Chromatographic material feedstock composition, chromatographic material and methods of making and using same, monolithic column and chromatography stack

Abstract

The application discloses a chromatography material raw material composition, a chromatography material and a preparation method and application thereof, a whole column and a chromatography pile. The chromatography material raw material composition, with the total weight of raw material A and pore former B being 100%, comprises the following components: 20%-70% of raw material A; 30%-80% of pore former B; 0.1 ‰-1 ‰ of catalyst C; and the raw material A comprises a substance containing a polycyclic epoxy group. The chromatography material composition of the application can solve the pain points of the application of the current chromatography microsphere filler on biological macromolecules. The chromatography material has the advantages of uniform internal structure, good preparation reproducibility, high toughness, modifiable functional ligand and high hydrophilicity, and is more suitable for the separation and purification of biological molecules.

Description

TECHNICAL FIELD

[0001] The present application particularly relates to a chromatography material raw material composition, a chromatography material and a preparation method and application thereof, a monolithic column and a chromatography stack. BACKGROUND

[0002] At present, the separation and purification of biomolecules are basically carried out by using chromatography microsphere medium for surface ligand modification to realize different functions of separation and purification, so as to obtain relatively pure molecules as medical, diagnostic reagents, biochemical process raw materials, food, cosmetic additives and the like. The chromatography microspheres are highlighted in the bottleneck of the field of purification of biological macromolecules (such as plasmid DNA, mRNA, viral vectors, exosomes, antibody conjugated drugs (ADC), complex high molecular proteins and the like). The specific defects are as follows:

[0003] 1. The pore distribution of the chromatography microspheres is small, which leads to that part of the macromolecules cannot effectively enter the middle and deep parts of the pores, the utilization rate of the ligand is low, and the processing capacity is low;

[0004] 2. The porous structure of the chromatography microspheres has obvious diffusion effect, and it is necessary to increase the retention time to fully diffuse to the pore surface of the porous microspheres to reach the theoretical specific surface value, and the process cycle is long;

[0005] 3. The internal pore structure of the chromatography microspheres is easy to produce vortex effect, thereby producing large shear force, and the porous structure of the chromatography microspheres contains dead hole structure, which produces the phenomenon of easy combination and difficult elution in local, and can lead to low yield of effective active biological macromolecules (such as slow virus, herpes virus, exosome and larger nucleic acid molecules), and the yield is even as low as 10-20%;

[0006] 4. During the use of the chromatography microspheres, packing is needed to prevent breakage and avoid pollution and other factors, which increases the cost of biological medicine processing and the risk of batch stability;

[0007] 5. The production cost of the chromatography microspheres is high, and the batch stability of the microspheres has always been a pain point of domestic chromatography fillers. Controlling the batch stability of the microspheres indirectly increases the processing cost of the microspheres, and finally increases the downstream production cost of the biological pharmaceutical companies.

[0008] The monolithic column is the fourth generation chromatography column, and its separation material has attracted wide attention in recent years. However, the existing polymer monolithic column is easy to form uneven internal structure in the preparation process, and still has the defects of uneven distribution of surface functional groups, poor adsorption performance, uneven structure and poor repeatability, thereby causing low column efficiency of the separation column and being limited in the application of analysis and separation.

[0009] Therefore, it is urgent to develop a new monolithic column material to overcome the above defects. SUMMARY

[0010] The technical problem to be solved by the present application is to overcome the defects of low processing capacity, long process cycle, low yield of effective active biomacromolecule, poor batch stability and high production cost of the existing chromatography microspheres in the field of biomacromolecule purification, and overcome the disadvantages of uneven internal structure and poor repeatability of the existing monolithic column, and provide a chromatography material raw material composition, a chromatography material and a preparation method and application thereof, a monolithic column and a chromatography stack.

[0011] The present application provides the following technical solutions to solve the above technical problems.

[0012] The present application provides a chromatography material raw material composition, which comprises the following components based on the total weight of raw material A and pore-forming agent B being 100%:

[0013] 20%-70% of raw material A;

[0014] 30%-80% of pore-forming agent B;

[0015] 0.1‰-1‰ of catalyst C;

[0016] The raw material A comprises a substance containing a plurality of epoxy groups.

[0017] In the present application, the substance containing a plurality of epoxy groups refers to a substance containing two or more epoxy groups in the structure.

[0018] In the present application, the substance containing a plurality of epoxy groups is preferably a plurality of epoxy group glycidyl ether substances, or a plurality of epoxy group glycidyl ester substances.

[0019] In the present application, the glycidyl ether substance can be one or more of the monomers satisfying the following structural formula I,

[0020]

[0021] In structural formula I, R 1 is selected from a hydrogen atom, a substituted or unsubstituted C1-C 10 alkyl group, or an epoxy group;

[0022] n is a positive integer of 0-10;

[0023] L 1 is selected from an oxygen atom or a nitrogen atom;

[0024] L 2 is selected from an oxygen atom or a nitrogen atom;

[0025] X is selected from a substituted or unsubstituted C1-C 10 alkylene group, a substituted or unsubstituted C3-C 10 cycloalkylene group,

[0026] or, a benzene ring.

[0027] Preferably, the glycidyl ether type substance is one or more of the following monomers (a) - (m) :

[0028]

[0029] The structure (a) is the structure of glycerol triglycidyl ether.

[0030] The structure (b) is the structure of pentaerythritol tetraglycidyl ether.

[0031] The structure (j) is the structure of 1,4-butanediol diglycidyl ether.

[0032] The structure (k) is the structure of trimethylolethane triglycidyl ether.

[0033] The structure (1) is the structure of bisphenol A diglycidyl ether.

[0034] The structure (m) is the structure of bisphenol F diglycidyl ether.

[0035] In the present application, the glycidyl ether type substance can be one or more of the following polymers satisfying the following structural formula II,

[0036]

[0037] In the structural formula II, R 2 is selected from a hydrogen atom, a substituted or unsubstituted C1-C 10 alkyl group, or,

[0038] m is a positive integer of 2 - 40.

[0039] Preferably, the glycidyl ether type substance is one or more of the following polymers (1) - (4) :

[0040]

[0041] In the present application, the glycidyl ester type substance can be one or more of the following monomers satisfying the following structural formula III.

