring at 25°C for 3h. Wash with pure water, requiring a conductivity of less than or equal to 10μs / cm. Dry and set aside.

[0092] (7) Coupling with 12-amine modified Oligo dT(25) primers: Add 0.15g of 12-amine modified Oligo dT(25) primers to 60mL of 0.2M carbonate buffer (pH=10) to form a mixture. Add 30g of the product from step (6) to the mixture, and then add 3g of sodium sulfate. React at 20℃ for 30h. Wash the product with pure water, requiring a conductivity of less than or equal to 10μs / cm, and dry it for later use.

[0093] (8) Capping: Add 0.03g of glutathione to 30mL of 0.2M carbonate buffer (pH=10) to form a mixture (pH=10). Mix 30g of the product from step (7) with the mixture and stir. React at 40℃ for 6h. Wash the product with pure water. The conductivity should be less than or equal to 10μs / cm. Dry and store in 20vol% ethanol solution.

[0094] Example 4

[0095] This embodiment provides a method for preparing a chromatography packing material for purifying mRNA. The specific steps and parameters are as follows:

[0096] (1) Pretreatment of polystyrene microspheres: The polystyrene microspheres after sieving were extracted with acetone (brand: macroporous ion exchange resin; particle size: 40-60μm; pore size: 150nm; source: Xi'an Lanxiao Technology New Material Co., Ltd.). 50g of the extracted polystyrene microspheres were added to 50g of water at 60℃ and stirred for 2h. After stirring, the mixture was dried. One cycle was defined as adding hot water until the mixture was dried. The cycle was repeated 3 times. The polystyrene microspheres were washed with acetone and pure water in turn until they were odorless. They were then dried for later use.

[0097] (2) Coupling of epoxy acrylate: 30g of resin dried in step (1), 25g of epoxy acrylate, 0.3g of azobisisobutyronitrile and 200mL of cyclohexane are mixed and stirred at 25℃ for 2h, reacted at 60℃ for 10h, washed 3 times with cyclohexane, washed 3 times with acetone, and washed 3 times with pure water until odorless, and dried in an oven at 50℃ until the product moisture content is less than 1%, and then packaged for later use.

[0098] (3) Coupling of triglycidyl isocyanurate and epichlorohydrin: Take 10g of the product from step (2) and 100g of dichloromethane and swell at 25°C for 5h. Add 0.1mL of boron trifluoride ether as an initiator and stir for 0.5h. Use a peristaltic pump to slowly add a mixture of triglycidyl isocyanurate and epichlorohydrin. The mixture consists of 10g of triglycidyl isocyanurate and 10g of epichlorohydrin. Add the mixture at a rate of 2g / h. After adding the mixture, react at 25°C for 5h. Wash the product after the reaction is complete with ethanol 3 times and pure water 3 times until the product is odorless. Dry the product for later use.

[0099] (4) Self-crosslinking: Mix 30g of the product from step (3) with 150g of 30wt% sodium hydroxide solution at 30℃ and stir for 1h. Increase the temperature to 50℃ and react for 15h. Wash the product with pure water and dry it for later use.

[0100] (5) Coupling of 1,2-epoxy-7-butene: Take 30g of the product from step (4), 9g of water, 60g of 45% sodium hydroxide solution, and 0.03g of potassium borohydride. Stir the above materials at 30°C for 2h. Add 24g of 1,2-epoxy-7-butene to the stirred mixture and react at 70°C for 13h. Wash the product after reaction with ethanol 3 times and then with pure water 3 times until it is odorless. Dry it for later use.

[0101] (6) Bromine addition: Take 30g of the product from step (5) and add 30g of water. Stir at 25°C for 0.5h, add 3g of bromine, and continue stirring at 25°C for 1h. After stirring, add 5g of sodium formate and continue stirring at 25°C for 3h. Wash with pure water, requiring a conductivity of less than or equal to 10μs / cm, and dry for later use.

[0102] (7) Coupling with 12-amine modified Oligo dT(20) primers: Add 0.15g of 12-amine modified Oligo dT(20) primers to 60mL of 0.2M carbonate buffer (pH=10) to form a mixture. Add 30g of the product from step (6) to the mixture, and then add 3g of sodium sulfate. React at 20℃ for 30h. Wash the product with pure water, requiring a conductivity of less than or equal to 10μs / cm, and dry it for later use.

