CN115894800A - High-loading multi-mode chromatography medium and preparation method and application thereof - Google Patents
High-loading multi-mode chromatography medium and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a high-load multi-mode chromatography medium, a preparation method and application thereof, wherein the chromatography medium comprises a chromatography matrix and a ligand, and the chromatography matrix is C with amino, hydroxyl, carboxyl, sulfydryl or chlorine substitution on the surface 6‑20 Porous microspheres of aryl groups; the ligand is hydrophobic alkenyl monomer and alkenyl monomer with ion exchange function, and the hydrophobic alkenyl monomer and the alkenyl monomer with ion exchange function are different. According to the invention, the hydrophobic vinyl compound and the ion exchange vinyl compound are grafted and polymerized on the surface of the chromatography matrix, the combination of more chromatography modes is realized through the hydrophobic vinyl monomers and the vinyl monomers with the ion exchange function in different types and different proportions, the hydrophobicity of the petunidin in the prepared chromatography medium and the number of the ion exchange groups can be accurately controlled, and the number of the petunidin binding protein is increased by regulating and controlling the proportion of the hydrophobic vinyl monomers and the vinyl monomers with the ion exchange function of the chromatography medium.
Description
Technical Field
The invention belongs to the technical field of preparation of chromatographic media, and particularly relates to a high-loading multi-mode chromatographic medium as well as a preparation method and application thereof.
Background
With the increasing expansion of the production scale of biopharmaceuticals, the downstream separation and purification efficiency is required to be higher and higher, and the development of a rapid and efficient purification method is urgent. Chromatographic separation is one of the mainstream purification technologies at present, and the purification efficiency of the method depends on the performance of the separation medium, wherein the loading capacity of the chromatographic medium is an important index influencing the separation efficiency. In addition, in view of the complexity of the biomacromolecule production process, the biomacromolecule production process is characterized in that the feed liquid component is complex, the target substance content is low, the requirement on biological activity is strict and the like, so that a product meeting the requirement can be obtained by a multi-step chromatography process, such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography and other multi-step chromatography combination, the recovery rate of each step of purification process is different, and the yield of the target substance is obviously reduced after the multi-step purification steps are superposed.
In the prior art, a chromatographic mode that a compound with both hydrophobicity and ion exchange functions is bonded to the surface of a material is mostly adopted to improve the separation effect, but the type and the chromatographic performance of the chromatographic mode of the medium are mainly limited by the properties of the bonded compound, the functional proportion of the hydrophobicity and the ion exchange cannot be accurately controlled, the protein molecules are denatured and inactivated due to strong hydrophobicity and the recovery rate is low when the ligand density is high, and the problem of low protein binding capacity is caused when the ligand density is low.
Therefore, the improvement of the recovery rate of the target protein and the separation efficiency, and the avoidance of the protein load of the sacrifice medium are problems to be solved urgently in the current large-scale biological pharmaceutical production.
Disclosure of Invention
In order to improve the technical problem, the invention provides a high-load multi-mode chromatography medium, which comprises a chromatography matrix and a ligand, wherein the chromatography matrix is C with amino, hydroxyl, carboxyl, sulfydryl or substituted by chlorine on the surface 6-20 Porous microspheres of aryl groups;
the ligand is hydrophobic alkenyl monomer and alkenyl monomer with ion exchange function, and the hydrophobic alkenyl monomer and the alkenyl monomer with ion exchange function are different;
the hydrophobic alkenyl monomer is selected from at least one of structural formulas shown as the following formulas 1 and 2,
C 4-18 alkylene-C (O) O-C 4-18 Alkylene group of formula 2
In the formula 1, R 1 、R 2 、R 3 Identical or different, independently of one another, from H, C 1-20 An alkyl group;
R 4 is selected from-C (= O) OR b Substituted or unsubstituted C 6-20 Aryl, substituent R a Is C 1-20 An alkyl group; r b Is selected from C 1-20 An alkyl group;
the alkenyl monomer with the ion exchange function is selected from at least one of structural formulas shown as the following formula 3,
in formula 3, R 5 、R 6 、R 7 Identical or different, independently of one another, from H, C 1-20 An alkyl group;
R 8 selected from substituted or unsubstituted C 6-20 Aryl group, -C (= O) OR c 、-C(＝O)N-R d 、-(CH 2 ) n -SO 3 H, substituent R e Is carboxyl, amino or sulfonic group; r is c Selected from H,
Wherein n is the same or different and is independently selected from an integer of 1 to 10, R 9 、R 10 、R 11 、R 12 、R 13 Identical or different, independently of one another, from H, C 1-10 An alkyl group; x is halogen;
R d selected from substituted or unsubstituted- (CH) 2 ) m -SO 3 H, m is an integer of 1-10, and the substituent is C 1-12 An alkyl group.