[0042]

[0043] In the structural formula III, Y is selected from a substituted or unsubstituted C1-C 10 alkylene group, a substituted or unsubstituted C3-C 10 cycloalkylene group, or a benzene ring.

[0044] Preferably, the glycidyl ester-based substance is one or more of the following monomers (A) and / or (B);

[0045]

[0046] In the present application, the substance containing multiple epoxy groups is preferably one or more of the following monomers (I) - (IV);

[0047]

[0048] In the present application, the substance containing multiple epoxy groups is preferably a multiple epoxy group glycidyl ether-based monomer and / or a multiple epoxy group glycidyl ether-based polymer.

[0049] The multiple epoxy group glycidyl ether-based monomer can be glycerol triglycidyl ether and / or pentaerythritol tetraglycidyl ether.

[0050] The multiple epoxy group glycidyl ether-based polymer can be polyglycerol glycidyl ether and / or poly-pentaerythritol tetraglycidyl ether.

[0051] In the present application, when the substance containing multiple epoxy groups is a mixture of the multiple epoxy group glycidyl ether-based monomer and the multiple epoxy group glycidyl ether-based polymer, the mixture volume ratio of the multiple epoxy group glycidyl ether-based monomer and the multiple epoxy group glycidyl ether-based polymer can be (0.1-9):1, for example 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 or 8:1.

[0052] In the present application, the molecular weight of the substance containing multiple epoxy groups can be 300-2000, for example 550 or 700.

[0053] In the present application, the weight percentage of the raw material A in the total weight of the raw material A and the porogen B is preferably 22%-70%, for example 25%, 30%, 35%, 40%, 45%, 50%, 55%, 58%, 65% or 68%.

[0054] In the present application, the raw material A can also include a substance containing a single epoxy group. The substance containing a single epoxy group refers to a substance containing one epoxy group in its structure.

[0055] The substance containing a single epoxy group is preferably a single epoxy group glycidyl ether-based substance, or a single epoxy group glycidyl ester-based substance.

[0056] The single epoxy group glycidyl ether-based substance can be a conventional substance containing one epoxy group in its structure in the art, and is preferably a phenyl glycidyl ether and / or a butyl glycidyl ether.

[0057] The monooxyglyceride substance can be one or more of phenyl glycidyl ether, butyl glycidyl ether, pentyl glycidyl ether, octyl glycidyl ether, octadecyl glycidyl ether, and naphthyl glycidyl ether, preferably phenyl glycidyl ether and / or butyl glycidyl ether.

[0058] In the present application, the porogen B is one or more of chemical solvents that are mutually soluble with the raw material A and the catalyst C, preferably one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO), and methyl tert-butyl ether, more preferably one or more of toluene, dioxane, and methyl tert-butyl ether, for example toluene and dioxane, toluene and methyl tert-butyl ether, or dioxane and methyl tert-butyl ether.

[0059] In the present application, when the porogen B is a mixture of two different substances, the volume ratio of the two different substances can be (0.1-9):1, for example 0.5:1, 1:1, 2:1, 3:1, 5:1, or 7:1.

[0060] In the present application, the weight percentage of the porogen B in the total weight of the raw material A and the porogen B is preferably 35%-80%, for example 40%, 45%, 50%, 60%, 65%, 70%, 75%, or 78%.

[0061] In the present application, the catalyst C can be a Lewis acid and / or a complex of a Lewis acid.

[0062] The Lewis acid can be one or more of aluminum trichloride, boron trifluoride, iron bromide, iron chloride, zinc chloride, niobium pentachloride, and sulfur trioxide, for example boron trifluoride.

[0063] The complex of the Lewis acid can be one or more of boron trifluoride diethyl ether, boron trifluoride acetonitrile, boron fluoride dimethyl carbonate, and boron trifluoride ethylamine.

[0064] In the present application, the weight percentage of the catalyst C in the total weight of the raw material A and the porogen B is preferably 0.3‰-1‰, for example 0.4‰, 0.5‰, 0.6‰, 0.7‰, 0.8‰, or 0.9‰.

[0065] In some preferred embodiments of the present application, the raw material A is a multi-oxyl group glycidyl ether monomer and / or a multi-oxyl group glycidyl ether polymer; and the porogen B is one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO), and methyl tert-butyl ether.

[0066] In some preferred embodiments of the present application, the starting material A is a polyglycerol triglycidyl ether and a mono-epoxy glycidyl ether monomer; the porogen B is one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO), and methyl tert-butyl ether.

[0067] In some preferred embodiments of the present application, the starting material A is a polyglycerol triglycidyl ether and a mono-epoxy glycidyl ether monomer; the porogen B is one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO), and methyl tert-butyl ether; the mono-epoxy glycidyl ether monomer is preferably a phenyl glycidyl ether and / or a butyl glycidyl ether.

[0068] In some preferred embodiments of the present application, the starting material A is a polyglycerol triglycidyl ether and a mono-epoxy glycidyl ether monomer; the porogen B is one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO), and methyl tert-butyl ether; the mono-epoxy glycidyl ether monomer is preferably a phenyl glycidyl ether and / or a butyl glycidyl ether.

[0069] In some preferred embodiments of the present application, the starting material A is a polyglycerol triglycidyl ether and a mono-epoxy glycidyl ether monomer; the porogen B is one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO), and methyl tert-butyl ether; the mono-epoxy glycidyl ether monomer is preferably a phenyl glycidyl ether and / or a butyl glycidyl ether.

[0070] 50-70% starting material A; the starting material A is a polyglycerol triglycidyl ether and a mono-epoxy glycidyl ether monomer; the mono-epoxy glycidyl ether monomer is a phenyl glycidyl ether and / or a butyl glycidyl ether;

[0071] 30-50% porogen B; the porogen B is "dioxane and methyl tert-butyl glycerol ether", or "toluene and dioxane".

[0072] In some preferred embodiments of the present application, the starting material A is a polyglycerol triglycidyl ether and a mono-epoxy glycidyl ether monomer; the porogen B is one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO), and methyl tert-butyl ether; the mono-epoxy glycidyl ether monomer is preferably a phenyl glycidyl ether and / or a butyl glycidyl ether.

