Chromatographic medium as well as preparation method and application thereof

Abstract

The invention discloses a chromatography medium, the structural formula of the chromatography medium is as shown in formula I. In the formula I, Q is a matrix; l comprises 1-10 carbon atoms and functional groups, and the functional groups comprise-NH-,-O-,-S-, covalent bonds and / or hydroxyl (-OH); r1 comprises a carboxylate radical (-COO-); r2 comprises an aromatic ring (-Ar); and the value range of n is 1-4. The chromatography medium disclosed by the invention has good effects of separating and purifying antibodies and proteins, and has excellent pressure resistance;

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological separation and purification, in particular to a chromatography medium and a preparation method and application thereof. BACKGROUND

[0002] In the field of biopharmaceuticals, large-scale purification of recombinant proteins and antibodies is a key link in the production process. In order to efficiently purify the target protein meeting the quality standards from a large amount of fermentation stock solution, while taking into account the economy of the overall process, the industry trend shows that using chromatography medium for capture and separation and purification is one of the most economical and effective strategies.

[0003] Taking monoclonal antibodies as an example, the traditional purification process usually includes three steps: first, capturing the target protein by affinity chromatography, and then sequentially performing cation exchange chromatography and anion exchange chromatography. However, this method has many steps and high production cost. In addition, the existing ion exchange chromatography medium usually covalently couples a single ligand to a polysaccharide matrix (such as agarose, cellulose), mainly relying on electrostatic interaction to capture target molecules. This way is prone to co-adsorption when facing impurities with similar charge properties in complex biological samples, which limits the resolution of purification and affects the purity and quality of the final product. SUMMARY

[0004] To solve the technical problems in the prior art, the present application provides a chromatography medium with both cation adsorption capacity and hydrophobic interaction. The chromatography medium not only has excellent compression resistance and high binding capacity, but also can effectively broaden its application range and be suitable for more scenarios. The structural formula of the chromatography medium is shown as formula I,

[0005]

[0006] In formula I, Q is a matrix;

[0007] L includes 1-10 carbon atoms and a functional group, the functional group including -NH-, -O-, -S-, a covalent bond and / or a hydroxyl group (-OH);

[0008] R1 includes a carboxylate group (-COO - );

[0009] R2 includes an aromatic ring (-Ar);

[0010] n is in the range of 1-4.

[0011] Further, the structure of R1 is -R3-COO - , and / or the structure of R2 is -R4-Ar.

[0012] Further, R3 is selected from covalently bonded, unsubstituted or substituted C1–C4 straight-chain or branched alkyl groups, unsubstituted or substituted C4–C6 cycloalkyl groups, and / or,

[0013] The R4 is selected from covalent, unsubstituted or substituted C1–C4 straight-chain or branched alkyl groups or -(CH2). m -O-, the value of m ranges from 1 to 4.

[0014] Furthermore, the aromatic ring (-Ar) is selected from unsubstituted or substituted phenyl, biphenyl, naphthalene or six-membered aromatic heterocycles;

[0015] Wherein, the substitution is performed by one or more C1–C4 straight-chain or branched alkyl groups, methoxy (-OCH3), methylthio (-SCH3), fluorine (-F), chlorine (-Cl), bromine (-Br), iodine (-I) or trifluoromethyl (-CF3);

[0016] Preferably, the six-membered aromatic heterocycle is selected from pyridine.

[0017] Furthermore, the matrix is ​​one or more of agarose microspheres, silica microspheres, polymethacrylate microspheres, dextran microspheres, glass beads, cellulose microspheres, polymer microspheres, or porous polymer microspheres.

[0018] Furthermore, the chromatography medium is selected from one of the following structures:

[0019]

[0020] Furthermore, the chromatography medium is formed by the coupling of a matrix and ligands;

[0021] The ligand is selected from one of the following ligands 1 to ligand 12 with the following structures.

[0022] Furthermore, the ligand density of the chromatography medium is 0.04–0.1 mmol / ml, preferably 0.049–0.094 mmol / ml.

