A high capacity temperature sensitive ion chromatography medium and a method for its preparation

Abstract

The application discloses a high-loading temperature-sensitive ion chromatography medium and a preparation method and application thereof. The chromatography medium is grafted with copolymer chains on the surface of a matrix. The copolymer chains are formed by double bond addition polymerization of monomer A and monomer B into random copolymers, wherein the molar ratio of monomer A to monomer B is (1-99):(99-1). The chromatography medium obtained by the application can realize ultra-high loading and fast elution under mild conditions through a reversible adjustment mechanism, thereby solving the technical contradiction between high loading and protein aggregation.

Description

Technical Field

[0001] This application belongs to the field of biological separation and purification, and in particular relates to a high-capacity temperature-sensitive ion chromatography medium and its preparation method. Background Technology

[0002] Human serum albumin (HSA) is a key protein in plasma for maintaining osmotic pressure and transporting substances, and it plays a strategically important role in the treatment of hemorrhagic shock, burns, and tumor ascites. Due to blood scarcity and safety concerns, the expression of recombinant human serum albumin (rHSA) using systems such as Pichia pastoris and rice cells has become the industry mainstream. Because the clinical single-dose dose of HSA is large (5-10 g), its pharmaceutical-grade purity requirement is extremely high (>99.999999%), leading to downstream purification processes typically involving multiple steps such as anion exchange, cation exchange, and hydrophobic chromatography, which is costly and cumbersome.

[0003] In industrial production, anion exchange chromatography is a crucial step. However, currently available commercial anion exchange chromatography media (such as the commonly used DEAE packing material) generally have low adsorption loadings, typically only 60–80 mg / mL, severely limiting production capacity. While existing technologies attempt to increase loading by improving the ligand density of the packing material, this strategy leads to excessively strong protein binding to the medium, requiring harsh conditions with high salt concentrations for complete elution. Studies have found that rHSA readily aggregates under high conductivity conditions, which not only reduces product yield but also introduces difficult-to-remove aggregated impurities into subsequent purification steps. Summary of the Invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a novel chromatography medium that can achieve ultra-high loading capacity and rapid elution under mild conditions through a reversible adjustment mechanism, thereby solving the technical contradiction between high loading capacity and protein aggregation.

[0005] In a first aspect, the present invention provides a high-load temperature-sensitive ion chromatography medium, wherein the chromatography medium is a matrix surface grafted with copolymer chains, wherein the copolymer chains are random copolymers formed by double bond addition polymerization of monomer A and monomer B, wherein the molar ratio of monomer A to monomer B is (1~99):(99~1).

[0006] Furthermore, the monomer A has the structure shown in Formula I:

[0007]

[0008] Formula I

[0009] Wherein, R1 is a hydrophilic group and R2 is a hydrophobic group;

[0010] R is selected from hydrogen or any substituted C1-C3 alkyl group;

[0011] R1 is selected from amide groups (including -CONH- or -N-CO- structures that are part of a lactam ring) or ether bonds (-O-).

[0012] R2 is selected from optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C3-C6 cycloalkyl-C1-C2-alkyl-, optionally substituted aryl, optionally substituted aryl-C1-C6-alkyl or optionally substituted heteroaryl-C1-C6-alkyl.

[0013] Alternatively, R1 and R2 may optionally form a ring. Preferably, when R1 is an amide group, the nitrogen atom in R1 and the terminal carbon atom of R2 can be connected to each other to form a 5- to 7-membered lactam ring.

[0014] Furthermore, the monomer B is composed of an active monomer C coupled with a functional group via a spacer arm, wherein the active monomer C contains a carbon-carbon double bond and a hydrophilic group;

[0015] Preferably, the hydrophilic group is an amide group (-CONH-) or an epoxy group (-CONH-). One or more of the following: , hydroxyl (-OH) or ether bond (-O-).

[0016] Furthermore, the spacer arm contains a reactive group and a linking group;

[0017] The reactive group contains one or more of carbon-carbon double bonds, epoxy groups, or halogens, and the linking group contains 2-15 carbon atoms, and also includes one or more hydroxyl groups, covalent bonds, and / or -NH-, -O-, or -S-.

[0018] Further, the functional group is an anion exchange functional group; preferably, the anion exchange functional group is a primary amino group (-NH2) or a secondary amino group (-NHR). 10 ), tertiary amino (-NR) 11 R 12 ) or quaternary ammonium group (-N⁺R 11 R 12 R 13 ), where R 10 R 11 R 12 R 13 Each is independently selected from one of the C1-C6 alkyl, hydroxyalkyl, or aminoalkyl groups.

[0019] Further, the matrix is ​​a hydrophilic matrix. Preferably, the hydrophilic matrix is ​​one or more of the following: hydrophilic modified polyacrylate microspheres, polymethacrylate microspheres, hydrophilic modified polystyrene-divinylbenzene microspheres, agarose microspheres, dextran microspheres, konjac syrup microspheres, or cellulose microspheres.

[0020] Secondly, the present invention provides a method for preparing the above-mentioned ion chromatography medium, comprising the following steps:

[0021] S1: Copolymerize temperature-sensitive monomer A and reactive monomer C onto the matrix;

[0022] S2: A spacer arm is grafted onto the product obtained in step S1;

[0023] S3: A functional group is coupled to the product obtained in step S2.

[0024] Further, step S1 involves the temperature-sensitive monomer A and the active monomer C containing carbon-carbon double bonds and hydrophilic groups forming a copolymer backbone on the matrix surface through a polymerization reaction under the action of an initiator.

[0025] Further, the molar ratio of monomer A to active monomer C is (1~99):(99~1); the mass ratio of temperature-sensitive monomer A to the matrix is ​​(0.1~3):1.

[0026] Thirdly, the present invention provides the application of the ion chromatography medium in the separation and purification of recombinant proteins, vaccines, antibodies, and human serum albumin.

[0027] Compared with the prior art, the present invention has the following significant advantages:

[0028] 1. The unique "comb-like" branched structure significantly reduces non-specific adsorption and greatly improves protein recovery rate.