[0073] 50-70% starting material A; the starting material A is a polyglycerol triglycidyl ether and a mono-epoxy glycidyl ether monomer; the mono-epoxy glycidyl ether monomer is a phenyl glycidyl ether and / or a butyl glycidyl ether;

[0074] 30-50% porogen B; the porogen B is "dioxane and methyl tert-butyl glycerol ether", or "toluene and dioxane".

[0075] 1% catalyst C.

[0076] In some preferred embodiments of the present application, the raw material composition of the chromatographic material comprises the following components, based on 100% of the total weight of raw material A and porogen B:

[0077] 60% of raw material A; the raw material A is polyglycerol tri- glycidyl ether with a molecular weight of 550 and butyl glycidyl ether, the volume ratio of the polyglycerol tri-glycidyl ether with a molecular weight of 550 and the butyl glycidyl ether being 2:1;

[0078] 40% of porogen B; the porogen B is dioxane and / or methyl tert-butyl glycerol ether; the volume ratio of the dioxane and the methyl tert-butyl glycerol ether being 1:1;

[0079] 1‰ of catalyst C.

[0080] In some preferred embodiments of the present application, the raw material composition of the chromatographic material comprises the following components, based on 100% of the total weight of raw material A and porogen B:

[0081] 60% of raw material A; the raw material A is polyglycerol tri- glycidyl ether with a molecular weight of 550 and butyl glycidyl ether, the volume ratio of the polyglycerol tri-glycidyl ether with a molecular weight of 550 and the butyl glycidyl ether being 2:1;

[0082] 40% of porogen B; the porogen B is dioxane and methyl tert-butyl glycerol ether; the volume ratio of the dioxane and the methyl tert-butyl glycerol ether being 1:1;

[0083] 1‰ of catalyst C; the catalyst is boron trifluoride etherate.

[0084] The present application also provides a chromatographic material comprising the raw material composition as described above;

[0085] The chromatographic material is provided with through holes, the average pore size of the through holes being 0.2-10 μm;

[0086] The porosity of the through holes is 30-80%;

[0087] The pore volume of the through holes is 0.5-2.5 mL / g;

[0088] The density of the epoxy ligand in the chromatographic material is 30-80 μmol / mL.

[0089] In the present application, the average pore size of the through holes is preferably 0.4-8 μm, for example 0.44 μm, 1.4 μm, 0.74 μm, 0.8 μm, 2 μm, 2.7 μm, 3.4 μm, 3.7 μm, 3.8 μm, 4.2 μm, 4.3 μm, 4.5 μm, 5.2 μm, 6 μm, 7 μm or 7.5 μm.

[0090] In the present application, the porosity of the through hole is preferably 40-68%, such as 45.9%, 46.5%, 46.8%, 48.8%, 49.7%, 59.7%, 58.6%, 60.1%, 60.4%, 65.1%, 65.9% or 66.3%.

[0091] In the present application, the pore volume of the through hole is preferably 0.5-1.8 mL / g, such as 0.53 mL / g, 0.58 mL / g, 0.59 mL / g, 0.62 mL / g, 0.86 mL / g, 1.2 mL / g, 1.33 mL / g, 1.39 mL / g, 1.42 mL / g, 1.5 mL / g, 1.6 mL / g, 1.7 mL / g or 1.78 mL / g;

[0092] In the present application, the density of the epoxy ligand in the chromatographic material is preferably 35-60 μmol / mL, such as 39 μmol / mL, 42 μmol / mL, 43 μmol / mL, 48 μmol / mL or 51 μmol / mL.

[0093] The present application also provides a preparation method of the chromatographic material, which comprises the following steps: pre-polymerizing the chromatographic material raw material composition as described above, and then shaping.

[0094] The temperature of the pre-polymerization is 0-10°C.

[0095] The temperature of the shaping is 25-60°C.

[0096] In the present application, the temperature of the pre-polymerization is preferably 2-8°C.

[0097] In the present application, the time of the pre-polymerization can be 15-60 min, preferably 20-40 min, such as 30 min.

[0098] In the present application, the temperature of the shaping is preferably 30-40°C.

[0099] In the present application, the time of the shaping can be 2-12 h, preferably 4-8 h.

[0100] In the present application, the shaping process is kept in a static state and is not affected by external force.

[0101] In the present application, the mold can be a conventional mold in the art, and the material of the mold can be a conventional material in the art, such as PE plastic. The shape of the mold can be a conventional shape in the art, such as a cylindrical shape.

[0102] In the present application, the shaping is preferably followed by post-treatment of the chromatographic material.

[0103] The post-treatment method can be conventional in the art, for example, the post-treatment is cleaning the chromatographic material with an alcohol organic reagent.

[0104] The alcohol organic reagent can be conventional in the art, for example, one or more of anhydrous methanol, ethanol and isopropyl alcohol.

[0105] The cleaning method can be conventional in the art, for example, vacuum cleaning or suction filtration leaching.

[0106] In some preferred embodiments of the present application, the preparation method of the chromatographic material comprises the following steps: after pre-polymerization of the chromatographic material raw material composition at 2-8°C, molding at 30-40°C, and then post-treatment.

[0107] In the chromatographic material raw material composition, the raw material A is a multi-epoxy group glycidyl ether polymer and a mono-epoxy group glycidyl ether monomer; and the pore-forming agent B is one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO) and methyl tert-butyl ether.

[0108] In some preferred embodiments of the present application, the preparation method of the chromatographic material comprises the following steps: after pre-polymerization of the chromatographic material raw material composition at 2-8°C, molding at 30-40°C, and then post-treatment.

[0109] In the chromatographic material raw material composition, the raw material A is a polyglycerol triglycidyl ether and a mono-epoxy group glycidyl ether monomer; and the pore-forming agent B is one or more of toluene, dioxane and methyl tert-butyl glycol ether; and the mono-epoxy group glycidyl ether monomer is preferably a phenyl glycidyl ether and / or a butyl glycidyl ether.

[0110] In some preferred embodiments of the present application, the preparation method of the chromatographic material comprises the following steps: after pre-polymerization of the chromatographic material raw material composition at 2-8°C, molding at 30-40°C, and then post-treatment.

