CN114989359B - Cation exchange chromatography medium and preparation method thereof - Google Patents
Cation exchange chromatography medium and preparation method thereof Download PDFInfo
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- CN114989359B CN114989359B CN202210712663.0A CN202210712663A CN114989359B CN 114989359 B CN114989359 B CN 114989359B CN 202210712663 A CN202210712663 A CN 202210712663A CN 114989359 B CN114989359 B CN 114989359B
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- 239000012501 chromatography medium Substances 0.000 title claims abstract description 56
- 238000005277 cation exchange chromatography Methods 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 239000000178 monomer Substances 0.000 claims abstract description 46
- XLPJNCYCZORXHG-UHFFFAOYSA-N 1-morpholin-4-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCOCC1 XLPJNCYCZORXHG-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000011159 matrix material Substances 0.000 claims abstract description 12
- 239000002253 acid Substances 0.000 claims abstract description 8
- 239000004005 microsphere Substances 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 239000008367 deionised water Substances 0.000 claims description 24
- 229910021641 deionized water Inorganic materials 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 17
- 229920000058 polyacrylate Polymers 0.000 claims description 10
- OZECDDHOAMNMQI-UHFFFAOYSA-H cerium(3+);trisulfate Chemical group [Ce+3].[Ce+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O OZECDDHOAMNMQI-UHFFFAOYSA-H 0.000 claims description 9
- 238000010559 graft polymerization reaction Methods 0.000 claims description 7
- 239000003999 initiator Substances 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 6
- 229920003053 polystyrene-divinylbenzene Polymers 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- HKVFISRIUUGTIB-UHFFFAOYSA-O azanium;cerium;nitrate Chemical compound [NH4+].[Ce].[O-][N+]([O-])=O HKVFISRIUUGTIB-UHFFFAOYSA-O 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims 1
- 238000001179 sorption measurement Methods 0.000 abstract description 17
- 230000015784 hyperosmotic salinity response Effects 0.000 abstract description 11
- 102000004169 proteins and genes Human genes 0.000 abstract description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 230000000052 comparative effect Effects 0.000 description 22
- 238000011068 loading method Methods 0.000 description 18
- 238000005342 ion exchange Methods 0.000 description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 238000004255 ion exchange chromatography Methods 0.000 description 11
- 150000003839 salts Chemical class 0.000 description 10
- 239000000523 sample Substances 0.000 description 9
- 239000012452 mother liquor Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000006116 polymerization reaction Methods 0.000 description 5
- 230000005526 G1 to G0 transition Effects 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000005341 cation exchange Methods 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 206010042674 Swelling Diseases 0.000 description 2
- 238000010560 atom transfer radical polymerization reaction Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 238000007334 copolymerization reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 125000003700 epoxy group Chemical group 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- UUORTJUPDJJXST-UHFFFAOYSA-N n-(2-hydroxyethyl)prop-2-enamide Chemical compound OCCNC(=O)C=C UUORTJUPDJJXST-UHFFFAOYSA-N 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000000379 polymerizing effect Effects 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 2
- 229920002554 vinyl polymer Polymers 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical group NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 229920000578 graft copolymer Polymers 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000002500 ions Chemical group 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000002685 polymerization catalyst Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F265/00—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00
- C08F265/04—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00 on to polymers of esters
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/264—Synthetic macromolecular compounds derived from different types of monomers, e.g. linear or branched copolymers, block copolymers, graft copolymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
- B01J39/08—Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
- B01J39/16—Organic material
- B01J39/18—Macromolecular compounds
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F257/00—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00
- C08F257/02—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00 on to polymers of styrene or alkyl-substituted styrenes
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- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Analytical Chemistry (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a cation exchange chromatography medium and a preparation method thereof. The cation exchange chromatography medium comprises a matrix, wherein the surface of the matrix is grafted with monomers, the monomers comprise 4-acryloylmorpholine monomer and 2-acrylamido-2-methyl-N-propane sulfonic acid monomer, and the mass ratio of the 4-acryloylmorpholine monomer to the 2-acrylamido-2-methyl-N-propane sulfonic acid monomer is (1:90) to (99:1). The cation exchange chromatography medium has good salt tolerance and high adsorption load, and can also show high adsorption load to antibody protein under the condition of high conductivity.
Description
Technical Field
The invention relates to the technical field of ion exchange chromatography media, in particular to a cation exchange chromatography medium and a preparation method thereof.
Background
Ion exchange chromatography is the most common method of purifying pharmaceutical proteins, peptides and other biological macromolecules, with the advantages of high loading, ease of handling, and the like, and almost all industrial purification processes involve one or more ion exchange steps. Chromatography media can be divided into two general categories, cationic and anionic, according to the type of functional groups that are ion exchanged. Among them, cation exchange is particularly widely used in the step of removing multimers of antibody proteins in purification.