[0029] This invention creatively employs a comb-like topology of "thermosensitive monomer and active monomer backbone - long spacer arms - functional groups". When the thermosensitive polymer backbone undergoes a phase transition (such as shrinkage / hydrophobication) to drive elution, it typically exposes strong hydrophobicity, easily leading to non-specific protein adsorption or denaturation. However, this invention, by grafting long spacer arms, effectively isolates the functionalized ligands (adsorption sites) from the thermosensitive backbone in space. This design constructs a "physical barrier," preventing adsorbed protein molecules (such as rHSA) from directly contacting the hydrophobic polymer backbone during elution, thereby significantly reducing irreversible protein adsorption and denaturation losses due to hydrophobic interactions and significantly improving the recovery rate of the target product.

[0030] 2. It achieves intelligent elution under "mild and low-salt" conditions, effectively solving the problem of protein aggregation.

[0031] Utilizing the temperature-sensitive properties of the main chain, the medium of this invention can achieve a conformational transition of the polymer chain through temperature regulation, characterized by "expansion-contraction." This volumetric phase change alters the spatial distribution density of ligands and the microenvironment, thereby generating a "repulsion" or "compression" effect that assists in the desorption of target proteins. This means that elution can be completed without the need for high-concentration salt solutions, fundamentally avoiding the risk of aggregation of sensitive proteins such as rHSA in high-conductivity environments, and ensuring the high purity and natural activity of the eluted products.

[0032] 3. The three-dimensional network adsorption layer provides ultra-high adsorption capacity.

[0033] This invention constructs a three-dimensional "polymer brush" layer on the surface of a medium using long-chain polymers formed through copolymerization. Compared to traditional planar grafting or short-chain modification, this structure provides more functionalization sites and a larger protein-accommodating space. Protein molecules can penetrate deep into the polymer chain for multi-point binding, thereby achieving high adsorption capacity and breaking through the 60-80 mg / mL capacity bottleneck of existing commercial products, significantly improving the efficiency of industrial production.

[0034] 4. Exhibits excellent mass transfer performance and elution kinetics.

[0035] The relaxed state of temperature-sensitive long chains is conducive to the rapid diffusion and capture of protein molecules; while during elution, the contraction of the chains not only provides physical driving force, but also rapidly reduces local binding force, resulting in sharp elution peaks and small volume, thus bringing about a significant concentration effect and reducing the processing pressure of subsequent processes. Attached Figure Description

[0036] Figure 1 The images show the binding loading of the chromatographic medium obtained in Example 1 at 20°C and 40°C.

[0037] Figure 2 This is a comparison of the dynamic loading of rHSA samples using the chromatography medium obtained in Example 2 and the commercially available PGMA (polyacrylate matrix) DEAE chromatography medium.

[0038] Figure 3 The images show the chromatograms and electrophoresis results of the purified rHSA obtained in Example 2 (lane 1 is the marker, lane 2 is the loaded sample, and lane 3 is the recovered sample).

[0039] Figure 4 The following are the chromatography and electrophoresis results of rHSA purified by commercially available PGMA (polyacrylate matrix) DEAE chromatography medium in Comparative Example 1 (lane 1 is the marker, lane 2 is the loaded sample, and lane 3 is the recovered sample).

[0040] Figure 5This is a comparison of HPLC chromatograms before and after purification of rHSA using the chromatographic medium obtained in Example 2. Detailed Implementation

[0041] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0042] The terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and not to describe a specific order or sequence. It should be understood that such use of data can be interchanged where appropriate so that embodiments of this application can be implemented in orders other than those illustrated or described herein, and the objects distinguished by "first," "second," etc., are generally of the same class and the number of objects is not limited; for example, a first object can be one or more. Furthermore, in the specification and claims, "and / or" indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] This invention creatively employs a comb-like topology of "thermosensitive monomer and active monomer main chain - long spacer arm - functional group". This specific structure allows the chromatography medium to achieve higher binding capacity while maintaining better recovery and improving the purity of the purified target.

[0045] Specifically, the high-load temperature-sensitive ion chromatography medium has copolymer chains grafted onto the matrix surface. These copolymer chains are formed by the addition polymerization of monomers A and B via double bonds to create a random copolymer. The molar ratio of monomer A to monomer B is (1~99):(99~1), preferably (50~99):(50~99:1), and more preferably (65~98):(2~35), such as (65~97):(3~35), (65~96):(4~35), (70~98):(2~30), (70~97):(3~30), (70~96):(4~30), (75~98):(2~25), ( 75~97):(3~25), (75~96):(4~25), (80~98):(2~20), (80~97):(3~20), (80~96):(4~20), (85~98):(2~15), (85~97):(3~15), (85~96):(4~15), for example 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4 or 97:3, but not limited to the listed values, other unlisted values ​​within this range also apply.

[0046] In one embodiment of the present invention, monomer A is a thermosensitive monomer, and monomer B is an active monomer composed of 'active monomer C-spacer arm-functional group', both of which are randomly distributed on the polymer backbone. The ratio of A to B is a key factor determining the performance of the medium.

[0047] If the content of A is too high, insufficient active sites will lead to a decrease in loading capacity; if the content of B is too high, the temperature-sensitive switching effect will be weakened, leading to incomplete elution or protein denaturation.

[0048] This invention cleverly balances the conflict between temperature-sensitive driving force and active capture capacity by constructing an A / B copolymer in a specific ratio. The polymer chains at this ratio not only possess sufficient adsorption sites for high loading, but also effectively shield the hydrophobic backbone from protein contact during elution through dramatic conformational contraction, thereby minimizing denaturation loss. Ultimately, this dual optimization of structure and composition enables the chromatography medium to achieve excellent target product recovery while maintaining high adsorption capacity.

[0049] In one embodiment of the present invention, the monomer A has the structure shown in Formula I:

[0050]

[0051] Formula I

[0052] Wherein, R1 is a hydrophilic group and R2 is a hydrophobic group;

[0053] R is selected from hydrogen or any substituted C1-C3 alkyl group;

[0054] R1 is selected from amide groups (including -CONH- or -N-CO- structures that are part of a lactam ring) or ether bonds (-O-).

[0055] R2 is selected from optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C3-C6 cycloalkyl-C1-C2-alkyl-, optionally substituted aryl, optionally substituted aryl-C1-C6-alkyl or optionally substituted heteroaryl-C1-C6-alkyl.

[0056] Alternatively, R1 and R2 may optionally form a ring. Preferably, when R1 is an amide group, the nitrogen atom in R1 and the terminal carbon atom of R2 can be connected to each other to form a 5- to 7-membered lactam ring.

