A high capacity multimodal chromatography media, its method of preparation and use

Abstract

The application discloses a high-loading multi-mode chromatography medium, a preparation method and application thereof. The chromatography medium comprises a chromatography matrix and a ligand. The chromatography matrix is a C 6‑20 porous microsphere with an amine group, a hydroxyl group, a carboxyl group, a sulfydryl group or a chlorine substitution on the surface; and the ligand is a hydrophobic alkenyl monomer and an alkenyl monomer with ion exchange function, and the hydrophobic alkenyl monomer and the alkenyl monomer with ion exchange function are different. The application grafts and polymerizes hydrophobic alkenyl compounds and ion exchange alkenyl compounds on the surface of the chromatography matrix. More chromatographic modes are combined by different types and different proportions of the hydrophobic alkenyl monomer and the alkenyl monomer with ion exchange function. The hydrophobicity of the ligand and the number of ion exchange groups in the prepared chromatography medium can be accurately controlled. The number of ligand binding proteins is increased by adjusting the proportion of the hydrophobic alkenyl monomer and the alkenyl monomer with ion exchange function of the chromatography medium.

Description

Technical Field

[0001] This invention belongs to the field of chromatography media preparation technology, specifically, it relates to a high-loading multimode chromatography media, its preparation method and application. Background Technology

[0002] With the increasing scale of biopharmaceutical production, the demand for downstream separation and purification efficiency is constantly growing, making the development of rapid and efficient purification methods an urgent priority. Chromatographic separation is one of the mainstream purification technologies currently available. The purification efficiency of this method depends on the performance of the separation medium, with the loading capacity of the chromatography medium being a crucial indicator affecting separation efficiency. Furthermore, given the complexity of biomacromolecule production processes—characterized by complex feed compositions, low target analyte content, and stringent bioactivity requirements—multi-step chromatographic processes are often necessary to obtain products that meet the requirements. These processes often combine affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography. However, the recovery rate of each purification step varies, and the superposition of multiple purification steps significantly reduces the yield of the target analyte.

[0003] In existing technologies, chromatographic modes that bond compounds with both hydrophobic and ion-exchange functions to the material surface are often used to improve separation. However, the types and performance of chromatographic modes for such media are mainly limited by the properties of the bonded compounds. The functional ratio of hydrophobicity and ion exchange cannot be precisely controlled. When the ligand density is high, protein molecules are denatured and inactivated due to strong hydrophobicity, resulting in low recovery rates. However, when the ligand density is low, the protein binding capacity is low.

[0004] Therefore, improving the recovery rate of target proteins and increasing separation efficiency while avoiding sacrificing the protein loading capacity of the medium are urgent problems to be solved in large-scale biopharmaceutical production. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a high-capacity multimode chromatography medium, comprising a chromatography matrix and ligands, wherein the chromatography matrix is ​​a C group with amino, hydroxyl, carboxyl, thiol groups, or chlorinated derivatives on its surface. 6-20 Aryl porous microspheres;

[0006] The ligand is a hydrophobic alkenyl monomer and an alkenyl monomer with ion exchange function, and the hydrophobic alkenyl monomer and the alkenyl monomer with ion exchange function are different;

[0007] The hydrophobic alkenyl monomer is selected from at least one of the structural formulas shown in Formula 1 and Formula 2 below.

[0008]

[0009] C 4-18 Alkylene-C(O)OC4-18 Alkylene 2

[0010] In Equation 1, R1, R2, and R3 may be the same or different, and are independently selected from H and C. 1-20 alkyl;

[0011] R4 is selected from -C(=O)OR b C, substituted or unsubstituted 6-20 Aryl, substituent R a C 1-20 Alkyl; R b Selected from C 1-20 alkyl;

[0012] The alkenyl monomer with ion exchange function is selected from at least one of the structural formulas shown in Formula 3 below.

[0013]

[0014] In Equation 3, R5, R6, and R7 may be the same or different, and are independently selected from H and C. 1-20 alkyl;

[0015] R8 is selected from substituted or unsubstituted C. 6-20 Aryl, -C(=O)OR c -C(=O)NR d -(CH2) n -SO3H, substituent R e It is a carboxyl, amino, or sulfonic acid group; R c Selected from H, Where n is the same or different, and is independently selected from integers 1-10, R9, R 10 R 11 R 12 R 13 Whether they are the same or different, they are independently selected from H and C. 1-10 Alkyl group; X is a halogen;

[0016] R d Selected from substituted or unsubstituted -(CH2) m -SO3H, m is an integer selected from 1 to 10, and the substituent is C. 1-12 alkyl.