[0023] On the other hand, the present invention provides a method for preparing the above-mentioned chromatography medium, comprising the following steps:

[0024] (1) Microsphere activation: The matrix, activator, and NaOH solution are reacted at 20-40℃ to obtain an activated microsphere suspension;

[0025] (2) Coupling ligands: The ligands are added to the activated microsphere suspension, the pH is adjusted to 10-12 and the reaction is carried out. The chromatography medium is obtained after the reaction is completed.

[0026] Further, the weight ratio of the matrix to the activator is (4-8):1, preferably (4-6):1;

[0027] The activator is selected from one or more combinations of epichlorohydrin, allyl glycidyl ether, allyl bromide, 4-vinylphenyl glycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, and neopentyl glycol diglycidyl ether.

[0028] Thirdly, the present invention provides the application of the above-mentioned chromatography medium in the separation and purification of antibodies and proteins.

[0029] In the chromatographic medium of this invention, R1 is a negatively charged group that can bind cations, thus performing cation chromatography; the aromatic ring in R2 has hydrophobic properties, allowing it to bind hydrophobic substances and perform hydrophobic chromatography; the sulfur atom is thiophilic; and some groups even have hydrogen bonding interactions. This invention constructs a multi-dimensional, high-intensity cation adsorption mechanism, significantly improving the separation efficiency and selectivity of cations in complex samples. It achieves high-load, high-selectivity purification of biomolecules, and is particularly suitable for the separation and purification of biomolecules (such as antibodies and recombinant proteins).

[0030] Simultaneously, this invention introduces linker arms (i.e., the structure shown in L and the structure composed of n -CH2- groups, etc.) on the surface of the matrix, which can reduce the steric hindrance of coupled macromolecules, improve the activity and utilization rate of macromolecules, and reduce non-specific interactions between macromolecules and the matrix surface. The linker arms include -NH-, -O-, -S-, covalent bonds and / or hydroxyl groups, which can then react with molecules containing thiol, amino, carboxyl groups, etc., significantly enhancing the reactivity of the matrix and overcoming the defect that traditional matrix active sites are difficult to utilize effectively.

[0031] In summary, the chromatography medium provided by this invention not only has excellent compressive strength and high binding capacity, but also achieves functionalization and efficient coupling through structural optimization, making it widely applicable, especially suitable for the separation and purification of macromolecular biopharmaceuticals. Attached Figure Description

[0032] Figure 1 This is a comparison chart of the pressure and flow rate of the chromatography medium prepared in Example 1 of the present invention with three other existing packing materials.

[0033] Figure 2 This is the breakthrough curve of the adsorption effect of the chromatographic medium prepared in Example 1 of the present invention on monoclonal antibodies. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] 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.

[0037] This application provides a chromatography medium that has an appropriate ligand density and good pressure resistance and binding capacity when separating and purifying antibodies and proteins.

[0038] Specifically, the structural formula of the chromatography medium is shown in Formula I.

[0039]

[0040] Wherein, Q represents the matrix; in one embodiment of the present invention, the matrix is ​​one or more of agarose microspheres, silica microspheres, polymethacrylate microspheres, dextran microspheres, glass beads, cellulose microspheres, polymer microspheres, or porous polymer microspheres. Agarose microspheres are preferred.

[0041] In one embodiment of the present invention, L is a spacer group comprising 2 to 8 carbon atoms and a functional group, wherein the functional group includes -NH-, -O-, -S-, covalent bonds and / or hydroxyl groups (-OH). Preferably, L is selected from -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2-, -O-CH2-CH(CH2-OH)-(O-CH2-CH(OH)-CH2)2-, -O-CH2-CH2-CH(CH2-OH)-(O-CH2-CH2-CH(OH)-CH2)2-, -O-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-, -O-CH2-CH(OH)-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH(OH)-CH2- or -CO-NH-C(CH3)2-CO-. More preferably, it is -O-CH2-, -O-CH2-CH(OH)-CH2- or -CO-NH-C(CH3)2-CO-.