[0057] In one embodiment of the present invention, the monomer B is composed of an active monomer C coupled with functional groups through a spacer arm, wherein the active monomer C contains a carbon-carbon double bond and a hydrophilic group;

[0058] Preferably, the hydrophilic group is an amide group (-CONH-) or an epoxy group (-CONH-). One or more of the following: , hydroxyl (-OH) or ether bond (-O-).

[0059] In one embodiment of the present invention, the spacer arm contains a reactive group and a linking group;

[0060] The reactive group contains one or more of carbon-carbon double bonds, epoxy groups, or halogens, and the linking group contains 2-15 carbon atoms, and also includes one or more hydroxyl groups, covalent bonds, and / or -NH-, -O-, or -S-.

[0061] In one embodiment of the present invention, the functional group is an anion exchange functional group; preferably, the anion exchange functional group is a primary amino group (-NH2) or a secondary amino group (-NHR). 10 ), tertiary amino (-NR) 11 R 12 ) or quaternary ammonium group (-N⁺R 11 R 12 R 13 ), where R 10 R 11 R 12 R 13 Each group is independently selected from one of C1-C6 alkyl, hydroxyalkyl, or aminoalkyl groups. Preferably, the anion exchange functional group is a primary amino, secondary amino, or tertiary amino group or its substitute.

[0062] In one embodiment of the present invention, the matrix is ​​a hydrophilic matrix. Preferably, the hydrophilic matrix is ​​one or more of the following: hydrophilic modified polyacrylate microspheres, polymethacrylate microspheres, hydrophilic modified polystyrene-divinylbenzene microspheres, agarose microspheres, dextran microspheres, konjac glycoside microspheres, or cellulose microspheres.

[0063] In one embodiment of the present invention, a high-loading temperature-sensitive ion chromatography medium, formed by the double-bond addition polymerization of monomers A and B to form a random copolymer, is grafted onto the matrix surface, as shown in Formula II:

[0064]

[0065] Formula II

[0066] In one embodiment of the present invention, M is a matrix;

[0067] In one embodiment of the present invention, R1 is an amide group (including -CONH- or -N-CO- structure as part of a lactam ring) or an ether bond (-O-).

[0068] In one embodiment of the present invention, R2 is an optionally substituted C1-C6 alkyl, an optionally substituted C3-C6 cycloalkyl, an optionally substituted C3-C6 cycloalkyl-C1-C2-alkyl-, an optionally substituted aryl, an optionally substituted aryl-C1-C6-alkyl, or an optionally substituted heteroaryl-C1-C6-alkyl.

[0069] Alternatively, when R1 is an amide group, the nitrogen atom in R1 can connect with the terminal carbon atom of R2 to form a 5- to 7-membered lactam ring.

[0070] In one embodiment of the present invention, R3 is one or more of an amide group (-CONH-), a hydroxyl group (-OH), or an ether bond (-O-), preferably one or more of an amide group (-CONH-) or an ether bond (-O-).

[0071] In one embodiment of the present invention, R4 is a spacer arm molecule; in another embodiment of the present invention, the spacer arm molecule comprises 2-15 carbon atoms, preferably 3-12 carbon atoms, and further comprises one or more hydroxyl groups, covalent bonds and / or -NH-, -O- or -S-. Preferably, R4 is selected from one or more of the molecules in Table 1.

[0072] Table 1

[0073]

[0074] In one embodiment of the present invention, R5 is a functional group; in another embodiment of the present invention, the functional group is an anion exchange functional group, such as an optionally substituted amino group (-N-), preferably, the functional group includes a primary amino group (-NH2) or a secondary amino group (-NHR). 10 ), tertiary amino (-NR) 11 R 12 ) or quaternary ammonium group (-N⁺R 11 R 12 R 13 ), where R 10 R 11 R 12 R 13 Each group is independently selected from one of C1-C6 alkyl, hydroxyalkyl, and aminoalkyl groups, preferably, the functional group is a tertiary amino (-NR) group. 11 R 12 ) or quaternary ammonium group (-N⁺R 11 R 12 R 13 ); preferably, R 10 R 11 R 12 R 13 Each is independently selected from one of the C1-C3 alkyl, hydroxyalkyl, or aminoalkyl groups.

[0075] In one embodiment of the present invention, n:m = (1~99):(99~1), preferably (50~99):(50~99:1), more preferably (65~98):(2~35), such as (65~97):(3~35), (65~96):(4~35), (70~98):(2~30), (70~97):(3~30), (70~96):(4~30), (75~98):(2~25), (75~97):(3~25), (75~96):(4~25), ( 80~98):(2~20), (80~97):(3~20), (80~96):(4~20), (85~98):(2~15), (85~97):(3~15), (85~96):(4~15), for example 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4 or 97:3, but not limited to the listed values, other unlisted values ​​within this range also apply.

[0076] In one embodiment of the present invention, the method for preparing the ion chromatography medium of the present invention includes the following steps:

[0077] S1: Copolymerize temperature-sensitive monomer A and reactive monomer C onto the matrix;

[0078] S2: A spacer arm is grafted onto the product obtained in step S1;

[0079] S3: A functional group is coupled to the product obtained in step S2.

[0080] In one embodiment of the present invention, the method for preparing the ion chromatography medium of the present invention includes the following steps:

[0081] S1: Copolymerize temperature-sensitive monomer A and reactive monomer C onto the matrix;

[0082] In one embodiment of the present invention, the temperature-sensitive monomer A contains carbon-carbon double bonds, hydrophilic groups and hydrophobic groups, and the hydrophilic-hydrophobic balance in its molecular structure can undergo a reversible change at a specific temperature, thereby endowing the polymer with low critical solution temperature (LCST) behavior.

[0083] Preferably, the hydrophilic group is an amide group (including -CONH- or -N-CO- structure as part of a lactam ring) or an ether bond (–O–); the hydrophobic group is an optionally substituted C1–C6 alkyl, optionally substituted C3–C6 cycloalkyl, optionally substituted C3–C6 cycloalkyl-C1–C2-alkyl, optionally substituted aryl, optionally substituted aryl-C1–C6-alkyl or optionally substituted heteroaryl-C1–C6-alkyl.