[0017] According to an embodiment of the present invention, X is F, Cl, Br or I.

[0018] According to an embodiment of the present invention, in Formula 1, R1, R2, and R3 may be the same or different, and are independently selected from H and C. 1-10 Alkyl group; preferably, R1, R2, and R3 may be the same or different, and are independently selected from H and C. 1-6alkyl.

[0019] According to an embodiment of the present invention, in formula 1, R4 is selected from -C(=O)OR b C, substituted or unsubstituted 6-14 Aryl, substituent R a C 1-10 Alkyl; R b Selected from C 1-10 Alkyl, preferably, R b Selected from C 1-6 alkyl.

[0020] According to an embodiment of the present invention, the structural formula shown in Formula 2 is preferably C 4-10 Alkylene-C(O)OC 4-10 Alkylene.

[0021] According to an embodiment of the present invention, in formula 3, R5, R6, and R7 may be the same or different, and are independently selected from H and C. 1-10 Alkyl group; preferably H or C 1-6 alkyl.

[0022] According to an embodiment of the present invention, in formula 3, R8 is selected from substituted or unsubstituted C. 6-14 Aryl, -C(=O)OR c -C(=O)NR d -(CH2) n -SO3H, substituent R e It can be a carboxyl, amino, or sulfonic acid group;

[0023] R c Selected from H, Where n is the same or different, and is independently selected from integers 1-6, R9, R 10 R 11 R 12 R 13 Whether they are the same or different, they are independently selected from H and C. 1-6 Alkyl group; X is Cl or Br;

[0024] R d Selected from substituted or unsubstituted -(CH2) m -SO3H, m is an integer selected from 1 to 6, and the substituent is C. 1-6 alkyl.

[0025] According to an embodiment of the present invention, the hydrophobic alkenyl monomer is selected, for example, from at least one of butyl methacrylate, octyl methacrylate, dodecyl methacrylate, ethylhexyl methacrylate, and styrene.

[0026] According to an embodiment of the present invention, the alkenyl monomer having ion exchange function is selected from at least one of dimethylaminoethyl methacrylate, methacryloyloxyethyltrimethylammonium chloride, methacrylic acid, methpropylene sulfonic acid, and 2-acrylamide-2-methylpropanesulfonic acid.

[0027] According to an embodiment of the present invention, the molar ratio of the hydrophobic alkenyl monomer and the alkenyl monomer with ion exchange function is 1:(0.5-20), preferably 1:(1-10), and exemplaryly 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0028] According to an embodiment of the present invention, the chromatographic matrix has a pore size of 20-3000 nm and a particle size of 20-200 μm. Exemplarily, the pore sizes are 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1500 nm, 2000 nm, 2500 nm, and 3000 nm; and the particle sizes are 20 μm, 50 μm, 80 μm, 100 μm, 120 μm, 180 μm, and 200 μm.

[0029] According to an embodiment of the present invention, the chromatography matrix is ​​selected from at least one of the following: polysaccharide microspheres with hydroxyl groups on the surface, polyacrylate microspheres with amine groups on the surface, polystyrene microspheres with carboxyl groups on the surface, polyacrylate microspheres with thiol groups on the surface, and polysaccharide microspheres with benzyl chloride groups on the surface.

[0030] According to an embodiment of the present invention, the mass ratio of the chromatographic matrix to the hydrophobic alkenyl monomer is 1:(0.04-20), preferably 1:(0.09-5), and exemplary ratios are 1:0.09, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5.

[0031] According to an embodiment of the present invention, the chromatography medium is a chromatography matrix surface in which hydrophobic vinyl monomers and ion-exchange functional vinyl monomers are grafted in polymer form.