According to an embodiment of the invention, X is F, cl, br or I.
According to an embodiment of the present invention, in formula 1, R 1 、R 2 、R 3 Identical or different, independently of one another, from H, C 1-10 An alkyl group; preferably, R 1 、R 2 、R 3 Identical or different, independently of one another, from H, C 1-6 An alkyl group.
According to an embodiment of the present invention, in formula 1, R 4 Selected from-C (= O) OR b Substituted or unsubstituted C 6-14 Aryl, substituent R a Is C 1-10 An alkyl group; r b Is selected from C 1-10 Alkyl, preferably, R b Is selected from C 1-6 An alkyl group.
According to an embodiment of the present invention, the structural formula shown in formula 2 is preferably C 4-10 alkylene-C (O) O-C 4-10 An alkylene group.
According to the inventionIn formula 3, R 5 、R 6 、R 7 Identical or different, independently of one another, from H, C 1-10 An alkyl group; preferably H and C 1-6 An alkyl group.
According to an embodiment of the present invention, in formula 3, R 8 Selected from substituted or unsubstituted C 6-14 Aryl, -C (= O) OR c 、-C(＝O)N-R d 、-(CH 2 ) n -SO 3 H, substituent R e Is a carboxyl group, an amino group or a sulfonic group;
R c selected from H,
Wherein n is the same or different and is independently selected from an integer of 1 to 6, R 9 、R 10 、R 11 、R 12 、R 13 Same or different and independently selected from H, C 1-6 An alkyl group; x is Cl or Br;
R d selected from substituted or unsubstituted- (CH) 2 ) m -SO 3 H, m is an integer of 1-6, and the substituent is C 1-6 An alkyl group.
According to an embodiment of the present invention, the hydrophobic ethylenic monomer is, for example, at least one selected from butyl methacrylate, octyl methacrylate, dodecyl methacrylate, ethylhexyl methacrylate, styrene.
According to an embodiment of the present invention, the alkenyl monomer having an ion exchange function is, for example, at least one selected from the group consisting of dimethylaminoethyl methacrylate, acyloxyethyltrimethyl ammonium methacrylate, methacrylic acid, methacryl sulfonic acid, 2-acrylamido-2-methylpropane sulfonic acid.
According to an embodiment of the present invention, the molar ratio of the hydrophobic ethylenic monomer and the ethylenic monomer having an ion exchange function is 1 (0.5-20), preferably 1 (1-10), exemplarily 1.
According to an embodiment of the invention, the chromatography matrix has a pore size of 20-3000nm and a particle size of 20-200um. Illustratively, the pore size is 20nm, 50nm, 100nm, 200nm, 500nm, 1000nm, 1500nm, 2000nm, 2500nm, 3000nm; the particle size is 20um, 50um, 80um, 100um, 120um, 180um, 200um.
According to an embodiment of the present invention, the chromatography matrix is, for example, at least one selected from polysaccharide microspheres having hydroxyl groups on the surface, polyacrylate microspheres having amine groups on the surface, polystyrene microspheres having carboxyl groups on the surface, polyacrylate microspheres having mercapto groups on the surface, and polysaccharide microspheres having benzyl chloride groups on the surface.
According to an embodiment of the invention, the mass ratio of the chromatography matrix to the hydrophobic ethylenic monomer is 1 (0.04-20), preferably 1 (0.09-5), exemplarily 1.
According to an embodiment of the invention, the chromatography medium is a chromatography matrix surface onto which hydrophobic vinyl monomers and ion-exchange functional vinyl monomers are grafted in the form of a polymer.
According to an embodiment of the invention, the grafting amount of the chromatography medium is between 0.2 and 1g/g, exemplarily 0.2g/g, 0.25g/g, 0.3g/g, 0.32g/g, 0.4g/g, 0.48g/g, 0.5g/g, 0.6g/g, 0.7g/g, 0.8g/g, 0.85g/g, 0.9g/g, 1g/g.