[0084] Preferably, the temperature-sensitive monomer A is selected from one or more of acrylamides, vinylamides, or (meth)acrylate temperature-sensitive monomers containing oligoethylene glycol side chains, such as: N-isopropylacrylamide (NIPAM), N-tert-butylacrylamide (NTBAM), N-n-propylacrylamide (NPAM), N-cyclohexylacrylamide (NCHAM), N,N-diethylacrylamide (DEAM), N-vinylcaprolactam (NVCL), or oligoethylene glycol methacrylate (such as tri(ethylene glycol) methyl ether methacrylate, TREGMA).

[0085] The preferred temperature-sensitive monomer is N-isopropylacrylamide (NIPAM).

[0086] In one embodiment of the present invention, the active monomer C contains a carbon-carbon double bond and a hydrophilic group; preferably, the hydrophilic group is an amide group (-CONH-) or an epoxy group (-CONH-). The active monomer is selected from one or more of hydroxyl (-OH) or ether bonds (-O-). More preferably, the active monomer is selected from one or more of methacrylate active monomers, methacrylamide active monomers, or acrylamide-derived monomers, such as hydroxyethyl methacrylate, hydroxypropyl methacrylate, 2-methyl-2-acrylate-2,3-dihydroxypropyl methacrylate, 2-hydroxybutyl methacrylate, glycidyl methacrylate, 2-hydroxypentyl methacrylate, 2,3-epoxybutyl methacrylate, polypropylene glycol methacrylate, hydroxyethyl acrylate (HEA), hydroxypropyl acrylate (HPA), N-(2-hydroxyethyl)- One or more of 2-methyl-2-acrylamide, N-(2-hydroxypropyl)methacrylamide, N,N-bis(2-hydroxyethyl)methacrylamide, N-[2-(2-hydroxyethoxy)ethyl]-2-methyl-2-acrylamide, N-(3-hydroxypropyl)-2-methylacrylamide, N-[2-(2-hydroxypropoxy)ethyl]-2-methylacrylamide, N,N-bis(3-hydroxypropyl)methacrylamide, N-(2-hydroxyethyl)acrylamide, N-hydroxymethylacrylamide (NMA) or N-(2-hydroxypropyl)acrylamide.

[0087] More preferably, the active monomer C is one or more of hydroxyethyl methacrylate, hydroxypropyl methacrylate, 2-methyl-2-acrylate-2,3-dihydroxypropyl methacrylate, 2-hydroxybutyl methacrylate, glycidyl methacrylate, N-(2-hydroxyethyl)-2-methyl-2-acrylamide, N-(2-hydroxypropyl)methacrylamide, N,N-bis(2-hydroxyethyl)methacrylamide, or N-[2-(2-hydroxyethoxy)ethyl]-2-methyl-2-acrylamide, more preferably 2-methyl-2-acrylate-2,3-dihydroxypropyl methacrylate, N-(2-hydroxyethyl)-2-methyl-2-acrylamide, or N-(2-hydroxyethyl)acrylamide.

[0088] S2: A spacer arm is grafted onto the product obtained in step S1;

[0089] In one embodiment of the present invention, the spacer arm contains a reactive group and a linking group; preferably, the reactive group contains one or more of a carbon-carbon double bond, an epoxy group, or a halogen; preferably, the linking group contains 2-15 carbon atoms, and further includes one or more hydroxyl groups, covalent bonds, and / or -NH-, -O-, or -S-.

[0090] More preferably, the spacer arm is selected from bifunctional spacer arm molecules containing epoxy groups and / or active spacer arm molecules containing allyl groups, such as epichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, glycerol triglycidyl ether, allyl glycidyl ether, 1,6-hexanediol diglycidyl ether, neopentyl glycol diglycidyl ether, glycerol diglycidyl ether, phenyl glycidyl ether, polyethylene glycol diglycidyl ether, 3,4-epoxy-1-butene, 1,2-epoxy-9-decene, or glycidyl methacrylate, allyl glycidyl ether, etc. The spacer arm molecule is selected from one or more of the following: hydroglycerol ether, methyl allyl glycidyl ether, 3-chloro-1-propene, allyl iodine, allyl chloride, 4-vinyl-1-cyclohexene dioxide, 4-bromo-1-butene, or 2-chloromethylpropene; more preferably, the spacer arm molecule is selected from one or more of the following: epichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, glycerol triglycidyl ether, allyl glycidyl ether, allyl iodine, 3,4-epoxy-1-butene, 1,2-epoxy-9-decene, or glycidyl methacrylate.

[0091] S3: The product obtained in step S2 reacts with a functionalizing reagent to couple functional groups.

[0092] In one embodiment of the present invention, the functionalizing reagent is an anion exchange reagent;

[0093] Preferably, the anion exchange reagent includes a weak anion exchange reagent and a strong anion exchange reagent, wherein the weak anion exchange reagent can introduce primary amino groups (-NH2) and secondary amino groups (-NHR). 10 ) or tertiary amino (-NR) 11 R 12 The strong anion exchange reagent can introduce quaternary ammonium groups (-N⁺R). 11 R 12 R 13 ); where R 10 R 11 R 12 R 13 Each is independently selected from one of C1-C6 alkyl, hydroxyalkyl, and aminoalkyl groups, preferably, R 10 R 11 R 12 R 13 Each is independently selected from one of the C1-C3 alkyl, hydroxyalkyl, or aminoalkyl groups.

[0094] More preferably, the anion exchange reagent can introduce a tertiary amino group (-NR). 11 R 12 ) or quaternary ammonium group (-N⁺R 11 R 12 R 13More preferably, the anion exchange reagent can introduce a tertiary amino group (-NR). 11 R 12 ), such as alkyl monoamine reagents (the molecule contains only one amino group (-NH2, -NHR)). 10 or -NR 11 R 12 Alkyl diamine reagents (containing two amino groups (which may be a combination of primary and secondary amino groups) and / or hydroxyl-containing alkylamine reagents (containing both an amino group and a hydroxyl (-OH) functional group). Examples include diethylamine, di-n-propylamine, N-methylcyclohexylamine, diisopropylamine, N,N-diethylethylenediamine, N,N-dimethylethylenediamine, N,N-diethylpropanediamine, N-methyl-N'-ethylethylenediamine, 3-diethylamino-1-propanol, 3-dimethylamino-1-propanol, 2-diethylaminoethanol, or 2-dimethylaminoethanol. More preferably, the functional group is selected from one or more of diethylamine, N,N-diethylethylenediamine, or 3-diethylamino-1-propanol.