[0032] According to an embodiment of the present invention, the grafting amount of the chromatography medium is 0.2-1 g / g, and exemplary amounts are 0.2 g / g, 0.25 g / g, 0.3 g / g, 0.32 g / g, 0.4 g / g, 0.48 g / g, 0.5 g / g, 0.6 g / g, 0.7 g / g, 0.8 g / g, 0.85 g / g, 0.9 g / g, and 1 g / g.

[0033] According to an embodiment of the present invention, the protein loading of the high-load multimode chromatography medium is 15-100 mg bovine serum albumin / mL, exemplarily 15 mg bovine serum albumin / mL, 20 mg bovine serum albumin / mL, 30 mg bovine serum albumin / mL, 40 mg bovine serum albumin / mL, 50 mg bovine serum albumin / mL, 60 mg bovine serum albumin / mL, 70 mg bovine serum albumin / mL, 78 mg bovine serum albumin / mL, 80 mg bovine serum albumin / mL, 85 mg bovine serum albumin / mL, 90 mg bovine serum albumin / mL, 97 mg bovine serum albumin / mL, and 100 mg bovine serum albumin / mL.

[0034] According to an embodiment of the present invention, the ion exchange capacity of the high-load multimode chromatography medium is 0.3-9 mmol / g, and exemplary values ​​are 0.3 mmol / g, 0.4 mmol / g, 0.75 mmol / g, 1 mmol / g, 1.45 mmol / g, 2.1 mmol / g, 3 mmol / g, 4 mmol / g, 5 mmol / g, 6 mmol / g, 7 mmol / g, 7.9 mmol / g, 8 mmol / g, and 9 mmol / g.

[0035] According to an embodiment of the present invention, the protein recovery rate of the high-load multi-mode chromatography medium is above 90%.

[0036] According to an embodiment of the present invention, the mechanical strength of the high-load multimode chromatography medium is 0.3-10 MPa, and exemplary values ​​are 0.3 MPa, 1 MPa, 2 MPa, 3 MPa, 4 MPa, 5 MPa, 6 MPa, 7 MPa, 8 MPa, 9 MPa, and 10 MPa.

[0037] This invention also provides a method for preparing high-loading multimode chromatography media, the method comprising the following steps:

[0038] The high-loading multimode chromatography medium is prepared by mixing and reacting a chromatography matrix, a hydrophobic alkenyl monomer, an alkenyl monomer with ion exchange function, and an initiator.

[0039] According to an embodiment of the present invention, the preparation method of the high-loading multimode chromatography medium is as follows:

[0040] 1) Mix the chromatography matrix with hydrophobic alkenyl monomers and alkenyl monomers with ion exchange function;

[0041] 2) Add an initiator to the mixed solution obtained in step 1) above to initiate polymerization and prepare a high-loading multimode chromatography medium.

[0042] According to embodiments of the present invention, the initiator includes, but is not limited to, at least one of cerium ammonium sulfate, cerium ammonium nitrate, potassium persulfate, ammonium persulfate, cuprous chloride, etc.

[0043] According to an embodiment of the present invention, a ligand, such as bipyridine, may also be added to the reaction system.

[0044] According to an embodiment of the present invention, the initiator mass is 0.5-5% of the chromatography matrix, preferably 1-3%, and exemplaryly 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8% or 3%.

[0045] According to an embodiment of the present invention, the reaction temperature is 15-60°C, and the reaction time is 6-24 hours. Exemplarily, the reaction temperature is 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C. Exemplarily, the reaction time is 6 hours, 10 hours, 12 hours, 16 hours, 18 hours, 20 hours, or 24 hours.

[0046] According to an embodiment of the present invention, the method further includes a post-processing step: washing the prepared product with water and / or ethanol and drying it.

[0047] According to an embodiment of the present invention, the high-load multimode chromatography medium includes, but is not limited to, at least one of hydrophobic / anion exchange, hydrophobic / cation exchange, size exclusion / hydrophobic / ion exchange, and reversed-phase / ion exchange.

[0048] The present invention also provides the application of the above-mentioned chromatography medium in the field of protein chromatography separation.