[0111] In the chromatographic material raw material composition, the raw material A is a polyglycerol triglycidyl ether and a mono-epoxy group glycidyl ether monomer; and the pore-forming agent B is one or more of toluene, dioxane and methyl tert-butyl glycol ether; and the mono-epoxy group glycidyl ether monomer is preferably a phenyl glycidyl ether and / or a butyl glycidyl ether.

[0112] In some preferred embodiments of the present application, the method for preparing the chromatographic material comprises the steps of: after pre-polymerization of the raw material composition of the chromatographic material at 2-8°C, molding at 30-40°C, and then drying, to obtain the chromatographic material.

[0113] In some preferred embodiments of the present application, the raw material composition of the chromatographic material comprises the following components: 50-70% of raw material A; the raw material A is polyglycerol triglycidyl ether and a mon-epoxy glycidyl ether monomer; the mon-epoxy glycidyl ether monomer is phenyl glycidyl ether and / or butyl glycidyl ether; 30-50% of porogen B; the porogen B is "dioxane and methyl tert-butyl glycerol ether", or "toluene and dioxane".

[0114] In some preferred embodiments of the present application, the method for preparing the chromatographic material comprises the steps of: after pre-polymerization of the raw material composition of the chromatographic material at 2-8°C, molding at 30-40°C, and then drying, to obtain the chromatographic material.

[0115] In some preferred embodiments of the present application, the raw material composition of the chromatographic material comprises the following components: 50-70% of raw material A; the raw material A is polyglycerol triglycidyl ether and a mon-epoxy glycidyl ether monomer; the mon-epoxy glycidyl ether monomer is phenyl glycidyl ether and / or butyl glycidyl ether; 30-50% of porogen B; the porogen B is "dioxane and methyl tert-butyl glycerol ether", or "toluene and dioxane".

[0116] In some preferred embodiments of the present application, the method for preparing the chromatographic material comprises the steps of: after pre-polymerization of the raw material composition of the chromatographic material at 2-8°C, molding at 30-40°C, and then drying, to obtain the chromatographic material.

[0117] In some preferred embodiments of the present application, the raw material composition of the chromatographic material comprises the following components: 50-70% of raw material A; the raw material A is polyglycerol triglycidyl ether and a mon-epoxy glycidyl ether monomer; the mon-epoxy glycidyl ether monomer is phenyl glycidyl ether and / or butyl glycidyl ether; 30-50% of porogen B; the porogen B is "dioxane and methyl tert-butyl glycerol ether", or "toluene and dioxane"; 1‰ of catalyst C.

[0118] In some preferred embodiments of the present application, the method for preparing the chromatographic material comprises the steps of: after pre-polymerization of the raw material composition of the chromatographic material at 2-8°C, molding at 30-40°C, and then drying, to obtain the chromatographic material.

[0119] The raw material composition of the chromatographic material comprises the following components, based on the total weight of raw material A and porogen B being 100%: 60% of raw material A; the raw material A is polyglycerol tri- glycidyl ether with a molecular weight of 550 and butyl glycidyl ether, the volume ratio of the polyglycerol tri-glycidyl ether with a molecular weight of 550 and the butyl glycidyl ether being 2:1; 40% of porogen B; the porogen B is dioxane and / or methyl tert-butyl glycerol ether; the volume ratio of the dioxane and the methyl tert-butyl glycerol ether being 1:1; 1 ‰ of catalyst C.

[0120] In a preferred embodiment of the present application, the preparation method of the chromatographic material comprises the following steps: after the raw material composition of the chromatographic material is pre-polymerized at 4℃, the raw material composition is molded at 35℃.

[0121] The raw material composition of the chromatographic material comprises the following components, based on the total weight of raw material A and porogen B being 100%: 60% of raw material A; the raw material A is polyglycerol tri- glycidyl ether with a molecular weight of 550 and butyl glycidyl ether, the volume ratio of the polyglycerol tri-glycidyl ether with a molecular weight of 550 and the butyl glycidyl ether being 2:1; 40% of porogen B; the porogen B is dioxane and methyl tert-butyl glycerol ether; the volume ratio of the dioxane and the methyl tert-butyl glycerol ether being 1:1; 1 ‰ of catalyst C; the catalyst is boron trifluoride ether.

[0122] The present application also provides a chromatographic material prepared by the preparation method as described above.

[0123] The present application also provides a monolithic column comprising the chromatographic material as described above.

[0124] The present application also provides a chromatography stack comprising the raw material composition as described above, the chromatographic material as described above, or the monolithic column as described above.

[0125] The chromatography stack in the present application refers to a chromatography separation material of a chromatographic material containing a plurality of epoxy groups or a plurality of hydroxyl groups.

[0126] The present application also provides an application of the raw material composition of the chromatographic material as described above, or the chromatographic material as described above, or the monolithic column as described above, or the chromatography stack as described above in the field of biological macromolecule purification.

[0127] In the present application, the biological macromolecule is generally a protein, a plasmid DNA, an mRNA, a viral carrier, an exosome, an ADC, or a complex high polymer protein.

[0128] On the basis of common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e. to obtain each preferred example of the present application.

[0129] The reagents and raw materials used in the present application are commercially available.

[0130] The positive progress effect of the present application is that:

[0131] 1. The raw materials contained in the chromatography material composition of the present application can be used to prepare chromatography materials with controllable through-pore channels (sub-micron-micron level) structure under the joint action of pore-forming agents and catalysts, and different ligand groups can be grafted through certain chemical coupling modification to realize chromatography function. The advantages can exactly solve the pain points of the current chromatography microsphere filler in the application of biological macromolecules, as follows:

[0132] (1) The pore channels of the chromatography material prepared by the present application are adjustable, sub-micron-micron level, and uniformly distributed, showing unimodal normal distribution, and the utilization rate of ligand is high;

[0133] (2) The pore channels of the chromatography material prepared by the present application are through-pores, without diffusion effect, without the need to increase the retention time, shorten the process cycle, without obvious vortex phenomenon, low shear force, and high effective activity yield of biological macromolecules;

[0134] (3) The chromatography material prepared by the present application has no influence on the loading of sample at high flow rate;

[0135] (4) The preparation process of the chromatography material of the present application is relatively simple, and batch stability is easy to achieve, without the need for column packing verification work, and can be directly used (plug and play type);

[0136] (5) The chromatography material prepared by the present application can be repeatedly used through batch verification, or can be used as disposable consumables, with high economic selectivity and low production cost.