[0095] In one embodiment of the present invention, the method for preparing the ion chromatography medium of the present invention includes the following steps:

[0096] S1 Matrix surface monomer copolymerization grafting backbone: Under the action of an initiator, temperature-sensitive monomer A and active monomer C undergo copolymerization reaction on the matrix surface to form a long polymer chain.

[0097] S2 Polymer Main Chain Branched Spacer Arms: Take the microspheres with polymer long chains on the surface prepared in step S1, add them together with the spacer arms into the solvent to react, and graft the spacer arm molecules onto the polymer main chain to form a highly branched brush-like side chain structure.

[0098] S3 Brush-like side chain coupling with functional groups: Take the microspheres with brush-like side chains obtained in step S2, add them to the solvent, and then add functionalizing reagents to react and complete the coupling of functional groups to obtain functionalized modified microspheres.

[0099] In one specific embodiment of the present invention, the method for preparing the ion chromatography medium of the present invention includes the following steps:

[0100] S1 Matrix surface monomer copolymerization grafting backbone: The matrix is ​​added to the solvent and reacted for a specific time at a set temperature and stirring rate. Under the action of the initiator, the temperature-sensitive monomer A and the active monomer C undergo a copolymerization reaction on the matrix surface to form a long polymer chain.

[0101] S2 Polymer Main Chain Branched Spacer Arms: Take the microspheres with polymer long chains on the surface prepared in step S1, add them together with the spacer arms to the solvent, and react under certain alkaline solution, temperature and stirring speed conditions to graft the spacer arm molecules onto the polymer main chain to form a highly branched brush-like side chain structure.

[0102] S3 Brush-like side chain coupling with functional groups: Take the microspheres with brush-like side chains obtained in step S2 and add them to the solvent. Then add the functionalizing reagent and stir evenly. Optionally, add alkali and react for a specific time at a set temperature and stirring rate to complete the coupling of functional groups and obtain functionalized modified microspheres.

[0103] In one embodiment of the present invention, the molar ratio of the temperature-sensitive monomer A to the active monomer C in step S1 is (1~99):(99~1), preferably (50~99):(50~99:1), and more preferably (65~98):(2~35), such as (65~97):(3~35), (65~96):(4~35), (70~98):(2~30), (70~97):(3~30), (70~96):(4~30), (75~98):(2~25), (75~97):(3~25), (7 5~96):(4~25), (80~98):(2~20), (80~97):(3~20), (80~96):(4~20), (85~98):(2~15), (85~97):(3~15), (85~96):(4~15), for example 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4 or 97:3, but not limited to the listed values, other unlisted values ​​within this range also apply.

[0104] In one embodiment of the present invention, the mass ratio of the temperature-sensitive monomer A to the matrix in step S1 is (0.1~3):1, preferably (0.5~2):1, more preferably (0.5~1):1, for example 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1 or 1:1, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0105] In one embodiment of the present invention, the set temperature in step S1 is 25~80℃, preferably 30~65℃, more preferably 40~65℃, and even more preferably 50~65℃, for example 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃ or 65℃, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0106] In one embodiment of the present invention, the specific duration mentioned in step S1 is 1 to 24 hours, preferably 1 to 10 hours, and more preferably 1 to 5 hours, such as 1 hour, 2 hours, 3 hours, 4 hours or 5 hours, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0107] In one embodiment of the present invention, the stirring speed in step S1 is 50~300 rpm, preferably 100~300 rpm, such as 100 rpm, 150 rpm, 200 rpm, 250 rpm or 300 rpm, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0108] In one embodiment of the present invention, the initiator described in step S1 is not limited, as long as it can provide an active center for the reaction and initiate the polymerization reaction of the monomer, it can be ammonium persulfate, sodium persulfate, potassium persulfate, benzoyl peroxide, cerium ammonium nitrate or cerium sulfate.

[0109] In one embodiment of the present invention, the solvent in step S1 is a polar solvent, which may be one or more of water, 1,4-dioxane, acetone, N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO).

[0110] In one embodiment of the present invention, the mass of the spacer arm in step S2 is 0.1 to 3 times that of the matrix, preferably 0.5 to 2 times, more preferably 0.5 to 1 times, for example 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times or 1 times, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0111] In one embodiment of the present invention, the alkaline solution mentioned in step S2 is not limited and can be a commonly used alkaline substance, such as sodium hydroxide (NaOH), potassium hydroxide (KOH), sodium carbonate (Na2CO3), or sodium bicarbonate (NaHCO3) solution.

[0112] In one embodiment of the present invention, the temperature in step S2 is 25~80°C, preferably 25~65°C, more preferably 25~40°C, for example 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0113] In one embodiment of the present invention, the specific duration mentioned in step S2 is 1 to 24 hours, preferably 1 to 10 hours, and more preferably 1 to 5 hours, such as 1 hour, 2 hours, 3 hours, 4 hours or 5 hours, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0114] In one embodiment of the present invention, the stirring speed in step S2 is 50~300 rpm, preferably 100~300 rpm, such as 100 rpm, 150 rpm, 200 rpm, 250 rpm or 300 rpm, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0115] In one embodiment of the present invention, the solvent in step S2 is a polar solvent, which may be one or more of water, dimethyl sulfoxide (DMSO), 1,4-dioxane, N,N-dimethylformamide (DMF), acetone, and tetrahydrofuran (THF).

[0116] In one embodiment of the present invention, the mass of the functionalizing reagent in step S3 is 0.1 to 3 times that of the matrix, preferably 0.1 to 1 times, more preferably 0.1 to 0.5 times, for example 0.1 times, 0.2 times, 0.3 times, 0.4 times or 5 times, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0117] In one embodiment of the present invention, the alkali mentioned in step S3 is not limited and can be a commonly used alkaline substance, such as sodium hydroxide (NaOH), potassium hydroxide (KOH), sodium carbonate (Na2CO3), or sodium bicarbonate (NaHCO3).

[0118] In one embodiment of the present invention, the temperature in step S3 is 25~80°C, preferably 25~65°C, more preferably 25~40°C, for example 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0119] In one embodiment of the present invention, the specific duration mentioned in step S3 is 1 to 24 hours, preferably 5 to 24 hours, and more preferably 10 to 24 hours, such as 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0120] In one embodiment of the present invention, the stirring speed in step S3 is 50~300 rpm, preferably 100~300 rpm, such as 100 rpm, 150 rpm, 200 rpm, 250 rpm or 300 rpm, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0121] Thirdly, the ion chromatography medium of the present invention is used in the separation and purification of recombinant proteins, vaccines, antibodies and human serum albumin, especially in the separation and purification of human serum albumin.