[0049] The beneficial effects of this invention are:

[0050] This invention grafts hydrophobic alkenes and ion-exchange alkenes onto the surface of a chromatography matrix. By using different types and ratios of hydrophobic alkenyl monomers and alkenyl monomers with ion-exchange functions, more combinations of chromatographic modes can be achieved. At the same time, the hydrophobicity of the ligands and the number of ion-exchange groups in the prepared chromatography medium can be accurately controlled. By adjusting the ratio of hydrophobic alkenyl monomers to alkenyl monomers with ion-exchange functions in the chromatography medium, the number of ligand-bound proteins can be increased, which can be used for efficient separation and purification of proteins and other biomacromolecules.

[0051] The method of this invention is simple and easy to operate, has a wide range of applications, and the prepared multi-mode chromatography medium has a higher protein loading capacity while ensuring a high protein recovery rate.

[0052] Terminology Definitions and Explanations

[0053] Term "C" 1-20 "Alkyl" should be understood as referring to a straight-chain or branched saturated monovalent hydrocarbon group having 1 to 20 carbon atoms. 1-12 "Alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. 1-6 "Alkyl" means a straight-chain or branched alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers.

[0054] Term "C" 4-18 "alkylene" should be understood as C 4-18 An alkyl group is formed by the loss of one hydrogen atom.

[0055] Term "C" 6-20 "Aryl" should be understood to preferably represent a monocyclic, bicyclic, or tricyclic hydrocarbon ring having 6 to 20 carbon atoms and possessing monovalent aromaticity or partial aromaticity, preferably "C". 6-14 "Aromatic". The term "C" 6-14 "Aryl" should be understood to preferably represent a monovalent aromatic or partially aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring ("C") having 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms. 6-14 Aryl), particularly a ring with 6 carbon atoms (“C6 aryl”), such as phenyl; or biphenyl, or a ring with 9 carbon atoms (“C9 aryl”), such as indenyl or indenyl, or a ring with 10 carbon atoms (“C9 aryl”). 10 Aryl groups, such as tetrahydronaphthyl, dihydronaphthyl, or naphthyl, or rings with 13 carbon atoms (“C”). 13 Aryl groups, such as fluorene groups, or rings with 14 carbon atoms (“C”). 14 Aryl), for example, anthracene. When the C 6-20 When the aryl group is substituted, it can be monosubstituted or polysubstituted. Furthermore, there are no restrictions on the substitution site; for example, it can be ortho, para, or meta substituted. Attached Figure Description

[0056] Figure 1 The images shown are scanning electron microscope images of the chromatography medium in Example 1 (A is a full view of the microspheres, and B is a magnified view of a local surface morphology of the microspheres).

[0057] Figure 2 The images shown are scanning electron microscope images of the chromatography medium in Example 2 (A is a full view of the microspheres, and B is a magnified view of a local surface morphology of the microspheres).

[0058] Figure 3 The images shown are scanning electron microscope images of the chromatography medium in Example 3 (A is a full view of the microspheres, and B is a magnified view of a local surface morphology of the microspheres).

[0059] Figure 4 The images shown are scanning electron microscope images of the chromatography medium in Example 4 (A is a full view of the microspheres, and B is a magnified view of a local surface morphology of the microspheres). Detailed Implementation

[0060] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0061] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0062] Grafting quantity measurement

[0063] The grafting amount of the multi-mode chromatography media prepared in Examples 1-5 was determined: the chromatography media prepared in Examples 1-5 were vacuum dried at 50°C for 24 h to constant weight, and the amount of grafted polymerization per gram of microspheres was calculated by mass method, using the following formula:

[0064]

[0065] Hydrophobic / Anion Exchange Medium Capacity Determination in Examples 1-2

[0066] Step 1: Hydroxytransformation

[0067] 1) Transfer the chromatography medium prepared in Examples 1-2 into the exchange column and let it settle slowly until it reaches a volume of 10 mL. Then, pass 50 mL of 1 mol / L sodium hydroxide solution through the column at a flow rate of 0.8-1.0 mL / min.

[0068] 2) Flow deionized water through the column until the effluent no longer shows color with phenolphthalein (approximately 4 hours).

[0069] Step 2: Acid-base neutralization

[0070] 1) Accurately measure 25 mL of standard hydrochloric acid solution using an acid burette, add it to the exchange column above, let it drip slowly, and collect it in an Erlenmeyer flask;

[0071] 2) Add 25 mL of 1 mol / L sodium chloride solution dropwise through the column and collect it in the Erlenmeyer flask mentioned above.