According to an embodiment of the invention, the protein loading of the high-loading multimode chromatography medium is 15-100mg bovine serum albumin/mL chromatography medium, illustratively 15mg bovine serum albumin/mL chromatography medium, 20mg bovine serum albumin/mL chromatography medium, 30mg bovine serum albumin/mL chromatography medium, 40mg bovine serum albumin/mL chromatography medium, 50mg bovine serum albumin/mL chromatography medium, 60mg bovine serum albumin/mL chromatography medium, 70mg bovine serum albumin/mL chromatography medium, 78mg bovine serum albumin/mL chromatography medium, 80mg bovine serum albumin/mL chromatography medium, 85mg bovine serum albumin/mL chromatography medium, 90mg bovine serum albumin/mL chromatography medium, 97mg bovine serum albumin/mL chromatography medium, 100mg bovine serum albumin/mL chromatography medium.
According to an embodiment of the invention, the high load multimodal chromatography medium has an ion exchange capacity of 0.3-9mmol/g, exemplarily 0.3mmol/g, 0.4mmol/g, 0.75mmol/g, 1mmol/g, 1.45mmol/g, 2.1mmol/g, 3mmol/g, 4mmol/g, 5mmol/g, 6mmol/g, 7mmol/g, 7.9mmol/g, 8mmol/g, 9mmol/g.
According to an embodiment of the invention, the high load multimodal chromatography medium has a protein recovery of above 90%.
According to an embodiment of the invention, the high-loading multimodal chromatography medium has a mechanical strength of 0.3-10MPa, exemplarily 0.3MPa, 1MPa, 2MPa, 3MPa, 4MPa, 5MPa, 6MPa, 7MPa, 8MPa, 9MPa, 10MPa.
The invention also provides a preparation method of the high-loading multi-mode chromatographic medium, which comprises the following steps:
mixing and reacting a chromatography matrix, a hydrophobic alkenyl monomer, an alkenyl monomer with an ion exchange function and an initiator to prepare the high-load multi-mode chromatography medium.
According to an embodiment of the present invention, the preparation method of the high-load multimode chromatography medium is:
1) Mixing a chromatography matrix with a hydrophobic alkenyl monomer and an alkenyl monomer with an ion exchange function;
2) Adding an initiator into the mixed solution obtained in the step 1) to initiate polymerization, and preparing the high-loading multi-mode chromatography medium.
According to an embodiment of the present invention, the initiator includes, but is not limited to, at least one of cerium ammonium sulfate, cerium ammonium nitrate, potassium persulfate, amine persulfate, cuprous chloride, and the like.
According to an embodiment of the present invention, a ligand, such as bipyridine, may also be added to the reaction system.
According to an embodiment of the invention, the mass of the initiator is 0.5-5%, preferably 1-3%, exemplarily 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8% or 3% of the chromatography matrix.
According to an embodiment of the present invention, the temperature of the reaction is 15 to 60 ℃ and the time of the reaction is 6 to 24 hours, and illustratively, the temperature of the reaction is 15 ℃,20 ℃, 25 ℃,30 ℃, 35 ℃,40 ℃, 45 ℃,50 ℃, 55 ℃ or 60 ℃. Illustratively, the reaction time is 6h, 10h, 12h, 16h, 18h, 20h, 24h.
According to an embodiment of the invention, the method further comprises a post-treatment step: washing the prepared product by water and/or ethanol, and drying.
According to embodiments of the present invention, the high-loading multimodal chromatography medium includes, but is not limited to, at least one of hydrophobic/anion exchange, hydrophobic/cation exchange, size exclusion/hydrophobic/ion exchange, reverse phase/ion exchange, and the like.
The invention also provides application of the chromatographic medium in the field of protein chromatographic separation.
The invention has the beneficial effects that:
according to the invention, the hydrophobic alkene compound and the ion exchange alkene compound are graft-polymerized on the surface of the chromatography matrix, more chromatographic modes can be combined by hydrophobic alkene monomers and alkene monomers with ion exchange functions in different types and different proportions, 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 can be increased by regulating and controlling the proportion of the hydrophobic alkene monomers and the alkene monomers with ion exchange functions of the chromatography medium, and the method can be used for efficiently separating and purifying biomacromolecules such as proteins.
The preparation process of the method is simple and easy to operate, the application range is wide, and the prepared multi-mode chromatography medium has higher protein loading capacity while ensuring higher protein recovery rate.
Definition and description of terms
The term "C 1-20 Alkyl "is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having from 1 to 20 carbon atoms. "C 1-12 Alkyl "denotes straight and branched chain alkyl groups having 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms," C 1-6 Alkyl "denotes straight and branched chain alkyl groups 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-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl, and the like, or isomers thereof.
The term "C 4-18 Alkylene is understood to mean C 4-18 A group formed after an alkyl group loses one H.