[0137] 2. The chromatography material of the present application has uniform internal structure, good preparation reproducibility, high toughness, modified functional ligand, and high hydrophilicity, and is more suitable for the separation and purification of biological molecules. BRIEF DESCRIPTION OF DRAWINGS

[0138] Figure 1 is a schematic view of the forming tool of the chromatography material, wherein, Figure 1 A is a schematic view of the cross section of the forming tool of the chromatography material; Figure 1 B is a schematic view of the longitudinal section of the forming tool of the chromatography material.

[0139] Figure 2 is a scanning electron microscope image of the chromatography material prepared in Example 1.

[0140] Figure 3 is a scanning electron microscope image of the chromatography material prepared in Example 3.

[0141] Figure 4 is a scanning electron microscope image of the chromatography material prepared in Example 6.

[0142] Figure 5 Pore size distribution of the chromatographic material prepared in Example 2.

[0143] Figure 6 Pore size distribution of the chromatographic material prepared in Example 7.

[0144] Figure 7 BSA loading of the chromatographic material prepared in Example 6 at different flow rates. DETAILED DESCRIPTION

[0145] The application will be further described in the following examples without limiting the application to the described examples. The experimental methods in the following examples, if not otherwise specified, are selected according to the conventional methods and conditions, or according to the commercial instructions.

[0146] The reagents used in the following examples and comparative examples are all commercially available products in the art, unless otherwise specified.

[0147] Example 1

[0148] 1. Accurately weigh 30% of raw material A (polyglycerol glycidyl ether, Mr=550), 70% of porogen B (toluene and dioxane mixed at a volume ratio of 1:1), and 1‰ of catalyst C (boron trifluoride), mix thoroughly, and stir at 4°C for 30 min to obtain a prepolymer;

[0149] 2. Pour the stirred prepolymer into a PE plastic mold (self-made mold, shaped as a concentric circular cylinder, and the structural diagram is shown in Figure 1 ), and stand for 4 hours at a temperature of 35°C, maintaining static stability during the molding process;

[0150] 3. After the reaction is completed, add anhydrous methanol organic reagent to perform vacuum cleaning or vacuum filtration to rinse the residual liquid in the pores;

[0151] 4. After cleaning, soak in a 20% ethanol solution and store at low temperature (4°C) for standby use.

[0152] In Examples 2-7, the conditions listed in Table 1 below are the same as those in Example 1, except for the conditions listed in Table 1.

[0153] Table 1

[0154]

[0155]

[0156]

[0157] Note: A%, B%, C% in Table 1 represent the weight percentage of raw material A, porogen B, catalyst C in the total amount of "A+B".

[0158] Effect Examples

[0159] (1) Field emission scanning electron microscope test

[0160] Test object: chromatography material prepared in Examples 1, 3 and 6.

[0161] Test method: The test method is a conventional method in the art.

[0162] Test equipment: Germany-Zeiss sigma300, Oxford energy spectrum.

[0163] Test result: As shown in Figure 2 , Figures 2-4 , the scanning electron microscope images of the chromatography material prepared in Examples 1, 3 and 6 are shown respectively. Figures 2-4 It is shown that the chromatography material prepared in Examples 1, 3 and 6 all have pore structures, wherein, Figure 2 It is shown that the pore of the chromatography material prepared in Example 1 is the largest, Figure 4 It is shown that the pore of the chromatography material prepared in Example 6 is the smallest. It can be seen that the proportion of raw material A has an effect on the size of the pore, and the pore can be adjusted by controlling the proportion of raw material A.

[0164] (2) Pore size distribution, pore volume and porosity test

[0165] Test object: chromatography material prepared in Examples 1-13 and Comparative Examples 1-10.

[0166] Test method: mercury intrusion method.

[0167] Test equipment: USA-Micromeritics-autopore V 9620.

[0168] Test result: The pore uniformity of the chromatography material of Examples 2 and 7 can be visually seen from Figures 5-6 . Figures 5-6 The original drawing of the equipment test is shown, the data in the figure is the original data of the test calculation, and the vertical coordinate in the figure is the logarithm of the differential mercury intrusion amount, and the horizontal coordinate is the pore size converted by the equipment analysis) The normal distribution of the pore shows a single peak distribution. Figures 5-6 It is shown that the pore size distribution of the chromatography material prepared in Examples 2 and 7 shows a good normal distribution, and there is no obvious double peak (i.e. no pore size double distribution phenomenon); and the average pore size result is consistent with the electron microscope result, with the increase of the addition proportion of raw material A, the pore size of the prepared chromatography material decreases, the average pore size of the chromatography material obtained in Example 2 is 4.2 μm micron-sized macropore, and the average pore size of the chromatography material prepared in Example 7 is 0.74 μm sub-micron-sized macropore.

[0169] The specific data measured for Examples 1-13 and Comparative Examples 1-6 are shown in Table 2 below.

[0170] Table 2

[0171]

[0172]

[0173] Note: The average pore size is positively correlated with the pore volume and the porosity, and the optimal range of the pore volume is 0.5-1.8 mL / g, the optimal range of the average pore size is 0.4-6 μm, and the optimal range of the porosity is 40-68%.

[0174] From Table 2, it can be seen that, overall, the pore volume of the chromatographic material prepared in Examples 1-13 can reach 0.53-1.7 mL / g, the average pore size can reach 0.51-5.2 μm, and the porosity can be 45.9-67.1%. By adjusting the type of raw material A, the type of porogen B, and the relative proportions of raw material A and porogen B, the pore volume, average pore size, and porosity of the chromatographic material prepared in Examples 1-13 can be changed. The pore volume, average pore size, and porosity of Examples 6-7 are not significantly different and are basically equivalent.

[0175] Compared with Example 1, in Comparative Example 1, the content of raw material A is higher, and the prepared chromatographic material has no pores, which cannot meet the application requirements.