[0072] Step 3: Collecting hydrochloric acid via back-drip

[0073] 1) Add 2-3 drops of phenolphthalein indicator to the liquid collected in the second step, and titrate with 0.1 mol / L sodium hydroxide standard solution until a faint red color is maintained for 15 seconds as the endpoint. Record the volume of alkali solution consumed.

[0074] 2) The ion exchange capacity (E) of the chromatography medium sample is calculated according to the following formula:

[0075] E = (C1V1 - C2V2) / V (mmol / mL)

[0076] Wherein, C1 – concentration of hydrochloric acid standard solution (mol / L); V1 – volume of hydrochloric acid standard solution (mL); C2 – concentration of sodium hydroxide standard solution (mol / L); V2 – volume of sodium hydroxide standard solution consumed during titration (mL); V – volume of medium in column (mL).

[0077] Hydrophobic / cation exchange medium capacity determination in Examples 3-5

[0078] Step 1: Hydrogen-based transition

[0079] 1) Transfer the chromatography medium prepared in Examples 3-5 into the exchange column and let it settle slowly until it reaches a volume of 10 mL. Then, pass 50 mL of 1 mol / L hydrochloric acid solution through the column at a flow rate of 0.8-1.0 mL / min.

[0080] 2) Flow deionized water through the column until the effluent does not change color to methyl orange (approximately 4 hours).

[0081] Step 2: Acid-base neutralization

[0082] 1) Accurately measure 25 mL of standard concentration sodium hydroxide solution using an acid burette, add it to the above exchange column, let it drip slowly, and collect it in an Erlenmeyer flask;

[0083] 2) Add 25 mL of 1 mol / L sodium chloride solution dropwise through the column and collect it in the Erlenmeyer flask mentioned above.

[0084] Step 3: Collecting sodium hydroxide by back-drip

[0085] 1) Add 2-3 drops of phenolphthalein indicator to the liquid collected in the second step, and titrate with 0.1 mol / L hydrochloric acid standard solution until the solution is colorless and remains colorless for 15 seconds as the endpoint. Record the volume of acid solution consumed.

[0086] 2) The ion exchange capacity (E) of the chromatography medium sample is calculated according to the following formula.

[0087] E = (C1V1 - C2V2) / V (mmol / mL)

[0088] Wherein, C1 is the concentration of sodium hydroxide standard solution (mol / L); V1 is the volume of sodium hydroxide standard solution (mL); C2 is the concentration of hydrochloric acid standard solution (mol / L); V2 is the volume of hydrochloric acid standard solution consumed during titration (mL); and V is the volume of medium in the column (mL).

[0089] Protein loading determination in hydrophobic / anion exchange chromatography media in Examples 1-2

[0090] The adsorbed protein sample was 1000 mL of bovine serum albumin (BSA) solution with a concentration of 2 mg / mL (pH = 8.0, 0.2 M NaCl, 20 mM Tris-HCl buffer); injection method A was used with a flow rate of 1 mL / min; mobile phase B was 1 M NaCl pH = 8.0 Tris-HCl buffer solution. The sample and mobile phase were used for the following chromatographic test steps.

[0091] 1) Test system dead volume

[0092] a. Prepare a 2.0 mg / mL BSA solution using a 1 M NaCl pH 8.0 Tris-HCl buffer solution; b. Connect the chromatographic column to the liquid chromatography system, inject the solution prepared in step a using a pump head, record the breakthrough time t0, test the system dead volume V0, and obtain the maximum UV absorbance value A0 of the sample.

[0093] 2) Test BSA dynamic load

[0094] a. Using a pump-injection method, record the 5% breakthrough time t with 1000 mL of 2 mg / mL BSA solution (pH = 8.0, 0.2 M NaCl, 20 mM Tris-HCl buffer). 5％ .

[0095] b. Calculate the dynamic adsorption capacity of BSA (Q) BSA The formula is as follows:

[0096]

[0097] Where F is the test flow rate, mL / min; C is the BSA sample concentration, mg / mL; and V is the volume of the chromatography medium, mL.