The term "C 6-20 Aryl "is to be understood as preferably meaning a mono-, bi-or tricyclic hydrocarbon ring of monovalent or partially aromatic character having from 6 to 20 carbon atoms, preferably" C 6-14 Aryl ". The term "C 6-14 Aryl "is to be understood as preferably meaning a mono-, bi-or tricyclic hydrocarbon ring having a monovalent or partially aromatic character with 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (" C 6-14 Aryl "), in particular a ring having 6 carbon atoms (" C 6 Aryl "), such as phenyl; or biphenyl, or is a ring having 9 carbon atoms ("C 9 Aryl), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C 10 Aryl radicals), such as tetralinyl, dihydronaphthyl or naphthyl, or rings having 13 carbon atoms ("C 13 Aryl radicals), such as the fluorenyl radical, or a ring having 14 carbon atoms ("C) 14 Aryl), such as anthracyl. When said C is 6-20 When the aryl group is substituted, it may be mono-or polysubstituted. The substitution site is not limited, and may be, for example, ortho-, para-or meta-substituted.
Drawings
FIG. 1 is a scanning electron microscope photograph of the chromatographic medium of example 1 (A is a full-scale image of the microspheres, and B is a partially enlarged surface topography of the microspheres).
FIG. 2 is a scanning electron microscope photograph of the chromatographic medium of example 2 (A is a full-scale image of the microspheres, and B is a partially enlarged surface topography of the microspheres).
FIG. 3 is a scanning electron microscope photograph of the chromatographic medium of example 3 (A is a full-scale image of the microspheres, and B is a partially enlarged topography image of the surface of the microspheres).
FIG. 4 is a scanning electron microscope photograph of the chromatographic medium of example 4 (A is a full-scale image of the microspheres, and B is a partially enlarged topography image of the surface of the microspheres).
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Graft amount measurement
The multimodal chromatography media prepared in examples 1-5 were subjected to graft determination: the chromatography media prepared in examples 1 to 5 were dried under vacuum at 50 ℃ for 24 hours to a constant weight, and the amount of graft polymerization per gram of microspheres was calculated by mass method as follows:
hydrophobic/anion exchange Medium Capacity determination in examples 1-2
The first step is as follows: transformation of hydroxyl form
1) The chromatography medium prepared in example 1-2 was transferred to an exchange column and slowly settled until reaching a volume of 10mL, passing through 50mL of 1mol/L sodium hydroxide solution at a flow rate of 0.8-1.0mL/min;
2) Deionized water was passed through the cartridge until the effluent was colorless to phenolphthalein (approximately 4 h).
The second step is that: acid-base neutralization
1) Accurately measuring 25mL of standard hydrochloric acid solution by using an acid burette, adding the standard hydrochloric acid solution into the exchange column, slowly dripping the solution, and collecting the solution into a triangular flask;
2) 25mL of 1mol/L sodium chloride solution was added dropwise through the column and collected in the above flask.
The third step: hydrochloric acid collected by back drip
1) Adding 2-3 drops of phenolphthalein indicator into the liquid collected in the second step, titrating to reddish with 0.1mol/L sodium hydroxide standard solution, keeping the color constant for 15s as an end point, and recording the volume of the consumed alkali liquor;
2) The ion exchange capacity (E) of the chromatography media sample is calculated according to the following formula:
E＝(C 1 V 1 -C 2 V 2 )/V(mmol/mL)
wherein, C 1 -hydrochloric acid standard solution concentration (mol/L); v 1 Hydrochloric acid standard solution volume (mL); c 2 -concentration of sodium hydroxide standard solution (mol/L); v 2 -volume of sodium hydroxide standard solution consumed at the time of titration (mL); v-volume of medium in cartridge (mL).
Hydrophobic/cation exchange Medium Capacity determination in examples 3-5
The first step is as follows: transformation of hydrogen form
1) Transferring the chromatographic medium prepared in the example 3-5 into an exchange column, slowly settling until the volume reaches 10mL, and passing through a 50mL 1mol/L hydrochloric acid solution at the flow rate of 0.8-1.0mL/min;
2) The column was run through with deionized water until the effluent did not color to methyl orange (approximately 4 h).
The second step is that: acid-base neutralization
1) Accurately measuring 25mL of sodium hydroxide solution with standard concentration by using an acid burette, adding the sodium hydroxide solution into the exchange column, slowly dripping the sodium hydroxide solution, and collecting the sodium hydroxide solution into a triangular flask;
2) Then 25mL of 1mol/L sodium chloride solution was added dropwise through the column and collected in the above flask.