[0042] In one embodiment of the present invention, R1 comprises a carboxylate group (–COO). - In one specific embodiment, the structure of R1 is -R3–COO. - Preferably, R3 is selected from covalently bonded, unsubstituted or substituted C1–C4 straight-chain or branched alkyl groups, and unsubstituted or substituted C4–C6 cycloalkyl groups. In one specific embodiment, when R3 is a substituted C4–C6 cycloalkyl group, its structure is -R5-R6-, wherein R5 is a C1–C4 straight-chain or branched alkyl group, and R6 is a C4–C6 cycloalkyl group. Preferably, R5 is a C1–C3 straight-chain alkyl group.

[0043] In one specific embodiment, the substitution of R3 is the substitution of one or more hydrogen atoms by any group, such as one or more C1–C4 straight-chain or branched alkyl groups, methoxy (-OCH3), methylthio (-SCH3), fluorine (-F), chlorine (-Cl), bromine (-Br), iodine (-I), or trifluoromethyl (-CF3).

[0044] In one specific embodiment, the C1–C4 straight-chain or branched alkyl groups in R3 and R5 are preferably C1–C3 straight-chain or branched alkyl groups, such as methyl, ethyl, or straight-chain or branched propyl.

[0045] The ranges C1–C4, C1–C3, C4–C6 mentioned in this invention include endpoints, that is, C1–C4 is C1, C2, C3 or C4; C4–C6 is C4, C5 or C6.

[0046] In one embodiment of the invention, R2 comprises an aromatic ring (-Ar); in a specific embodiment, the structure of R2 is -R4–Ar; preferably, R4 is selected from covalently bonded, unsubstituted or substituted C1–C4 straight-chain or branched alkyl groups or -(CH2). m -O-, the value of m ranges from 1 to 4, preferably from 1 to 3, for example 1, 2 or 3.

[0047] Preferably, R4 is selected from covalently bonded, unsubstituted C1–C4 straight-chain alkyl groups or -CH2-O-;

[0048] Preferably, Ar is selected from unsubstituted or substituted phenyl, biphenyl, naphthalene or six-membered aromatic heterocycles;

[0049] Wherein, the substitution is that one or more hydrogen atoms on the ring structure are replaced by one or more C1–C4 straight-chain or branched alkyl, methoxy (-OCH3), methylthio (-SCH3), fluorine (-F), chlorine (-Cl), bromine (-Br), iodine (-I) or trifluoromethyl (-CF3);

[0050] Preferably, the heteroatom of the six-membered aromatic heterocycle is N, and preferably, the six-membered aromatic heterocycle is selected from pyridine.

[0051] Specifically, the chromatography medium is formed by the coupling of a matrix and ligands, and more specifically, the coupling can be achieved through a spacer group L.

[0052] The ligands can be obtained by purchasing or by synthesizing using existing processes.

[0053] Taking the preparation method of ligand 1 as an example, the following preparation method can be used:

[0054] (1) Synthesis of intermediate 1: 1-methyl-2-vinyldisulfide (25 g), methanol (25 ml) and bromine (45.0 g) were added to a 250 ml three-necked flask, and the mixture was stirred and reacted at 28 °C for 2 hours. Then, ammonia (9.88 g) was added to the reaction system, and the mixture was stirred at 25 °C for 18 hours.

[0055] After the reaction was completed, the reaction mixture was poured into a round-bottom flask, the water was removed by rotary evaporation, and then extracted with dichloromethane. The crude product after the organic phase was dried by rotary evaporation was purified by column chromatography to obtain intermediate 1 (23.35 g).

[0056] (2) Synthesis of intermediate 2: Intermediate 1 (23.35 g), methanol (50 ml), and 4-methoxybenzaldehyde (29.17 g) were added to a 500 ml three-necked flask. Stirring was started, and the mixture was cooled to 0 °C to allow it to dissolve completely. Then, NaBH3CN (13.46 g) was added to the reaction system in portions, and the reaction mixture was heated to room temperature and stirred for 4 hours.