[0098] For the determination of protein loading in the hydrophobic / cation exchange chromatography media prepared in Examples 3, 4, and 5, the adsorbed protein sample was 1000 mL of lysozyme (Lyz) solution with a concentration of 2 mg / mL (pH = 7.0, 0.2 M NaCl, 20 mM PB buffer); injection method A was used with a flow rate of 1 mL / min; mobile phase B was 1 M NaCl pH = 7.0 PB buffer solution. The sample and mobile phase were used for the following chromatographic test steps.

[0099] 1) Test system dead volume

[0100] a. Prepare a 2.0 mg / mL Lyz solution using 1 M NaCl pH=7.0 PB buffer solution; b. Connect the chromatographic column to the liquid chromatography system, inject the solution prepared in step a using the pump head, record the breakthrough time t0, test the system dead volume V0, and obtain the maximum UV absorbance value A0 of the sample.

[0101] 2) Test Lyz dynamic load

[0102] a. Using a pump-injection method, record the 5% breakthrough time t using 1000 mL of 2 mg / mL Lyz solution (pH = 7.0, 0.2 M NaCl in PB buffer). 5％ .

[0103] b. Calculate the dynamic adsorption capacity of Lyz (Q) Lyz The formula is as follows:

[0104]

[0105] Where F is the test flow rate, mL / min; C is the Lyz sample concentration, mg / mL; and V is the volume of the chromatography medium, mL.

[0106] Protein recovery determination using hydrophobic / anion exchange chromatography media in Examples 1-2

[0107] The test protein sample was 5 mL of a 2 mg / mL bovine serum albumin (BSA) solution (pH 8.0, 0.2 M NaCl, 50 mM Tris-HCl buffer); a 5 mL quantitative loop was used at a flow rate of 1 mL / min; mobile phase A was 0.2 M NaCl pH 8.0 Tris-HCl buffer; mobile phase B was 1 M NaCl pH 8.0 Tris-HCl buffer. The sample and mobile phase were used for the following chromatographic test steps:

[0108] (1) Use mobile phase A to wash the system until baseline equilibrium is reached. Take the prepared BSA solution and inject 5 mL of protein sample. Record the empty peak area.

[0109] (2) Connect the chromatography column containing 1 mL of chromatography medium to the chromatography pipeline system, and equilibrate the chromatography column with mobile phase A until the UV absorption curve and conductivity curve are stable, then inject 5 mL of BSA sample.

[0110] (3) Use the above mobile phase B as the eluent to elute the chromatography column after the adsorption of protein. The flow rate is 1 mL / min. Record the elution peak area.

[0111] (4) The formula for calculating protein recovery rate is as follows:

[0112]

[0113] Protein recovery determination in hydrophobic / cation exchange chromatography media in Examples 3-5

[0114] The test protein sample consisted of 5 mL of a 2 mg / mL lysozyme (Lyz) solution (pH 7.0, 0.2 M NaCl, 50 mM PB buffer); a 5 mL quantitative loop was used at a flow rate of 1 mL / min; mobile phase A was 0.2 M NaCl pH 7.0 PB buffer solution; mobile phase B was 1 M NaCl pH 7.0 PB buffer solution. The sample and mobile phase were used for the following chromatographic test steps:

[0115] (1) Use mobile phase A to wash the system until baseline equilibrium is reached. Take the prepared Lyz solution and inject 5 mL of protein sample. Record the empty peak area.

[0116] (2) Connect the chromatography column containing 1 mL of chromatography medium to the chromatography pipeline system, and equilibrate the chromatography column with mobile phase A until the UV absorption curve and conductivity curve are stable, then inject 5 mL of Lyz sample.

[0117] (3) Use the above mobile phase B as the eluent to elute the chromatography column after the adsorption of protein. The flow rate is 1 mL / min. Record the elution peak area.

[0118] (4) The formula for calculating protein recovery rate is as follows:

[0119]

[0120] Example 1

[0121] Experimental Step 1: Polysaccharide microspheres (Sepharose-4FF, average pore size 20 nm, particle size range 50-120 μm) with hydroxyl groups on the surface were mixed with butyl methacrylate and dimethylaminoethyl methacrylate in an ethanol solution. The mass ratio of microspheres to butyl methacrylate was 100:9, and the molar ratio of butyl methacrylate to dimethylaminoethyl methacrylate was 1:10.