The third step: back drip collected sodium hydroxide
1) Adding 2-3 drops of phenolphthalein indicator into the liquid collected in the second step, titrating with 0.1mol/L hydrochloric acid standard solution until the solution is colorless, keeping the coloration for 15s as an end point, and recording the volume of the consumed acid liquid;
2) The ion exchange capacity (E) of the chromatography medium sample is calculated as follows.
E＝(C 1 V 1 -C 2 V 2 )/V(mmol/mL)
Wherein, C 1 -sodium hydroxide standard solution concentration (mol/L); v 1 -volume of sodium hydroxide standard solution (mL); c 2 -concentration of hydrochloric acid standard solution (mol/L); v 2 -volume of hydrochloric acid standard solution consumed at the time of titration (mL); v-volume of medium in cartridge (mL).
Protein Loading determination of hydrophobic/anion exchange chromatography Medium in examples 1-2
The adsorbed protein sample was 1000mL (pH =8.0,0.2M NaCl,20mM Tris-HCl buffer) of Bovine Serum Albumin (BSA) solution with a concentration of 2 mg/mL; a, pump head sample introduction mode is adopted, and the flow rate is 1mL/min; the mobile phase B was a Tris-HCl buffer solution of 1M NaCl ph=8.0, the sample and mobile phase were used for the following chromatographic test steps;
1) Dead volume of test system
a. Preparing 2.0mg/mL BSA solution by using 1M NaCl pH =8.0 Tris-HCl buffer solution; b. connecting chromatographic column to liquid phase system, using solution prepared in step a by means of pump head sample introduction, recording initial penetration time t 0 Test System dead volume V 0 And obtaining the maximum ultraviolet absorption value A of the sample 0 。
2) Testing BSA dynamic Loading
a. The 5% breakthrough time t was recorded in pumphead format with 1000mL of 2mg/mL BSA solution (pH =8.0,0.2M NaCl,20mM Tris-HCl buffer) 5％ 。
b. The amount of BSA dynamically adsorbed (Q) was calculated BSA ) The formula is as follows:
wherein, F, testing flow rate, mL/min; c, BSA sample concentration, mg/mL; v, chromatography medium volume, mL.
Protein load determination of hydrophobic/cation exchange chromatography media prepared in examples 3, 4, and 5 the adsorbed protein sample was 1000mL of lysozyme (Lyz for short) solution (pH =7.0,0.2m nacl,20mm PB buffer) at a concentration of 2 mg/mL; a, pump head sample introduction mode is adopted, and the flow rate is 1mL/min; mobile phase B was a PB buffer solution of 1M NaCl ph =7.0, and the sample and mobile phase were used in the following respective chromatography test steps;
1) Dead volume of test system
a. Preparing a 2.0mg/mL Lyz solution by using a 1M NaCl pH =7.0 PB buffer solution; b. connecting chromatographic column to liquid phase system, feeding the solution prepared in step a by pump head sample feeding mode, and recording the starting penetration time t 0 Test System dead volume V 0 And obtaining the maximum ultraviolet absorption value A of the sample 0 。
2) Testing Lyz dynamic Capacity
a. The 5% breakthrough time t was recorded in 1000mL Lyz solution (pH =7.0,0.2M NaCl PB buffer) at 2mg/mL using a pump injection 5％ 。
b. Calculation of the amount of dynamic adsorption (Q) of Lyz Lyz ) The formula is as follows:
wherein, F, testing flow rate, mL/min; c, lyz sample concentration, mg/mL; v, chromatography medium volume, mL.