[0057] After the reaction was completed, the reaction mixture was poured into ice water under stirring, and then extracted with ethyl acetate. The crude product after the organic phase was dried by rotary evaporation was purified by column chromatography to obtain oily intermediate 2 (32.49 g).

[0058] (3) Synthesis of ligand 1: Add intermediate 2 (32.49 g), sodium carbonate (6.95 g), tetrahydrofuran (50 ml), and bromoacetic acid (27.30 ml) to a 500 ml three-necked flask, and start stirring at 40 °C for 5 hours.

[0059] After the reaction was completed, the reaction mixture was poured into ice water under stirring, and then extracted with dichloromethane. The crude product after the organic phase was dried by rotary evaporation was purified by column chromatography to obtain ligand 1 (35.53 g).

[0060] Taking the preparation method of ligand 2 as an example, the following preparation method can be used:

[0061] (1) Synthesis of intermediate 1: 1-methyl-2-vinyldisulfide (25 g), methanol (50 ml) and bromine (45.07 g) were added to a 250 ml three-necked flask, and the mixture was stirred and reacted at 28 °C for 2 hours. Subsequently, m-trifluorotoluidine (50.36 g) was added to the reaction system, and the mixture was stirred at 55 °C for 18 hours.

[0062] After the reaction was completed, the reaction mixture was poured into a round-bottom flask, the water was removed by rotary evaporation, and then extracted with dichloromethane. The crude product after the organic phase was dried by rotary evaporation was purified by column chromatography to obtain intermediate 1 (37.69 g).

[0063] (2) Synthesis of ligand 2: Intermediate 1 (37.69 g), carbonic acid (17.45 g), DCM (dichloromethane) (50 ml), DCC (N,N'-dicyclohexylcarbodiimide) (5.82 g) were added to a 250 ml three-necked flask, and the mixture was stirred at 25 °C for 18 hours.

[0064] After the reaction was completed, the reaction mixture was poured into ice water under stirring, and then extracted with dichloromethane. The crude product after the organic phase was dried by rotary evaporation was purified by column chromatography to obtain ligand 2 (34.13 g).

[0065] Other ligands can be prepared using the same methods as ligand 1 or ligand 2, or other methods commonly used in the field.

[0066] The synthesis route can be found in Table 1.

[0067] Table 1. Structure and synthetic route of ligands

[0068]

[0069]

[0070]

[0071] The specific embodiments and comparative examples of the present invention are listed below, but the present invention is not limited to the following examples.

[0072] The preparation method of the chromatography medium described in the examples is as follows:

[0073] (1) Microsphere activation: Agarose microspheres, epichlorohydrin and NaOH solution were reacted at 25-30℃ for 3 hours. After the reaction, the microspheres were washed with water to obtain an activated microsphere suspension.

[0074] The ratio of agarose microspheres to epichlorohydrin was 1g:0.2g, the NaOH solution was 19M, and the mass ratio of agarose microspheres to NaOH solution was 1:0.13.

[0075] (2) Ligand coupling: The ligand was added to the above activated microsphere suspension, and the pH was adjusted to 11 by adding NaOH solution (2M). The reaction mixture was reacted at 40-45℃ for 13 hours. After the reaction was completed, the mixture was washed with water to obtain the chromatography medium. The mass ratio of activated microspheres to ligands was 1:0.05.

[0076] Table 2. Structure of the chromatography medium of the present invention and corresponding ligands.

[0077]

[0078]

[0079] Effect verification 1 : Ligand density test

[0080] The testing method is as follows:

[0081] (1) Load the chromatography medium into the chromatography column and connect it to the peristaltic pump.

[0082] (2) Saturate the chromatography column with 100 mL of 1 mol / L sodium chloride + 0.1 mol / L sodium hydroxide.

[0083] (3) Wash the chromatography column with 100 mL of 1 mmol / L sodium hydroxide solution.

[0084] (4) Use 20 mL of 0.1 mol / L hydrochloric acid for ion exchange and collect the effluent in a 50 mL graduated cylinder.