[0176] Compared with Example 2, in Comparative Example 2, the content of catalyst C is higher, and the prepared chromatographic material has no pores, which cannot meet the application requirements.

[0177] In Comparative Examples 3-4, raw material A only uses glycidyl ether monomers with a single epoxy group (butyl glycidyl ether in Comparative Example 3 and phenyl glycidyl ether in Comparative Example 4), and the prepared chromatographic material is in a viscous state, which cannot meet the application requirements.

[0178] Compared with Example 6, in Comparative Example 5, the temperature of the prepolymerization reaction is lower, and the prepared chromatographic material cracks, which cannot meet the application requirements.

[0179] Compared with Example 6, in Comparative Example 6, the temperature of the prepolymerization reaction is higher, and the prepared chromatographic material falls off, which cannot meet the application requirements.

[0180] Compared with Example 6, in Comparative Example 7, the temperatures of the prepolymerization reaction and molding are the same, both being 25°C, and the prepared chromatographic material falls off, which cannot meet the application requirements.

[0181] Compared with Example 6, in Comparative Example 8, the temperatures of the prepolymerization reaction and molding are the same, both being 5°C, and the prepared chromatographic material is in a viscous state, which cannot meet the application requirements.

[0182] Compared with Example 6, in Comparative Example 9, the molding temperature is lower, the prepared chromatographic material is peeled off, and cannot meet the application requirements.

[0183] Compared with Example 6, in Comparative Example 10, the molding temperature is higher, the prepared chromatographic material is non-porous, and cannot meet the application requirements.

[0184] (3) Epoxy ligand density test

[0185] Test object: chromatographic material prepared from Example 1-13 and Comparative Example 1-10 modified by epichlorohydrin.

[0186] Since the chromatographic material itself contains a small amount of epoxy ligand, direct determination cannot accurately quantify, and therefore, the chromatographic material needs to be first subjected to "epichlorohydrin modification".

[0187] "Epichlorohydrin modification" is a pretreatment of chromatographic material of Example 1-13 and Comparative Example 1-10 with epichlorohydrin before the test of epoxy ligand density. In the pretreatment, the reagents used and their amounts are as follows: epichlorohydrin: 10wt%; deionized water: 44wt%; dimethyl sulfoxide (DMSO): 44wt%; sodium hydroxide: 2wt%, the percentages are the percentages of each reagent in the total weight of the reagents; the mass ratio of chromatographic material to epichlorohydrin is 1:1; the pretreatment reaction temperature is 45°C; and the time is 10h. Except for the conditions specifically described above, other conditions can use the conventional conditions for the pretreatment in the art.

[0188] Test method: sodium thiosulfate-hydrochloric acid titration method.

[0189] After the chromatographic material of Example 1-13 and Comparative Example 1-10 modified by epichlorohydrin is washed clean with deionized water, it is placed in a sand core funnel and vacuum dried for 10min, then each 1 piece (about 0.5g) is placed in a ground mouth conical flask, 3mL of 1.3mol / L sodium thiosulfate and 1-2 drops of phenolphthalein indicator are added, the conical flask is sealed, and then placed at room temperature for 1h of reaction. The supernatant solution after reaction is titrated with 0.1mol / L hydrochloric acid standard solution until the red solution becomes colorless. According to the volume of hydrochloric acid standard solution consumed, the following formula is used to calculate the epoxy ligand density:

[0190] S = M HCl (V0-V1)*p / W

[0191] S: epoxy ligand density, mol / L;

[0192] M HCl : hydrochloric acid concentration, mol / L;

[0193] V0, V1: volume of HCl before and after titration, mL;

[0194] p: density of medium (1.2 g / mL);

[0195] W: mass of the whole block weighed.

[0196] Test equipment: acid-base titration device.

[0197] Test results: as shown in Table 3 below.

[0198] Table 3

[0199]

[0200]

[0201] Note: " / " in Table 3 represents that since the structure of the chromatographic material does not meet the requirements (for example, the chromatographic material has no through holes; the chromatographic material is in a viscous state; the chromatographic material falls off or drops slag; the chromatographic material has no holes, etc.), it is meaningless to continue to determine the data of the epoxy ligand density, so the data of the epoxy ligand density is not determined.

[0202] As shown in the data of Table 3 above, after modification by the conventional epoxidation modification method of epichlorohydrin, the epoxy group density on the prepared chromatographic material is generally at the level of 35-60 pmol / mL, which is comparable to the epoxy group density of chromatographic column filler microspheres, and can meet the use of chromatographic separation and analysis; and the size of the epoxy group density is positively correlated with the amount of raw material A used in the preparation process of the chromatographic material, and the epoxy value of the polymer or monomer in the used raw material A. By adjusting the type of raw material A, the type of porogen B, the relative proportion of the amount of raw material A and porogen B, the amount of catalyst, etc., the epoxy group ligand density of the chromatographic material prepared in Examples 1-13 will change. Among them, the epoxy group ligand density of Examples 6-7 is not much different, and is basically comparable. Specifically:

[0203] Compared with Example 1, in Comparative Example 1, the content of raw material A is higher, and the epoxy group ligand density of the prepared chromatographic material is only 6 pmol / mL, which cannot meet the application requirements.

[0204] Compared with Example 2, in Comparative Example 2, the content of catalyst C is higher, and the epoxy group ligand density of the prepared chromatographic material is only 8 pmol / mL, which cannot meet the application requirements.

[0205] In Comparative Examples 3-4, raw material A only uses glycidyl ether monomers with a single epoxy group (butyl glycidyl ether in Comparative Example 3 and phenyl glycidyl ether in Comparative Example 4), and the prepared chromatographic material is in a viscous state, and the epoxy group ligand density data cannot be measured.

[0206] In comparison with Example 6, in Comparative Example 5, the temperature of the prepolymerization is lower, the prepared chromatographic material is cracked, and the epoxy ligand density data thereof cannot be measured.

[0207] In comparison with Example 6, in Comparative Example 6, the temperature of the prepolymerization is higher, the prepared chromatographic material is peeled off, and the epoxy ligand density data thereof cannot be measured.

[0208] In comparison with Example 6, in Comparative Example 7, the temperature of the prepolymerization and the molding are both 25°C, the prepared chromatographic material is peeled off, and the epoxy ligand density data thereof cannot be measured.