Protein recovery assay for hydrophobic/anion exchange chromatography media in examples 1-2
The test protein sample is Bovine Serum Albumin (BSA) solution with the concentration of 2mg/mL, 5mL (pH =8.0,0.2M NaCl,50mM Tris-HCl buffer solution); 5mL of quantitative ring, and the flow rate is 1mL/min; the mobile phase A is a Tris-HCl buffer solution with 0.2M NaCl pH= 8.0; mobile phase B was a Tris-HCl buffer solution of 1M NaCl ph=8.0, samples and mobile phases were used for the following chromatographic test steps:
(1) Using a mobile phase A to flush the system until the baseline is balanced, taking the prepared BSA solution, injecting a sample of 5mL of protein sample, and recording the area of the no-load peak;
(2) Connecting a chromatographic column filled with 1mL of chromatographic medium into a chromatographic pipeline system, balancing the chromatographic column by using a mobile phase A until an ultraviolet absorption curve and a conductivity curve are stable, and injecting a sample of 5mL of BSA (bovine serum albumin);
(3) Eluting the chromatographic column after adsorbing the protein by using the mobile phase B as an eluent at the operation flow rate of 1mL/min; recording the elution peak area;
(4) The protein recovery rate calculation formula is as follows:
determination of protein recovery Rate for hydrophobic/cation exchange chromatography Medium in examples 3-5
The test protein sample is 5mL of lysozyme (Lyz for short) solution with the concentration of 2mg/mL (pH =7.0,0.2M NaCl,50mM PB buffer); 5mL of quantitative ring, and the flow rate is 1mL/min; the mobile phase A is a PB buffer solution with 0.2M NaCl pH= 7.0; mobile phase B was a PB buffer solution of 1M NaCl ph=7.0, samples and mobile phases were used for the following various chromatography testing steps:
(1) Using a mobile phase A washing system to balance a base line, taking a prepared Lyz solution, injecting a sample 5mL of protein sample, and recording the area of a no-load peak;
(2) Connecting a chromatographic column filled with 1mL of chromatographic medium into a chromatographic pipeline system, balancing the chromatographic column by using a mobile phase A until an ultraviolet absorption curve and a conductivity curve are stable, and injecting a sample of 5mL of Lyz;
(3) Eluting the chromatographic column after adsorbing the protein by using the mobile phase B as an eluent at the operation flow rate of 1mL/min; recording the elution peak area;
(4) The protein recovery rate calculation formula is as follows:
example 1
Experiment step 1: mixing polysaccharide microspheres (Sepharose-4 FF, the average pore diameter is 20nm, the particle size range is 50-120 um) with hydroxyl, butyl methacrylate and dimethylaminoethyl methacrylate in an ethanol solution, wherein the mass ratio of the microspheres to the butyl methacrylate is 100, and the molar ratio of the butyl methacrylate to the dimethylaminoethyl methacrylate is 1.
Experiment step 2: and (2) adding ammonium ceric sulfate (the mass ratio of the ammonium ceric sulfate to the microspheres is 3%) into the reaction solution obtained in the step (1), reacting at 30 ℃ for 12 hours, after the reaction is finished, performing suction filtration, and sequentially cleaning the microspheres with ethanol and water until the microspheres are colorless to prepare a hydrophobic/weak anion exchange chromatography medium, wherein the grafting amount of the hydrophobic/weak anion exchange chromatography medium is 0.25g/g, the ion exchange capacity is 1.45mmol/g, the protein loading amount is 100mg/mL, and the protein recovery rate is 97% by weight method.
FIG. 1 is a scanning electron micrograph of a chromatography medium in example 1.
Example 2
Experimental step 1: the superporous polyacrylate microsphere (FastSep-NH) with amino on the surface 2 Average pore diameter of 3000nm, average particle size of 200 um) with octyl methacrylate and acyloxyethyltrimethyl ammonium chloride in an ethanol solution, wherein the mass ratio of the microspheres to the octyl methacrylate is 1.
Experiment step 2: adding ammonium persulfate (the mass ratio of the ammonium persulfate to the microspheres is 3%) into the reaction liquid obtained in the step 1, reacting for 12 hours at 50 ℃, after the reaction is finished, performing suction filtration, and sequentially cleaning the microspheres with ethanol and water until the microspheres are colorless to prepare a hydrophobic/anion exchange chromatography medium, wherein the grafting amount of the hydrophobic/anion exchange chromatography medium is 0.32g/g, the ion exchange capacity is 0.4mmol/g, the protein loading amount is 15mg/mL, and the protein recovery rate is 96% through gravimetric method determination.
FIG. 2 is a scanning electron micrograph of the chromatography medium of example 2.
Example 3
Experiment step 1: mixing macroporous polystyrene microspheres (FastSep-OH, the average pore diameter is 200nm, the particle size range is 50-100 um) with carboxyl on the surface with dodecyl methacrylate and methacrylic acid in an ethanol solution, wherein the mass ratio of the microspheres to the dodecyl methacrylate is 1.
Experiment step 2: and (2) adding ammonium ceric nitrate (the mass ratio of the ammonium ceric nitrate to the microspheres is 3%) into the reaction solution obtained in the step (1), reacting for 12 hours at 50 ℃, after the reaction is finished, performing suction filtration, and sequentially cleaning the microspheres to be colorless by using ethanol and water to prepare a hydrophobic/weak cation exchange chromatography medium, wherein the grafting amount of the hydrophobic/weak cation exchange chromatography medium is 0.85g/g, the ion exchange capacity is 7.9mmol/g, the protein loading amount is 78mg/mL, and the protein recovery rate is 97% by weight method.