[0103] (8) End-capping: Add 0.03g serine to 30mL of 0.2M carbonate buffer (pH=10) to form a mixture (pH=10). Mix 30g of the product from step (7) with the mixture and stir. React at 40℃ for 6h. Wash the product with pure water. The conductivity should be less than or equal to 10μs / cm. Dry the product and store it in 20vol% ethanol solution.

[0104] Experimental Example 1

[0105] The linear flow rate of each chromatographic packing material was recorded using high-pressure chromatography equipment and chromatography columns.

[0106] The chromatography packing materials used were the chromatography packing material prepared in Example 1 and the commercially available chromatography packing material POROS Oligo(dT)25 from Thermo Fisher Scientific, USA. The specific experimental results are shown in Table 1.

[0107] Pressure resistance flow rate test:

[0108] Equipment: Incyte chromatography system;

[0109] Column specifications: 16 / 200, actual packing size 100mm;

[0110] Mobile phase medium: 0.1M sodium chloride.

[0111] Experimental group: The chromatography packing material prepared in Example 1, the specific results are shown in Table 1 and... Figure 2 .

[0112] Table 1. Pressure and flow rate data of the chromatography packing material prepared in Example 1.

[0113]

[0114]

[0115] Combining Table 1 and Figures 2-3 It can be seen that, in a mobile phase medium of 0.1M sodium chloride, the chromatography packing material provided in this embodiment of the invention has a greater flow rate than the commercially available chromatography packing material POROS Oligo(dT)25 under the same pressure.

[0116] Experimental Example 2

[0117] The dynamic loading of each chromatographic packing material was determined by circulating 1M hydrochloric acid as the mobile phase for 48 hours using a high-pressure chromatography equipment and column.

[0118] The dynamic loading of each chromatographic packing material was determined by circulating 0.5M sodium hydroxide solution as the mobile phase for 48 hours using a high-pressure chromatography equipment and column.

[0119] The chromatography packing materials used were those from Example 1 and the commercially available Thermo Fisher Scientific POROS Oligo(dT) 25. Specific experimental results are shown in Table 2. The chromatography packing material prepared in Example 1 served as the experimental group, while the commercially available Thermo Fisher Scientific POROS Oligo(dT) 25 served as the control group.

[0120] Equipment: Incyte chromatography system;

[0121] Column specifications: 1mL pre-packed column;

[0122] Sample: mRNA stock solution (2000 nt);

[0123] Test method:

[0124] 1. Use 1M hydrochloric acid as the mobile phase and circulate for 48 hours. Measure the dynamic loading rate every 12 hours and observe the decay of the dynamic loading rate.

[0125] 2. Use 0.5M sodium hydroxide solution as the mobile phase and circulate for 48 hours. Measure the dynamic load every 12 hours and observe the decay of the dynamic load.

[0126] Sample loading conditions:

[0127] Loading Buffer: 10mM Tris-HCl, 1mM EDTA, 0.5M NaCl, 0.1% SDS, pH 7.4;

[0128] Load at a flow rate of 0.5 ml / min, and stop loading after Q10 (the total loading volume of protein concentration flowing through to 10% of the total loading concentration).

[0129] Elution buffer: 10 mM Tris-HCl, 1 mM EDTA;

[0130] Regeneration solution: 0.1M NaOH;

[0131] The specific results are shown in Table 2.

[0132] Table 2. Decrease in dynamic loading of the experimental and control groups in 1M hydrochloric acid and 0.5M NaOH (unit: mg / ml)

[0133]

[0134] According to the data in Table 2, the experimental group showed better acid and alkali resistance than the control group. After 48 hours of acid-alkali cycling, the dynamic load was still maintained at more than 80% of the initial load, while the control group dropped to about 70% of the initial load.

[0135] Experimental Example 3

[0136] The 2000nt mRNA stock solution was purified using chromatography packing materials, and the dynamic loading and recovery rate of each chromatography packing material were recorded.