[0085] (5) Wash the chromatography column with 50 mL of ultrapure water and collect the eluent in a 50 mL graduated cylinder.

[0086] (6) Dilute the collected solution to a 100 mL volumetric flask with ultrapure water, then transfer it to a 250 mL Erlenmeyer flask. Add 3 drops of methyl red indicator and titrate with a standardized 0.1 mol / L sodium hydroxide standard solution until the solution changes from pink to colorless and remains unchanged for 30 seconds. This is the titration endpoint. Record the volume of sodium hydroxide standard solution consumed, Vsample.

[0087] (7) Blank titration: Pipette 20 mL of 0.1 mol / L hydrochloric acid into an Erlenmeyer flask, dilute to 100 mL with ultrapure water, add 3 drops of methyl red indicator, and titrate with 0.1 mol / L sodium hydroxide standard solution. The titration endpoint is when the solution changes from pink to colorless and remains unchanged for 30 s. Record the volume V blank required for titration.

[0088] Calculate the carboxyl group density using the following formula, and use the carboxyl group density as the ligand density:

[0089]

[0090] C NaOH : The concentration of the standard sodium hydroxide solution obtained by calibration, in mol / L;

[0091] V 样品 : The volume of sodium hydroxide consumed when the titration of the sample reaches the endpoint, in mL;

[0092] V 空白 : mL;

[0093] V 填料 Volume of the compacted packing material, mL.

[0094] The test results are shown in Table 3 below:

[0095] Table 3

[0096]

[0097]

[0098] Ligand density refers to the number of immobilized ligands per unit volume or unit mass of a medium. It directly reflects the binding potential and separation efficiency of the medium for target molecules and is one of the core parameters for chromatography medium design.

[0099] The ligand density in a chromatography medium is a core indicator of its potential to bind target molecules, directly reflecting its binding capacity, separation selectivity, mass transfer efficiency, and process economy. High ligand density media are suitable for capturing high-capacity, low-concentration target analytes, while low ligand density media are suitable for high-precision, high-purity separation. In practical applications, an appropriate ligand density must be selected based on the characteristics of the target analyte (charge, molecular weight), process requirements (separation accuracy, recovery rate), and cost constraints to balance separation performance and economy.

[0100] The ligand density of the chromatography medium obtained by this invention is between 0.04 and 0.10 mmol / ml, which is considered "low to medium ligand density". This balances binding capacity and impurity removal, making it suitable for fine purification (such as removing host protein HCP) and avoiding co-elution of strongly bound impurities.

[0101] Effect verification 2: Determination of the resistance to compression

[0102] The chromatography media obtained in Examples 1-12 were compared with three other existing packing materials (from Bailinco (Lanzhou) New Materials Co., Ltd.). Existing packing material 1 was selected from... HR affinity packing series, existing packing 2 selected from Ion exchange packing series, the existing packing 3 is selected from The HR hydrophobic packing series was compared using pressure and flow rate, and the results are as follows: Figure 1 (The chromatography packing material of this patent is compared with the chromatography medium prepared in Example 1 and existing packing materials) and Table 4.

[0103] Table 4

[0104]

[0105]

[0106] The chromatography medium provided by this invention exhibits significant advantages in terms of mechanical strength, hydrodynamic properties, and chemical stability. The "compression resistance" of the chromatography medium is manifested as follows: at the same flow rate, the lower the pressure (or the higher the flow rate at the same pressure), the smaller the "flow resistance" the medium experiences in the fluid (water, sodium chloride solution), and the better its compression resistance; conversely, the higher the pressure, the weaker the compression resistance.

[0107] Combination Figure 1 As can be seen from the pressure-flow rate curves and the specific data in Table 4, under the same flow rate conditions, the operating pressure of the chromatography medium of this invention in both pure water and 1M NaCl solution is significantly lower than that of existing packing materials, indicating that it has superior mechanical strength and higher bed permeability. This characteristic not only helps to reduce the pressure resistance requirements of the chromatography system pump and reduce energy consumption, but also makes it possible to apply large-size chromatography columns, as its high permeability can effectively alleviate the problem of increased bed resistance during scale-up.