FIG. 3 is a scanning electron micrograph of the chromatography medium of example 3.
Example 4
Experiment step 1: mixing macroporous polyacrylate microspheres (FastSep-SH, the average pore diameter is 100nm, and the average particle size is 20 microns) with sulfydryl, ethylhexyl methacrylate and methacrylic sulfonic acid in a dimethylformamide solution, wherein the mass ratio of the microspheres to the ethylhexyl methacrylate is 1.
Experiment step 2: adding potassium persulfate (the mass ratio of the potassium persulfate to the microspheres is 3%) into the reaction solution obtained in the step 1, reacting for 12 hours at 50 ℃, after the reaction is finished, performing suction filtration, and sequentially cleaning the microspheres with water and ethanol until the microspheres are colorless to prepare a hydrophobic/strong cation exchange chromatography medium, wherein the grafting amount of the hydrophobic/strong cation exchange chromatography medium is 0.48g/g, the ion exchange capacity is 2.1mmol/g, the protein loading amount is 85mg/mL, and the protein recovery rate is 98% through gravimetric method determination.
FIG. 4 is a scanning electron micrograph of a chromatography medium in example 4.
Example 5
Experiment step 1: mixing polysaccharide microspheres (Sepharose-CH, average pore diameter of 50nm and particle size range of 30-150 um) with surface containing benzyl chloride groups, styrene and 2-acrylamide-2-methylpropanesulfonic acid in a dimethylformamide solution, wherein the mass ratio of the microspheres to the styrene is 1.
Experiment step 2: and (2) after the reaction solution in the step (1) is filled with nitrogen and oxygen is discharged for 1h, cuprous chloride and bipyridyl (the mass ratio of the cuprous chloride to the bipyridyl is 1 percent to 3 percent) are added to react for 10h at 40 ℃, after the reaction is finished, the reaction solution is subjected to suction filtration, and the microspheres are washed by water and ethanol in sequence until the microspheres are colorless, so that a hydrophobic/strong cation exchange chromatography medium is prepared, and the grafting amount is 0.30g/g, the ion exchange capacity is 0.75mmol/g, the protein loading amount is 97mg/mL, and the protein recovery rate is 98 percent by weight.
The invention prepares the multi-mode chromatography medium by a surface-initiated free radical graft polymerization method, wherein the grafted polymer chain consists of random copolymers with different hydrophobicity and ion exchange functions, and vinyl monomers with different hydrophobicity and vinyl monomers with different ion exchange functions can be combined as required, so that the precise control of the hydrophobicity and the ion exchange function is realized, and the multi-mode chromatography medium has higher protein loading capacity.
The embodiments of the present invention have been described above by way of example. However, the scope of the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims (10)
1. The high-loading multi-mode chromatography medium is characterized by comprising a chromatography matrix and a ligand, wherein the chromatography matrix is C with amino, hydroxyl, carboxyl, sulfydryl or substituted by chlorine on the surface 6-20 Porous microspheres of aryl groups;
the ligand is hydrophobic alkenyl monomer and alkenyl monomer with ion exchange function, and the hydrophobic alkenyl monomer and the alkenyl monomer with ion exchange function are different;
the hydrophobic alkenyl monomer is selected from at least one of structural formulas shown as the following formula 1 and formula 2,
C 4-18 alkylene-C (O) O-C 4-18 Alkylene group of formula 2
In the formula 1, R 1 、R 2 、R 3 Identical or different, independently of one another, from H, C 1-20 An alkyl group;
R 4 is selected from-C (= O) OR b Substituted or unsubstituted C 6-20 Aryl, substituent R a Is C 1-20 An alkyl group; r b Is selected from C 1-20 An alkyl group;
the alkenyl monomer with the ion exchange function is selected from at least one of structural formulas shown as the following formula 3,
in formula 3, R 5 、R 6 、R 7 Identical or different, independently of one another, from H, C 1-20 An alkyl group;
R 8 selected from substituted or unsubstituted C 6-20 Aryl group, -C (= O) OR c 、-C(＝O)N-R d 、-(CH 2 ) n -SO 3 H, substituent R e Is a carboxyl group, an amino group or a sulfonic group; r is c Selected from H,
Wherein n is the same or different and is independently selected from an integer of 1 to 10, R 9 、R 10 、R 11 、R 12 、R 13 Same or different and independently selected from H, C 1-10 An alkyl group; x is halogen;
R d selected from substituted or unsubstituted- (CH) 2 ) m -SO 3 H, m is an integer of 1-10, and the substituent is C 1-12 An alkyl group.