[0122] The specific embodiments and comparative examples of the present invention are listed below, but the present invention is not limited to the following examples.

[0123] Example 1

[0124] Schematic diagram of chromatography media preparation process:

[0125]

[0126] Step 1

[0127]

[0128] Step 2

[0129]

[0130] Step 3

[0131] Specific preparation process:

[0132] (1) Copolymerizing temperature-sensitive monomers and reactive monomers onto a matrix

[0133] 50 g of matrix-modified hydrophilic polyacrylate microspheres (60 μm) were weighed and added to 110 g of purified water. After stirring evenly, N2 was bubbled through for 30 min to remove oxygen. Then, 0.5 g of sodium persulfate initiator was added. After 5 min, 40 g (0.3535 mol) of N-isopropylacrylamide and 2.5 g (0.0156 mol) of 2-methyl-2-acrylate-2,3-dihydroxypropyl ester were added. The mass ratio of N-isopropylacrylamide, 2-methyl-2-acrylate-2,3-dihydroxypropyl ester to matrix was 8:0.5:10. The reaction was carried out under N2 protection at 60 °C and 200 rpm for 3 h. After the reaction, the microspheres were washed with water and anhydrous ethanol and then dried to obtain MonoPMPolyIAMDHM microspheres.

[0134] (2) Grafting spacer arms onto MonoPM PolyIAMDHM microspheres

[0135] Weigh 40g of purified water and 1.3g of NaOH, dissolve them, add 40g of DMSO, stir evenly, and then add the mixture to the MonoPM PolyIAMDHM microspheres obtained in step (1). After stirring, add 22g of epichlorohydrin dropwise at 35℃ and 200 rpm for about 1 hour. After the addition is completed, continue the reaction under these conditions for 3 hours. After the reaction is completed, wash with water and anhydrous ethanol and then dry to obtain MonoPM PolyEpo microspheres.

[0136] (3) Coupling functional groups

[0137] Take the MonoPM PolyEpo microspheres obtained in step (2), add 60 g of purified water and 10 g of N,N-diethylethylenediamine, stir evenly, and react at 30℃ and 150 rpm for 18 h. After the reaction is completed, wash with water and anhydrous ethanol and then dry to obtain the final MonoPM PolyDEAE microspheres, a high-load temperature-sensitive ion chromatography medium.

[0138] Example 2

[0139] Schematic diagram of chromatography media preparation process:

[0140]

[0141] Step 1

[0142]

[0143] Step 2

[0144]

[0145] Step 3

[0146] Specific preparation process:

[0147] (1) Copolymerizing temperature-sensitive monomers and reactive monomers onto a matrix

[0148] 50 g of matrix-modified hydrophilic polyacrylate microspheres (60 μm) were weighed and added to 110 g of purified water. After stirring evenly, N2 was bubbled through the mixture for 30 min to remove oxygen. Then, 0.5 g of sodium persulfate initiator was added. After 5 min, 30 g (0.2651 mol) of N-isopropylacrylamide and 5 g (0.0387 mol) of N-(2-hydroxyethyl)-2-methyl-2-acrylamide were added. The mass ratio of N-isopropylacrylamide, N-(2-hydroxyethyl)-2-methyl-2-acrylamide to matrix was 6:1:10. The reaction was carried out under N2 protection at 60 °C and 200 rpm for 3 h. After the reaction, the microspheres were washed with water and anhydrous ethanol and then dried to obtain MonoPM PolyIAMHME microspheres.

[0149] (2) Grafting spacer arms onto MonoPM PolyIAMHME microspheres

[0150] Weigh 40g of purified water and 1.6g of NaOH, dissolve them, add 40g of DMSO, stir evenly, and then add the mixture to the MonoPM PolyIAMHME microspheres obtained in step (1). After stirring, add 35g of ethylene glycol diglycidyl ether dropwise at 28℃ and 200 rpm for about 1 hour. After the addition is completed, continue the reaction under these conditions for 4 hours. After the reaction is completed, wash with water and anhydrous ethanol and then dry to obtain MonoPM PolyEpo microspheres.

[0151] (3) Coupling functional groups

[0152] Take the MonoPM PolyEpo microspheres obtained in step (2), add 60 g of purified water and 7.3 g of diethylamine, stir evenly, and react at 30℃ and 150 rpm for 3 h. Then add 30 g of 1.5 M NaOH and continue to react under the same conditions for 15 h. After the reaction is completed, wash with water and anhydrous ethanol and then dry to obtain the final MonoPM PolyDEAE microspheres, a high-load temperature-sensitive ion chromatography medium.

[0153] Example 3

[0154] Schematic diagram of chromatography media preparation process:

[0155]

[0156] Step 1

[0157]

[0158] Step 2

[0159]

[0160] Step 3

[0161] Specific preparation process:

[0162] (1) Copolymerizing temperature-sensitive monomers and reactive monomers onto a matrix

[0163] Weigh 50 g of matrix-modified hydrophilic polyacrylate microspheres (60 μm), add 110 g of purified water, stir evenly, and then purge with N2 for 30 min to remove oxygen. Then add 0.5 g of sodium persulfate initiator, and after 5 min, add 35 g (0.2515 mol) of N-vinylcaprolactam and 15 g (0.1303 mol) of N-(2-hydroxyethyl)acrylamide. The mass ratio of N-vinylcaprolactam, N-(2-hydroxyethyl)acrylamide to matrix is ​​7:3:10. Under N2 protection, the reaction is carried out at 60 °C and 200 rpm for 3 h. After the reaction, wash with water and anhydrous ethanol and then dry to obtain MonoPM PolyVCAHEM microspheres.

[0164] (2) Grafting spacer arms onto MonoPM PolyIVCAHEM microspheres

[0165] Weigh 40 g of purified water and 1.6 g of NaOH, dissolve them, add 40 g of DMSO, stir evenly, and then add the mixture to the MonoPM PolyVCAHEM microspheres obtained in step (1). After stirring, add 35 g of allyl iodine dropwise at 28 °C and 200 rpm for about 1 h. After the addition is completed, continue the reaction under these conditions for 4 h. After the reaction is completed, wash with water and anhydrous ethanol and then dry to obtain MonoPM PolyEne microspheres.