[0122] Experimental Step 2: Add cerium ammonium sulfate (3% by mass of microspheres) to the reaction solution in Step 1, and react at 30℃ for 12 h. After the reaction is complete, filter and wash the microspheres with ethanol and water sequentially until colorless to prepare a hydrophobic / weak anion exchange chromatography medium. The grafting amount was determined to be 0.25 g / g, the ion exchange capacity was 1.45 mmol / g, the protein loading was 100 mg / mL, and the protein recovery rate was 97% by gravimetric method.

[0123] Figure 1 This is a scanning electron microscope image of the chromatography medium in Example 1.

[0124] Example 2

[0125] Experimental Step 1: Ultraporous polyacrylate microspheres (FastSep-NH2, average pore size 3000nm, average particle size 200um) with amine groups on the surface were mixed with octyl methacrylate and methacryloyloxyethyltrimethylammonium chloride in an ethanol solution. The mass ratio of microspheres to octyl methacrylate was 1:2, and the molar ratio of octyl methacrylate to methacryloyloxyethyltrimethylammonium chloride was 2:1.

[0126] Experimental Step 2: Add ammonium persulfate (3% by mass of microspheres) to the reaction solution in Step 1, and react at 50℃ for 12 h. After the reaction is complete, filter and wash the microspheres with ethanol and water sequentially until colorless to prepare a hydrophobic / anion exchange chromatography medium. The grafting amount was determined to be 0.32 g / g, the ion exchange capacity was 0.4 mmol / g, the protein loading was 15 mg / mL, and the protein recovery rate was 96% by gravimetric method.

[0127] Figure 2 This is a scanning electron microscope image of the chromatography medium in Example 2.

[0128] Example 3

[0129] Experimental Step 1: Macroporous polystyrene microspheres (FastSep-OH, average pore size 200nm, particle size range 50-100um) with carboxyl groups on the surface were mixed with dodecyl methacrylate and methacrylic acid in an ethanol solution. The mass ratio of microspheres to dodecyl methacrylate was 1:1, and the molar ratio of dodecyl methacrylate to methacrylic acid was 1:9.

[0130] Experimental Step 2: Add cerium ammonium nitrate (3% by mass of microspheres) to the reaction solution in Step 1, and react at 50℃ for 12 h. After the reaction is complete, filter and wash the microspheres with ethanol and water sequentially until colorless to prepare a hydrophobic / weak cation exchange chromatography medium. The grafting amount was determined to be 0.85 g / g, the ion exchange capacity was 7.9 mmol / g, the protein loading was 78 mg / mL, and the protein recovery rate was 97% by gravimetric method.

[0131] Figure 3 This is a scanning electron microscope image of the chromatography medium in Example 3.

[0132] Example 4

[0133] Experimental Step 1: Macroporous polyacrylate microspheres (FastSep-SH, average pore size 100nm, average particle size 20um) with thiol groups on the surface were mixed with ethylhexyl methacrylate and methacrylsulfonic acid in dimethylformamide solution. The mass ratio of microspheres to ethylhexyl methacrylate was 1:1, and the molar ratio of ethylhexyl methacrylate to methacrylsulfonic acid was 1:5.

[0134] Experimental Step 2: Add potassium persulfate (3% by mass of microspheres) to the reaction solution in Step 1, and react at 50℃ for 12 h. After the reaction is complete, filter and wash the microspheres with water and ethanol sequentially until colorless to prepare a hydrophobic / strong cation exchange chromatography medium. The grafting amount was determined to be 0.48 g / g, the ion exchange capacity was 2.1 mmol / g, the protein loading was 85 mg / mL, and the protein recovery rate was 98% by gravimetric method.

[0135] Figure 4 This is a scanning electron microscope image of the chromatography medium in Example 4.

[0136] Example 5

[0137] Experimental Step 1: Polysaccharide microspheres (Sepharose-CH, average pore size 50 nm, particle size range 30-150 μm) with benzyl chloride groups on the surface were mixed with styrene and 2-acrylamide-2-methylpropanesulfonic acid in dimethylformamide solution. The mass ratio of microspheres to styrene was 1:1, and the molar ratio of styrene to 2-acrylamide-2-methylpropanesulfonic acid was 1:1.