[0209] In comparison with Example 6, in Comparative Example 8, the temperature of the prepolymerization and the molding are both 5°C, the prepared chromatographic material is in a viscous state, and the epoxy ligand density data thereof cannot be measured.

[0210] In comparison with Example 6, in Comparative Example 9, the temperature of the molding is lower, the prepared chromatographic material is peeled off, and the epoxy ligand density data thereof cannot be measured.

[0211] In comparison with Example 6, in Comparative Example 10, the temperature of the molding is higher, the prepared chromatographic material is non-porous, and the epoxy ligand density data thereof cannot be measured.

[0212] (4) Test of yield of biological macromolecules

[0213] Test object: chromatographic materials prepared in Examples 6 and 7.

[0214] Test method: The hydrophobic effect, chromatographic binding, and yield of bovine serum albumin after elution of the chromatographic materials prepared in Examples 6 and 7 were determined by using a chromatographic device and a chromatographic column (a 1 mL monolithic column device of BIA Company was used, and the chromatographic materials prepared in Examples 6 and 7 were arranged therein), and a lorry method for determining the protein content (i.e., an experimental method for determining the protein content by using a Folin-phenol reagent method). The chromatographic mobile phase was phosphate buffer PB (pH = 6.5) + 0.8 M ammonium sulfate for equilibrium liquid A and phosphate buffer PB (pH = 6.5) for elution liquid B. The flow rate of the chromatography was 1 mL / min. After 10 mL of the equilibrium liquid A was balanced, 30 mL of a sample (bovine serum albumin dissolved in the equilibrium liquid, with a concentration of 2 mg / mL) was injected, 5-10 mL of the equilibrium liquid was used to flush the chromatographic column, the elution liquid B was used for elution, the elution volume was 3-4 mL, and the elution liquid was collected for determination of the protein concentration.

[0215] Test device: Akata explore 100 chromatographic device.

[0216] Test result: As shown in Table 4 below.

[0217] Table 4

[0218]

[0219] From the data in Table 4, it can be seen that the chromatographic materials prepared in Examples 6 and 7 have a bovine serum albumin chromatographic yield of more than 90% (a yield of more than 90% in a single step is generally desirable); the bovine serum albumin yield of the chromatographic material prepared in Example 6 is 95%, and the bovine serum albumin yield of the chromatographic material prepared in Example 7 is 98%. This is because the hydrophobic effect of the phenyl group is stronger than that of the butyl group, i.e., under the same mobile phase, the phenyl group has stronger binding, a larger elution volume, and stronger elution strength, so under the same elution conditions, the butyl group has a higher yield. This indicates that the non-specific irreversible adsorption of the chromatographic material prepared in Example 7 is lower than that of the chromatographic material prepared in Example 6.

[0220] (5) Test of sample loading capacity at different flow rates

[0221] Test object: the chromatographic material prepared in Example 6.

[0222] Test method: The protein content was determined by the chromatographic device and the chromatographic column (a 1 mL monolithic column kit from BIA was used, and the chromatographic material prepared in Example 6 was arranged inside) and the lorry method. The amount of bovine serum albumin adsorbed by the chromatographic material prepared in Example 6 at different flow rates (i.e., the loading capacity) was determined. The mobile phase and the chromatographic method were the same as in (4) Test of biological macromolecule yield.

[0223] Test device: Akata explore 100 chromatographic device.

[0224] Test results: As shown in Table 5 and Figure 7 .

[0225] Table 5

[0226]

[0227] Note: DBC refers to the dynamic binding capacity of bovine serum albumin on the chromatographic material.

[0228] As can be seen from Table 5 and Figure 7 , an increase in flow rate has little effect on protein adsorption. Generally, a conventional chromatographic microsphere medium has a high flow rate within a retention time of 2 min (120 s), and generally needs to be retained for 3-4 min or even longer. The chromatographic material prepared in Example 6 has a shorter retention time, and in addition, when the flow rate is increased, the loading capacity of the conventional chromatographic microsphere packing decreases exponentially, while the change in the loading capacity of the chromatographic material prepared in Example 6 is relatively small.

[0229] (6) Test of batch stability

[0230] Test object: the chromatographic material prepared in Example 7.

[0231] Test method: by chromatography equipment and chromatography column (use BIA company 1 mL whole column equipment, inner device made of example 7 chromatography material for testing). Test the batch stability of chromatography process. The test sample is a mixture of bovine serum albumin (Yancheng Saibao Biological Technology Co., Ltd.) and human immunoglobulin IgG (Guangdong Shuanglin Biological Pharmaceutical Co., Ltd.) (prepared with the above equilibrium liquid A, the final concentration of the two proteins is 2 mg / mL), the mobile phase and chromatography method are the same as in (4) biological macromolecule yield test.

[0232] Test equipment: Akata explore 100 chromatography equipment.

[0233] Test results: as shown in table 6.

[0234] Table 6

[0235]

[0236] From the data in table 6, it can be seen that the chromatography process is stable between batches, the normal distribution curve is basically consistent, and the repeatability is high. The peaks within 0-8 min retention time are the characteristic peaks of bovine serum albumin, and the peaks within 8-13.5 min retention time are the characteristic peaks of human immunoglobulin IgG. Because the hydrophobicity of human immunoglobulin IgG is greater than that of bovine serum albumin, the peak is later. The peak time and peak size overlap well between batches (see table 6 for details), and the data in table 6 is slightly biased due to manual sampling during production. Automated sampling and chromatography process can eliminate this error.