[0166] (3) Coupling functional groups

[0167] Take the MonoPM PolyEne microspheres obtained in step (2), add 100 g of purified water and 6 g of bromine, stir at 150 rpm for 2 h at room temperature, wash away the unreacted bromine with water and then dry, add 60 g of purified water and 7.3 g of 3-diethylamino-1-propanol, stir evenly, react at 30 °C and 150 rpm for 3 h, add 30 g of 1.5 M NaOH, and continue to react under these conditions for 15 h. After the reaction is completed, wash with water and anhydrous ethanol and then dry to obtain the final MonoPM PolyDEAE microspheres, a high-load temperature-sensitive ion chromatography medium.

[0168] Example 4

[0169] In step (1) of Example 2, the amounts of the thermosensitive monomer N-isopropylacrylamide and the active monomer N-(2-hydroxyethyl)-2-methyl-2-acrylamide were adjusted to 32g (0.2828mol) and 3g (0.023mol), respectively, with a molar ratio of approximately 92.5:7.5. Other process steps and conditions were the same as in Example 2.

[0170] Example 5

[0171] In step (1) of Example 2, the amounts of the thermosensitive monomer N-isopropylacrylamide and the active monomer N-(2-hydroxyethyl)-2-methyl-2-acrylamide were adjusted to 27g (0.2386mol) and 8g (0.062mol), respectively, with a molar ratio of approximately 79.4:20.6. Other process steps and conditions were the same as in Example 2.

[0172] Comparative Example 1

[0173] Commercially available anion exchange chromatography media, such as Bio-Rad (Macro-Prep DEAE Resin), Tosoh Bioscience (TOYOPEARL GigaCap DEAE-650), and Sepax Technologies (Generik DEAE, Monomix HC60-DEAE), are all prepared using methacrylate polymers (partially hydroxylated and hydrophilically modified) as the backbone, bonded with DEAE (diethylaminoethyl) functional groups. The commercially available DEAE (TOYOPEARL GigaCap DEAE-650) packing material was selected as Comparative Example 1. The structural diagram of the medium is shown below:

[0174]

[0175] Comparative Example 2

[0176] In step (1) of Example 2, the amounts of the thermosensitive monomer N-isopropylacrylamide and the active monomer N-(2-hydroxyethyl)-2-methyl-2-acrylamide were adjusted to 20g (0.1747mol) and 15g (0.1161mol), respectively, with a molar ratio of approximately 60.3:39.7. Other process steps and conditions were the same as in Example 2.

[0177] Comparative Example 3

[0178] In step (1) of Example 2, the amounts of the thermosensitive monomer N-isopropylacrylamide and the active monomer N-(2-hydroxyethyl)-2-methyl-2-acrylamide were adjusted to 34g (0.300mol) and 0.5g (0.004mol), respectively, with a molar ratio of approximately 98.7:1.3. Other process steps and conditions were the same as in Example 2.

[0179] Table 2 Comparison of raw materials and dosages between the examples and comparative examples

[0180]

[0181] The effectiveness of the chromatography media obtained in the examples and comparative examples was verified:

[0182] 1. Considering load capacity

[0183] (1) Determine the temperature sensitivity of the chromatography medium obtained in Example 1 (binding loading DBC at different temperatures)

[0184] The test conditions were as follows: column size: 4.6 × 50 mm ID; loading buffer: 20 mM Tris, pH 8.0; elution buffer: 20 mM Tris + 1 M NaCl, pH 8.0; sample: 3 mg / mL BSA dissolved in the loading buffer; the column and buffer were placed in a column oven at 20℃ or a water bath at 40℃ for constant temperature testing. Flow rate: 1 mL / min; detection wavelength: 280 nm.

[0185] Results: The binding loading of the chromatography medium obtained in Example 1 at 20°C and 40°C was as follows: Figure 1 It can be seen that the binding force of the filler changes significantly with temperature. When the temperature is below LCST, the surface polymer chains of this temperature-sensitive filler hydrophilically expand, exposing active sites to bind the target protein; when the temperature is above LCST, the polymer chains hydrophobically shrink, reducing the effective charge sites and achieving rapid desorption of the target protein. The medium of this invention can achieve the conformational transformation of the polymer chains through temperature regulation.

[0186] (2) The binding loading of rHSA by the chromatography media obtained in other examples and the commercially available anion exchange chromatography media of the comparative example was determined.

[0187] The test conditions were as follows: column size: 8.0 × 100 mm ID; equilibration buffer: 20 mM HAc-NaAc, pH 5.7; elution buffer: 150 mM HAc-NaAc, pH 5.7; regeneration buffer: 50 mM HAc-NaAc + 0.5 M NaCl, pH 5.7; CIP: 0.5 M NaOH; sample: 28 mg / mL HSA dissolved in the loading buffer. Flow rate: 5 min residence time; detection wavelength: 280 nm. 10% DBC: the loading volume was calculated based on 10% of the maximum flow-through UV absorbance.

[0188] Result: As Figure 2 (Comparison of test results of market fillers in Example 2 and Comparative Example 1) and Table 3.

[0189] 2. Purity (detected by high performance liquid chromatography (HPLC) and SDS-PAGE electrophoresis)

[0190] The test conditions were as follows: column size: 8.0 × 100 mm ID; equilibration buffer: 20 mM HAc-NaAc, pH 5.7; elution buffer: 150 mM HAc-NaAc, pH 5.7; sample: 28 mg / mL HSA dissolved in the loading buffer. The loading volume was 80% of 10% DBC. Flow rate: 5 min residence time; detection wavelength: 280 nm. During elution, the column oven was adjusted to 40℃, and the elution buffer was placed in a 40℃ constant temperature water bath.

[0191] Result: As Figures 3-5 And Table 3.

[0192] Table 3

[0193]

[0194] Recovery rate = (rHSA peak area in eluent * elution volume) / (rHSA peak area in loading solution * loading volume) * 100%

[0195] As per the instruction manual Figure 2 As shown in Table 3, compared with existing commercially available packing materials (Comparative Example 1), the chromatography medium prepared by the present invention exhibits a significantly higher dynamic binding capacity; at the same time, the target protein recovery rate is greatly improved during the elution stage, proving that the chromatography medium of the present invention has excellent mass transfer performance and adsorption / desorption efficiency.