[0138] Experimental Step 2: After purging the reaction solution from Step 1 with nitrogen gas for 1 hour to remove oxygen, cuprous chloride and bipyridine (mass ratio of the two to 1:3) (mass ratio of the two to the microspheres is 3%) were added. The reaction was carried out at 40°C for 10 hours. After the reaction was completed, the microspheres were filtered and washed successively with water and ethanol until they were colorless, thus preparing a hydrophobic / strong cation exchange chromatography medium. The grafting amount was determined by gravimetric method to be 0.30 g / g, the ion exchange capacity was 0.75 mmol / g, the protein loading was 97 mg / mL, and the protein recovery rate was 98%.

[0139] This invention prepares a multi-mode chromatography medium by surface-initiated free radical grafting polymerization. The grafted polymer chains are composed of random copolymers with different hydrophobicity and ion exchange functions. Vinyl monomers with different hydrophobicity and vinyl monomers with different ion exchange functions can be combined as needed to achieve precise control of hydrophobicity and ion exchange function, resulting in higher protein loading capacity.

[0140] The embodiments of the present invention have been described above by way of example. However, the scope of protection of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A high-load multi-mode chromatography medium, characterized in that, The chromatography medium includes a chromatography matrix and a ligand. The chromatography matrix is ​​selected from at least one of the following: polysaccharide microspheres with hydroxyl groups on the surface, polyacrylate microspheres with amine groups on the surface, polystyrene microspheres with carboxyl groups on the surface, polyacrylate microspheres with thiol groups on the surface, and polysaccharide microspheres with benzyl chloride groups on the surface. The pore size of the chromatography matrix is ​​20-3000 nm, and the particle size is 20-200 μm. The ligand is a hydrophobic alkenyl monomer and an alkenyl monomer with ion exchange function, and the hydrophobic alkenyl monomer and the alkenyl monomer with ion exchange function are different; The molar ratio of the hydrophobic alkenyl monomer to the alkenyl monomer with ion exchange function is 1:(0.5-20). The hydrophobic alkenyl monomer is selected from at least one of butyl methacrylate, octyl methacrylate, dodecyl methacrylate, and ethylhexyl methacrylate; The alkenyl monomer with ion exchange function is selected from at least one of dimethylaminoethyl methacrylate, methacryloyloxyethyltrimethylammonium chloride, methacrylic acid, methpropylene sulfonic acid, and 2-acrylamide-2-methylpropanesulfonic acid. Furthermore, the mass ratio of the chromatographic matrix to the hydrophobic alkenyl monomer is 1:(0.04-20); The chromatography medium is formed by grafting hydrophobic vinyl monomers and ion-exchange functional vinyl monomers onto the surface of the chromatography matrix in the form of polymers.

2. The chromatography medium according to claim 1, characterized in that, The grafting amount of the high-load multimode chromatography medium is 0.2-1 g / g.

3. The chromatography medium according to claim 1, characterized in that, The protein loading of the high-capacity multi-mode chromatography medium is 15-100 mg bovine serum albumin / mL chromatography medium.

4. The chromatography medium according to claim 1, characterized in that, The ion exchange capacity of the high-load multimode chromatography medium is 0.3-9 mmol / g.

5. The chromatography medium according to claim 1, characterized in that, The protein recovery rate of the high-load multi-mode chromatography medium is over 90%.

6. The chromatography medium according to claim 1, characterized in that, The mechanical strength of the high-load multimode chromatography medium is 0.3-10 MPa.

7. The method for preparing high-loading multimode chromatography media according to any one of claims 1-6, characterized in that, The method includes the following steps: The high-loading multimode chromatography medium is prepared by mixing and reacting a chromatography matrix, a hydrophobic alkenyl monomer, an alkenyl monomer with ion exchange function, and an initiator.

8. The method according to claim 7, characterized in that, The initiator is selected from at least one of cerium ammonium sulfate, cerium ammonium nitrate, potassium persulfate, ammonium persulfate, and cuprous chloride; The initiator mass is 0.5-5% of the chromatography matrix.

9. The method according to claim 7, characterized in that, The reaction temperature is 15-60 °C, and the reaction time is 6-24 h.

10. The application of the high-capacity multi-mode chromatography medium according to any one of claims 1-6 in the field of protein chromatography separation.

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