Claims

1. A method for preparing a chromatographic material, characterized by, The steps include: forming the chromatographic material raw material composition after pre-polymerization of the chromatographic material raw material composition, and then shaping, and the chromatographic material raw material composition is shaped at a temperature of 30-40 DEG C. The pre-polymerization temperature is 2-8 DEG C. The shaping temperature is 30-40 DEG C. The chromatographic material raw material composition, with the total weight of the raw material A and the pore-forming agent B being 100%, comprises the following components: 20-70% of the raw material A; 30-80% of the pore-forming agent B; 0.1-1‰ of the catalyst C; The raw material A comprises a multi-epoxy group-containing substance and a single-epoxy group-containing substance. The multi-epoxy group-containing substance is a multi-epoxy group glycidyl ether substance; the multi-epoxy group glycidyl ether substance is polyglycerol glycidyl ether. The single-epoxy group-containing substance is a single-epoxy group glycidyl ether substance; the single-epoxy group glycidyl ether substance is phenyl glycidyl ether and / or butyl glycidyl ether. The pore-forming agent B is "toluene and dioxane", "toluene and methyl tert-butyl ether", or "dioxane and methyl tert-butyl ether"; the pore-forming agent B is a mixture of two different substances, and the volume ratio of the two different substances is 1:

1. The catalyst C is boron trifluoride etherate.

2. The method for preparing a chromatographic material according to claim 1, wherein The molecular weight of the multi-epoxy group-containing substance is 300-2000.

3. The method of preparing a chromatographic material of claim 1, wherein, The weight percentage of the raw material A in the total weight of the raw material A and the pore-forming agent B is 22-70%.

4. The method of preparing a chromatographic material of claim 1, wherein, The weight percentage of the pore-forming agent B in the total weight of the raw material A and the pore-forming agent B is 35-80%.

5. The method of preparing a chromatographic material of claim 1, wherein, The weight percentage of the catalyst C in the total weight of the raw material A and the pore-forming agent B is 0.3-1‰.

6. The method of preparing a chromatographic material of claim 1, wherein, The molecular weight of the multi-epoxy group-containing substance is 550 or 700.

7. The method of preparing a chromatographic material of claim 1, wherein, The weight percentage of the raw material A in the total weight of the raw material A and the pore-forming agent B is 25%, 30%, 35%, 40%, 45%, 50%, 55%, 58%, 65% or 68%.

8. The method of preparing a chromatographic material of claim 1, wherein, The weight percentage of the pore-forming agent B in the total weight of the raw material A and the pore-forming agent B is 40%, 45%, 50%, 60%, 65%, 70%, 75% or 78%.

9. The method of preparing a chromatographic material of claim 1, wherein, The weight percentage of the catalyst C in the total weight of the raw material A and the pore-forming agent B is 0.4‰, 0.5‰, 0.6‰, 0.7‰, 0.8‰ or 0.9‰.

10. The method of preparing a chromatographic material of claim 1, wherein, The chromatographic material raw material composition, with the total weight of the raw material A and the pore-forming agent B being 100%, comprises the following components: 50-70% of the raw material A; the raw material A is polyglycerol triglycidyl ether and "phenyl glycidyl ether and / or butyl glycidyl ether"; 30-50% of the pore-forming agent B; the pore-forming agent B is "dioxane and methyl tert-butyl glycidyl ether", or "toluene and dioxane".

11. The method of preparing a chromatographic material of claim 1, wherein, The chromatography material raw material composition includes the following components, taking the total weight of raw material A and porogen B as 100%: 60% of raw material A; the raw material A is polyglycerol triglycidyl ether with a molecular weight of 550 and butyl glycidyl ether, the volume ratio of the polyglycerol triglycidyl ether with a molecular weight of 550 and the butyl glycidyl ether is 2:1; 40% of porogen B; the porogen B is dioxane and methyl tert-butyl glycidyl ether; the volume ratio of the dioxane and the methyl tert-butyl glycidyl ether is 1:1; 1‰ of catalyst C; the catalyst is boron trifluoride etherate.

12. The method of preparing a chromatographic material of claim 1, wherein, The pre-polymerization time is 15-60 min.

13. The method for preparing a chromatography material according to claim 1, wherein: The molding time is 2-12 h.

14. The method for preparing a chromatography material according to claim 1, wherein: The molding process is kept in a static state and is not affected by external force.

15. The method for preparing a chromatography material according to claim 1, wherein: The chromatography material preparation method includes the following steps: after pre-polymerization at 2-8°C, the chromatography material raw material composition is molded at 30-40°C, and the process is completed; in the chromatography material raw material composition, the raw material A is polyglycerol triglycidyl ether and "phenyl glycidyl ether and / or butyl glycidyl ether"; the porogen B is "toluene and dioxane", "toluene and methyl tert-butyl ether", or "dioxane and methyl tert-butyl ether".

16. The method of preparing a chromatographic material of claim 1, wherein, The pre-polymerization time is 20-40 min.

17. The method for preparing a chromatography material according to claim 1, wherein: The molding time is 4-8 h.

18. The method of preparing a chromatographic material of claim 1, wherein, The chromatography material preparation method includes the following steps: after pre-polymerization at 2-8°C, the chromatography material raw material composition is molded at 30-40°C, and the process is completed; in the chromatography material raw material composition, taking the total weight of raw material A and porogen B as 100%, the chromatography material raw material composition includes the following components: 50-70% of raw material A; the raw material A is polyglycerol triglycidyl ether and "phenyl glycidyl ether and / or butyl glycidyl ether"; 30-50% of porogen B; the porogen B is "dioxane and methyl tert-butyl glycidyl ether", or "toluene and dioxane".

19. The method for preparing a chromatography material according to claim 1, wherein: The chromatography material preparation method includes the following steps: after pre-polymerization at 2-8°C, the chromatography material raw material composition is molded at 30-40°C, and the process is completed; in the chromatography material raw material composition, taking the total weight of raw material A and porogen B as 100%, the chromatography material raw material composition includes the following components: 60% of raw material A; the raw material A is polyglycerol triglycidyl ether with a molecular weight of 550 and butyl glycidyl ether, the volume ratio of the polyglycerol triglycidyl ether with a molecular weight of 550 and the butyl glycidyl ether is 2:1; 40% of porogen B; the porogen B is dioxane and methyl tert-butyl glycidyl ether; the volume ratio of the dioxane and the methyl tert-butyl glycidyl ether is 1:1; 1‰ of catalyst C; the catalyst is boron trifluoride etherate.

20. A chromatography material prepared by the preparation method in any one of claims 1-19.

21. A monolithic column characterized by, It includes the chromatography material in claim 20.

22. A chromatic stack, characterized in that, It includes the chromatography material in claim 20, or the monolithic column in claim 21.

23. Use of a chromatographic material according to claim 20 in the field of purification of biological macromolecules.

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