2. The chromatography media of claim 1, wherein R in formula 1 is 1 、R 2 、R 3 Same or different and independently selected from H, C 1-10 An alkyl group;
in the formula 1, R 4 Selected from-C (= O) OR b Substituted or unsubstituted C 6-14 Aryl, substituent R a Is C 1-10 An alkyl group; r is b Is selected from C 1-10 An alkyl group;
preferably, the structural formula shown in formula 2 is C 4-10 alkylene-C (O) O-C 4-10 An alkylene group.
3. The chromatography medium according to claim 1 or 2, wherein R in formula 3 is 5 、R 6 、R 7 Identical or different, independently of one another, from H, C 1-10 An alkyl group.
In the formula 3, R 8 Selected from substituted or unsubstituted C 6-14 Aryl, -C (= O) OR c 、-C(＝O)N-R d 、-(CH 2 ) n -SO 3 H, substituent R e Is carboxyl, amino or sulfonic group;
R c selected from H,
Wherein n is the same or different and is independently selected from an integer of 1 to 6, R 9 、R 10 、R 11 、R 12 、R 13 Same or different and independently selected from H, C 1-6 An alkyl group; x is Cl or Br;
R d selected from substituted or unsubstituted- (CH) 2 ) m -SO 3 H, m is an integer of 1-6, and the substituent is C 1-6 An alkyl group.
4. The chromatography media of any of claims 1-3, wherein the hydrophobic ethylenic monomer is selected from at least one of butyl methacrylate, octyl methacrylate, dodecyl methacrylate, ethylhexyl methacrylate, styrene.
Preferably, the alkenyl monomer having an ion exchange function is selected from at least one of dimethylaminoethyl methacrylate, acyloxyethyltrimethyl ammonium methacrylate, methacrylic acid sulfonic acid, and 2-acrylamido-2-methylpropanesulfonic acid.
Preferably, the molar ratio of the hydrophobic alkenyl monomer to the alkenyl monomer having an ion exchange function is 1 (0.5-20).
5. A chromatography media according to any of claims 1-4, wherein the chromatography matrix has a pore size of 20-3000nm and a particle size of 20-200um.
Preferably, the chromatography matrix is at least one selected from polysaccharide microspheres with hydroxyl groups on the surface, polyacrylate microspheres with amino groups on the surface, polystyrene microspheres with carboxyl groups on the surface, polyacrylate microspheres with sulfhydryl groups on the surface and polysaccharide microspheres with benzyl chloride groups on the surface.
Preferably, the mass ratio of the chromatography matrix to the hydrophobic alkenyl monomer is 1 (0.04-20).
Preferably, the chromatography medium is prepared by grafting hydrophobic vinyl monomer and vinyl monomer with ion exchange function on the surface of the chromatography matrix in the form of polymer.
6. The chromatography media of any one of claims 1-5, wherein the high-loading multimodal chromatography media has a grafting amount of 0.2-1g/g.
Preferably, the protein load of the high-load multi-mode chromatography medium is 15-100mg bovine serum albumin/mL chromatography medium.
Preferably, the high-loading multimodal chromatography medium has an ion exchange capacity of 0.3 to 9mmol/g.
Preferably, the high load multimode chromatography media has a protein recovery of 90% or more.
Preferably, the high-loading multimode chromatographic medium has a mechanical strength of 0.3 to 10MPa.
7. A method of preparing an elevated load multi-modal chromatography media as recited in any of claims 1-6, wherein the method comprises the steps of:
mixing and reacting a chromatography matrix, a hydrophobic alkenyl monomer, an alkenyl monomer with an ion exchange function and an initiator to prepare the high-load multi-mode chromatography medium.
8. The method of claim 7, wherein the initiator is selected from at least one of cerium ammonium sulfate, cerium ammonium nitrate, potassium persulfate, ammonium persulfate, cuprous chloride.
Preferably, the mass of the initiator is 0.5-5% of the chromatography matrix.
9. The method according to claim 7, wherein the reaction temperature is 15-60 ℃ and the reaction time is 6-24h.
10. Use of the high load multimodal chromatography medium according to any of claims 1-6 in the field of protein chromatographic separations.
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