[0108] Furthermore, the operating pressure of all media under high ionic strength (e.g., 1M NaCl) conditions was higher than that under pure water conditions, indicating that high-salt solutions reduce the media's pressure resistance to some extent. However, the media of this invention maintained a lower pressure in sodium chloride solution, demonstrating excellent chemical tolerance and structural stability. The pressure resistance under aqueous conditions directly determines the physical stability of the media during high-flow-rate purification, while its performance under sodium chloride conditions relates to its tolerance in the high-salt elution stage. This is particularly crucial for removing weakly bound impurities and ensuring the binding efficiency of target molecules in the low-salt adsorption stage. In the purification processes of biomolecules such as antibodies, the media often needs to withstand both high flow rates (e.g., 300 cm / h) and high salt concentrations (e.g., 3M NaCl). The media of this invention exhibits superior overall performance under these dual challenges, not only improving the controllability and operating window of the purification process but also contributing to increased recovery rate and process repeatability of the target product.

[0109] In summary, the chromatography packing material of this invention is significantly superior to existing products in terms of pressure resistance, permeability, and chemical stability. It is particularly suitable for efficient and large-scale downstream purification processes of biomolecules under high flow rate and high salt concentration environments, and has important industrial application value.

[0110] Effect verification 3: Determination of the dynamic binding capacity (DBC)

[0111] The adsorption effect of the chromatographic medium on the monoclonal antibody (human 1gG) was characterized by 10% DBC. The DBC of the chromatographic medium obtained in Example 1 was measured. The detection procedure is as follows:

[0112] 1. Preparation before the experiment

[0113] Reagent preparation:

[0114] Preparation of equilibrium solution: Accurately prepare 20 mmol / L NaH2PO4 + 0.15 mol / L sodium chloride buffer solution, adjust the pH to 7.4 with acid or base, and degas by sonication before use.

[0115] Preparation of sample loading solution: Take human IgG1 sample, dilute it with the above equilibration solution, and ensure that the sample solution is consistent with the equilibration solution system, and set aside.

[0116] Instrument and column preparation:

[0117] Select a 4.7 mL chromatography column (specifications: column height 10 cm, inner diameter 7.7 mm), pack the column according to the instructions (if it is a pre-packed column, use it directly), and connect it to the high performance liquid chromatography system or chromatography system.

[0118] Install the UV280nm ultraviolet absorption detector, ensure that the detector is properly connected to the chromatography system signal, and preheat the instrument to a stable state.

[0119] 2. Column equilibration

[0120] The flow rate of the chromatography system was set to 0.78 mL / min.

[0121] The column was flushed with the above equilibration solution until the UV detector baseline stabilized (280nm absorbance fluctuation ≤ ±2mAu), and the stable baseline value was recorded.

[0122] 3. Sample loading and breakthrough curve monitoring

[0123] Maintaining a constant flow rate, the prepared human IgG1 sample solution was continuously injected into the chromatographic column, while the UV detector was turned on to monitor and record the absorbance changes at 280 nm in real time, generating a breakthrough curve (corresponding to...). Figure 2 ).

[0124] During the process, the flow-through fluid is continuously collected, and the corresponding absorbance values ​​are recorded at fixed volume intervals (e.g., every 2 mL) to ensure that the data points clearly reflect the breakthrough trend.

[0125] 4. Key Steps in Dynamic Load Calculation

[0126] Identify the 10% flow-through point on the breakthrough curve: Mark the time point when the UV absorbance reaches 10% of the baseline value after equilibration.

[0127] Calculate the corresponding loading volume: Based on the marked time point and the set flow rate (flow rate × time), obtain the loading volume corresponding to the 10% flow-through point. This volume is the key parameter for calculating the dynamic loading capacity of the chromatographic column (dynamic loading capacity = loading volume × IgG1 concentration in the loading solution / column volume, the column volume is known to be 4.7 mL).