[0137] The purification of mRNA stock solution (2000 nt) was carried out using the chromatography packing material prepared in Example 1 and the commercially available Thermo Fisher Scientific POROS Oligo(dT) 25 chromatography packing material. The specific steps are as follows:

[0138] Equipment: Incyte chromatography system;

[0139] Column specifications: 1mL pre-packed column;

[0140] Sample and processing: mRNA stock solution (2000nt), denatured at 65℃ for 10min, then quickly placed in an ice bath for 3min.

[0141] Sample loading amount: 8 mg;

[0142] The experimental procedure is shown in Table 3.

[0143] Table 3. mRNA purification steps

[0144] step Buffer Flow rate (mL / min) Column volume Cleaning Deionized water 1 10 Balanced buffer 1 10mM Tris-HCl, 500mM NaCl, 1mMEDTA 1 10 Sample 10mM Tris-HCl, 500mM NaCl, 1mM EDTA 0.3 / Balanced Buffer 2 10mM Tris-HCl, 250mM NaCl, 1mM EDTA 1 / Washout 10 mM Tris-HCl, 1 mM EDTA 1 / Cleaning Deionized water 1 10 regeneration 0.1M NaOH 1 5

[0145] Test results are available Figure 4-6 See Table 4-5.

[0146] Table 4. Dynamic loading capacity of Q10 for different packing materials

[0147] filler Q10 dynamic load (mg / ml) Chromatographic packing material prepared in Example 1 3.22 Commercially available POROS Oligo(dT) 25 2.25

[0148] Table 5 Elution recovery results for different samples

[0149]

[0150]

[0151] The dynamic load test results are shown below. Figures 4-5 As shown in Table 4, the dynamic loading capacity of the chromatography packing material Q10 provided in the embodiments of the present invention is significantly higher than that of commercially available POROS Oligo(dT)25.

[0152] The elution recovery rate test results are shown below. Figure 6 As shown in Table 5, the chromatography packing material provided in the embodiments of the present invention has a high recovery rate. In summary, the chromatography column provided in the embodiments of the present invention has significant advantages in both dynamic loading and recovery rate.

[0153] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for producing a chromatographic packing for purifying mRNA, characterized in that, Includes the following steps, Styrene resin is coupled with a first coupling agent to obtain a first intermediate, wherein the first coupling agent is a compound having the chemical formula shown in formula (I) or (II). In formula (Ⅰ), n takes the value of 0 to 6, and in formula (Ⅱ), R1 is independently selected from hydrogen atoms or C1-C6 alkyl groups; The first intermediate, the second coupling agent, and the third coupling agent are coupled together to obtain the second intermediate, wherein the second and third coupling agents are independently selected from compounds having the chemical formula shown in formula (III). Wherein, R2 is independently selected from hydroxyl, halogen atom, At least one of them, In the structural formula of either the second or third coupling agent, R2 is selected from a halogen atom; The second intermediate is self-crosslinked to obtain the third intermediate; The third intermediate is coupled with the fourth coupling agent to obtain the fourth intermediate, wherein the fourth coupling agent is an epoxy compound containing a double bond; The fourth intermediate was bromine-coated with a 12-amine modified primer and capped to obtain a purified mRNA chromatography packing material, wherein the primer was oligothymine.

2. The method for preparing the chromatographic packing material for purifying mRNA according to claim 1, characterized in that, In equation (Ⅰ), n can take the values ​​0, 2, or 6. In formula (II), R1 is selected from methyl; The fourth coupling agent includes at least one of allyl glycidyl ether, glycidyl methacrylate, 1,2-epoxy-5-hexene, and 1,2-epoxy-7-butene.

3. The method for preparing the chromatographic packing material for purifying mRNA according to claim 1, characterized in that, The first coupling agent includes at least one of glycidyl methacrylate, 3,4-epoxy-1-butene, 1,2-epoxy-9-decene, 1,2-epoxy-5-hexene, and glycidyl acrylate.

4. The method for preparing the chromatographic packing material for purifying mRNA according to claim 1, characterized in that, The second and / or third coupling agent comprises at least one of glycidyl ether, epichlorohydrin, epibromopropane, glycerol triglycidyl ether, and triglycidyl isocyanate. One of the second or third coupling agents is at least one of epichlorohydrin and epibromopropane.