[0196] By comparing the instruction manual appendix Figure 3 (Example 2) and Figure 4 The chromatograms and SDS-PAGE results of the commercially available product show that, in Example 2, the eluted fraction purified by the corresponding chromatographic medium (lane 3) exhibits only a clear, single main band at the target molecular weight position, with a clean background and no obvious impurities; while the commercially available product still shows obvious impurities. Therefore, the chromatographic medium described in Example 2 can significantly improve the protein purity of the eluted fraction, effectively remove non-target impurities, and its purification effect is significantly better than that of the commercially available product in Comparative Example 1.

[0197] Figure 5The comparative HPLC chromatograms before and after HSA purification using the chromatographic medium in Example 2 further corroborate this result: the medium of the present invention can significantly improve the monomer purity of the target protein and effectively reduce the content of impurities such as polymers. Specifically, the target protein monomer peak is located at the retention time RT = 8.046 min in the chromatogram. After purification, the sample contains only this target monomer peak, and the peak areas of all impurity peaks are significantly reduced, demonstrating a clear impurity removal effect.

[0198] Examples 1-3 use different raw materials to prepare chromatographic media with different structures. As shown in Table 3, each medium exhibits good binding capacity, purity and recovery rate.

[0199] This invention further investigated the effect of the molar ratio of temperature-sensitive monomers and reactive monomers in the final chromatographic medium on its performance. Table 3 shows that when the molar ratio of temperature-sensitive monomers to reactive monomers is controlled within the range of (65~98):(2~35) (as in Examples 1~5), the resulting chromatographic medium exhibits good performance in terms of binding capacity, purity, and recovery rate. However, when the proportion of temperature-sensitive monomers is too high and the amount of reactive monomers is too low (as in Comparative Example 3), the resulting medium has an extremely low binding capacity, making effective chromatographic separation impossible. Conversely, when the proportion of temperature-sensitive monomers is too low (as in Comparative Example 2), although the binding capacity of the medium increases, the purification effect deteriorates, polymer residues are high, and the recovery rate decreases significantly.

[0200] In summary, by adjusting the ratio of temperature-sensitive monomers to active monomers, the chromatographic medium can be effectively endowed with temperature-sensitive properties, enabling it to achieve high binding capacity, excellent purification effect, and high recovery rate under mild conditions.

[0201] The chromatography medium provided by this invention possesses the triple advantages of high binding capacity, high recovery rate, and high purity, and performs particularly well in removing key impurities such as polymers. Furthermore, thanks to its unique temperature-sensitive mechanism, this medium can achieve rapid elution under mild conditions, effectively protecting the activity of biomolecules. The overall process is simple and quick, possessing significant industrial application value.

[0202] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A high-capacity temperature-sensitive ion chromatography medium, characterized in that, The chromatography medium has copolymer chains grafted onto the matrix surface. The copolymer chains are random copolymers formed by the double bond addition polymerization of monomer A and monomer B, wherein the molar ratio of monomer A to monomer B is (1~99):(99~1).

2. The ion chromatography medium according to claim 1, characterized in that, The monomer A has the structure shown in Formula I: Formula I Wherein, R1 is a hydrophilic group and R2 is a hydrophobic group; R is selected from hydrogen or any substituted C1-C3 alkyl group; R1 is selected from amide groups (including -CONH- or -N-CO- structures that are part of a lactam ring) or ether bonds (-O-). R2 is selected from optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C3-C6 cycloalkyl-C1-C2-alkyl-, optionally substituted aryl, optionally substituted aryl-C1-C6-alkyl or optionally substituted heteroaryl-C1-C6-alkyl. Alternatively, R1 and R2 may optionally form a ring. Preferably, when R1 is an amide group, the nitrogen atom in R1 and the terminal carbon atom of R2 can be connected to each other to form a 5- to 7-membered lactam ring.

3. The ion chromatography medium according to claim 1, characterized in that, The monomer B is composed of an active monomer C coupled with functional groups through a spacer arm, and the active monomer C contains a carbon-carbon double bond and a hydrophilic group. Preferably, the hydrophilic group is an amide group (-CONH-) or an epoxy group (-CONH-). One or more of the following: , hydroxyl (-OH) or ether bond (-O-).

4. The ion chromatography medium according to claim 3, characterized in that, The spacer arm contains reactive groups and linking groups; The reactive group contains one or more of carbon-carbon double bonds, epoxy groups, or halogens, and the linking group contains 2-15 carbon atoms, and also includes one or more hydroxyl groups, covalent bonds, and / or -NH-, -O-, or -S-.

5. The ion chromatography medium according to claim 4, characterized in that, The functional group is an anion exchange functional group; preferably, the anion exchange functional group is a primary amino group (-NH2) or a secondary amino group (-NHR). 10 ), tertiary amino (-NR) 11 R 12 ) or quaternary ammonium group (-N⁺R 11 R 12 R 13 ), where R 10 R 11 R 12 R 13 Each is independently selected from one of the C1-C6 alkyl, hydroxyalkyl, or aminoalkyl groups.

6. The ion chromatography medium according to claim 1, characterized in that, The matrix is ​​a hydrophilic matrix. Preferably, the hydrophilic matrix is ​​one or more of the following: hydrophilic modified polyacrylate microspheres, polymethacrylate microspheres, hydrophilic modified polystyrene-divinylbenzene microspheres, agarose microspheres, dextran microspheres, konjac syrup microspheres, or cellulose microspheres.

7. The method for preparing the ion chromatography medium according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Copolymerize temperature-sensitive monomer A and reactive monomer C onto the matrix. S2: A spacer arm is grafted onto the product obtained in step S1; S3: A functional group is coupled to the product obtained in step S2.

8. The method for preparing the ion chromatography medium according to claim 7, characterized in that, Step S1 involves the polymerization reaction of temperature-sensitive monomer A and active monomer C containing carbon-carbon double bonds and hydrophilic groups on the matrix surface under the action of an initiator to form a copolymer backbone.

9. The method for preparing the ion chromatography medium according to claim 8, characterized in that, The molar ratio of monomer A to active monomer C is (1~99):(99~1); the mass ratio of temperature-sensitive monomer A to the matrix is ​​(0.1~3):

1.

10. The use of the ion chromatography medium according to any one of claims 1 to 6, or the ion chromatography medium prepared by the preparation method according to any one of claims 7 to 9, in the separation and purification of recombinant proteins, vaccines, antibodies, and human serum albumin.

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