[0128] As can be seen from Table 5, the novel chromatography packing material provided by this invention can adsorb up to 83 mg / ml of 10% DBC for human 1gG, which fully demonstrates its superior high loading capacity and high efficiency.

[0129] Table 5 Calculation formula:

[0130]

[0131] C: Concentration of the corrected IgG1 standard, mg / mL

[0132] V 10％ : Sample loading volume when breakthrough value is 10%, mL

[0133] Vt Column volume, mL

[0134] Dynamic binding capacity (DBC) refers to the maximum amount of target molecules that a chromatography medium can stably bind under actual separation conditions (such as specific flow rate, pH, temperature, and buffer composition). The chromatography medium obtained in this invention has a 10% dynamic binding capacity as high as 83 mg / ml, which is relatively high among chromatography media, demonstrating the medium's high binding capacity and stability, making it suitable for the separation of large molecular targets.

[0135] 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 chromatography medium, characterized in that, The structure of the chromatography medium is shown in formula I: In formula I, Q is a matrix; L includes 1-10 carbon atoms and a functional group, the functional group including -NH-, -O-, -S-, a covalent bond and / or a hydroxyl group (-OH); R1includes a carboxylate (-COO - ); R2 includes an aromatic ring (-Ar); n is in the range of 1-4.

2. The chromatography medium of claim 1, wherein, The structure of R1 is -R3-COO - and / or the structure of R2 is -R4-Ar.

3. The chromatography medium of claim 2, wherein, R3 is selected from a covalent bond, an unsubstituted or substituted C1-C4 linear or branched alkyl group, an unsubstituted or substituted C4-C6 cycloalkyl group, and / or, said R4is selected from the group consisting of a covalent bond, unsubstituted or substituted C1-C4straight or branched alkyl or -(CH2) m -O-, m has a value ranging from 1 to 4.

4. The chromatography medium of claim 3, wherein, the aromatic ring (-Ar) is selected from an unsubstituted or substituted phenyl group, a biphenyl group, a naphthalene group or a six-membered aromatic heterocyclic ring; wherein the substitution is substituted by one or more C1-C4 linear or branched alkyl groups, a methoxy group (-OCH3), a methylthio group (-SCH3), fluorine (-F), chlorine (-Cl), bromine (-Br), iodine (-I) or a trifluoromethyl group (-CF3); Preferably, the six-membered aromatic heterocyclic ring is selected from a pyridine.

5. The chromatography medium of claim 4, wherein, The matrix is one or more of agarose microspheres, silica gel microspheres, polymethacrylate microspheres, dextran microspheres, glass beads, cellulose microspheres, polymer microspheres or porous polymer microspheres.

6. The chromatography medium of claim 5, wherein, The chromatography medium is selected from one of the following structures:

7. The chromatography medium according to any one of claims 1 to 6, characterized in that, The chromatography medium is formed by a matrix and a ligand coupling; The ligand is selected from one of ligand 1-ligand 12 of the following structures, 8. The chromatography medium of claim 7, wherein, The ligand density of the chromatography medium is 0.04-0.1 mmol / ml.

9. A method for the preparation of a chromatography medium according to any one of claims 1 to 8, characterized in that, The method comprises the following steps: (1) Microsphere activation: the matrix is reacted with an activating agent and a NaOH solution at 20-40°C to obtain an activated microsphere suspension; (2) Coupling ligand: the ligand is added to the activated microsphere suspension, the pH value is adjusted to 10-12 and then reacted, and the chromatography medium is obtained after the reaction is completed; Preferably, the weight ratio of the matrix to the activating agent is (4-8):1; Preferably, the activating agent is selected from one or more of the following: epichlorohydrin, allyl glycidyl ether, allyl bromide, 4-vinylphenyl glycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether and neopentyl glycol diglycidyl ether.

10. Use of the chromatography medium of any one of claims 1-8 or the chromatography medium prepared by the method of claim 9 in separating and purifying antibodies and proteins.

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