5. The method for preparing the chromatographic packing material for purifying mRNA according to claim 1, characterized in that, In the formation of the first intermediate, styrene resin and the first coupling agent are reacted at a reaction temperature of 60-80℃ for 10-15 hours in the presence of the first initiator and organic solvent. The ratio of the styrene resin, the first coupling agent, the first initiator, and the organic solvent is 1:0.7-1.0:0.01-0.05:5-10, in g:g:g:mL. The organic solvent includes at least one of amide compounds, aromatic hydrocarbons, cycloalkanes, halogenated hydrocarbons, or ethers. The first initiator includes an azo initiator and benzoyl peroxide.

6. The method for preparing the chromatographic packing material for purifying mRNA according to claim 5, characterized in that, In the formation of the second intermediate, a mixture of the second and third coupling agents is added dropwise to a swelling solution containing the second initiator and the first intermediate. The reaction is carried out at 25-40°C for 5-10 hours at a dropping rate of 1.25-2 g / h. The mass ratio of the first intermediate to the swelling agent is 1:20-30. The mass ratio of the first intermediate, the second initiator, the second coupling agent, and the third coupling agent is 1:0.01-0.08:1-1.5:1-4.5, in g:mL:g:g. The second initiator includes boron trifluoride diethyl ether; and / or, The azo initiator includes at least one of azobisisobutyronitrile and azobisisoheptanenitrile; The organic solvent includes at least one of N,N-dimethylformamide, toluene, dimethyl sulfoxide, nitrobenzene, benzene, cyclohexane, dichloroethane, dioxane, and xylene.

7. The method for preparing the chromatographic packing material for purifying mRNA according to claim 1, characterized in that, In the formation of the third intermediate, the second intermediate is stirred with an alkaline solution at 25-40°C for 1-3 hours, and then reacted at 50-70°C for 15-20 hours. The mass ratio of the second intermediate to the alkaline solution is 1:5-8, and the concentration of the alkaline substance in the alkaline solution is 20-50 wt%.

8. The method for preparing the chromatographic packing material for purifying mRNA according to claim 1, characterized in that, In the formation of the fourth intermediate, the third intermediate and the reducing agent are mixed in the presence of an alkaline solution, and the fourth coupling agent is added. The mixture is then reacted at 40-70°C for 13-18 hours. The mass ratio of the third intermediate, reducing agent, and fourth coupling agent is 1:0.001-0.008:0.8-1.

2. The reducing agent includes at least one of potassium borohydride, sodium borohydride, and sodium cyanoborohydride. When a third intermediate and / or a fourth intermediate is formed, the alkaline solution comprises an inorganic base and an organic base, wherein the inorganic base comprises at least one of sodium hydroxide and potassium hydroxide, and the organic base comprises at least one of sodium alkoxides or potassium alkoxides.

9. The method for preparing the chromatographic packing material for purifying mRNA according to claim 8, characterized in that, The step of bromine-adding the fourth intermediate specifically involves mixing the fourth intermediate with bromine in the presence of water for 1-3 hours, then adding a bromine removal agent and reacting for 3-6 hours. The bromine removal agent includes a sodium salt. The mass ratio of the fourth intermediate, bromine, and bromine removal agent is 1:0.1-0.5:0.5-1.0; The fourth intermediate of bromination was mixed with a buffer solution containing primers modified with 12-amine, and reacted at 20-30°C for 30-50 h in the presence of a neutral salt. The fourth intermediate of the bromine, containing a primer modified with 12 amine and a neutral salt, has a mass ratio of 1:0.005-0.01:0.1-0.

5.

10. The method for preparing the chromatographic packing material for purifying mRNA according to claim 9, characterized in that, The specific capping step involves coupling a fourth intermediate of a 12-amine modified primer with a capping agent in a buffer solution at a temperature of 40-60°C for 6-8 hours. The capping agent includes at least one selected from Tris base, ethanolamine, glutathione, serine, and threonine. The mass ratio of the fourth intermediate coupled with the 12-amine modified primer to the capping agent is 1:0.001-0.005; and / or, The sodium salt includes at least one of sodium formate, sodium acetate, sodium bicarbonate, and sodium carbonate; and / or, The neutral salt includes at least one of sodium chloride, potassium sulfate, and sodium sulfate; and / or, The oligomeric thymine in the primers is 18-25 bp in length.

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