The principle of ion exchange chromatography is to use the interaction between charged molecules in a sample and oppositely charged functional groups in the stationary phase of the chromatographic medium. The mobile phase has lower electric conductivity (low salt concentration) when the sample is loaded, the interaction force of ion exchange charges is strong, and the sample is adsorbed on the chromatographic medium stationary phase. When the mobile phase conductivity is increased (salt solubility increases), the ion exchange charge effect of the sample and the stationary phase is masked and can be eluted. The isoelectric points (PI values) of different biomolecules are different, so that the binding force with the ion exchange stationary phase is different, and the chromatographic separation effect can be achieved by gradient elution of different salt concentrations.
The adsorption capacity of conventional ion exchange chromatography media is very sensitive to the conductance of the loaded sample solution and is very poor in salt tolerance. When the conductance in the sample solution exceeds 10ms/cm, the ion exchange adsorption capacity is significantly reduced. For biomolecule sample solutions with higher salt concentrations, it is often necessary to desalt or dilute the sample prior to the ion exchange step to load the sample, thereby increasing the cost and steps of chromatographic purification. Thus, a salt tolerant ion exchange chromatography media that can maintain higher adsorption loadings at higher conductivities is preferred.
Surface graft polymerization (tentacle grafting) is a commonly used prior art (prior art) ion exchange chromatography media for preparing high adsorption loadings, and ion exchange packing materials obtained by graft polymerizing the corresponding ion exchange functionalized monomer onto the surface of a different chromatography media have long been known from W.M U.S. chromatography,1990,510,133-140. In addition, the preparation of ion exchange chromatography media by graft copolymerization is disclosed in patent EP0337144B1 or in US patent US5453186a, which are intended for chromatographic separation processes of biomolecules. These ion exchange chromatography media comprise graft copolymerization of two or more monomers at the surface of the media. The monomer contains a cation exchange group or an anion exchange group or a neutral monomer. These surface graft copolymerized ion exchange chromatographic media can provide higher adsorption loadings or better selectivity for target biomolecule separation.
CN108276526B discloses a high-loading large-aperture polymer cation exchange chromatography medium and its preparation. The cation exchange chromatography medium takes polyacrylate or polystyrene microsphere as a matrix, and vinyl functional monomers are grafted on the microsphere surface. The preparation method comprises the following steps: swelling polyacrylate or polystyrene microsphere in vinyl functional monomer solution; adding an atom transfer radical polymerization catalyst ligand system into the microspheres after the swelling treatment, carrying out graft polymerization, removing unreacted monomers after the polymerization, and carrying out vacuum drying to obtain the cation exchange chromatography medium. The cation exchange chromatography medium prepared by the method has high ion exchange capacity, high protein binding load and high operation flow rate, and is suitable for rapid separation and purification of biological macromolecules.
CN112871147a discloses a preparation method of a chromatography medium for removing multimers in monoclonal antibodies, which comprises the steps of mixing oil phase components containing two reaction monomers and a cross-linking agent uniformly, adding the mixture into a water phase, stirring, heating and polymerizing to obtain a ternary polymerization porous material; initiating surface grafting of the ternary polymerization porous material by using an acrylamide monomer through atom transfer radical polymerization, so as to obtain polyacrylamide long-chain molecules on the surface of the ternary polymerization porous material; and opening the ring of the epoxy group on the surface of the ternary polymerization porous material to enable the epoxy group to be derived into a cation exchange functional group with negative charge, thereby preparing the chromatography medium for removing the multimer in the monoclonal antibody. The prepared chromatographic medium has the functions of volume exclusion and cation exchange, can rapidly and efficiently remove the multimers in the monoclonal antibody, and has important application significance in improving the yield-resistant efficiency of the monoclonal antibody.
However, the graft copolymer ion chromatography medium prepared by the prior art is as sensitive to the conductance of the loading solution as the conventional ion exchange chromatography medium, the solubility of salt in the solution is slightly high, and the ion exchange adsorption capacity can be obviously reduced.
Therefore, it is necessary to develop a cation exchange chromatography medium with salt tolerance and high loading adsorption and a preparation method thereof.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a cation exchange chromatography medium and a preparation method thereof, wherein the cation exchange chromatography medium has good salt tolerance and high adsorption load, and can also show high adsorption load to antibody protein under the condition of high electric conductivity of more than 8 ms/cm.
To achieve the purpose, the invention adopts the following technical scheme:
a cation exchange chromatography medium comprising a substrate having a surface grafted with monomers comprising 4-acryloylmorpholine monomer and 2-acrylamido-2-methyl-N-propanesulfonic acid monomer, the mass ratio of the 4-acryloylmorpholine monomer to the 2-acrylamido-2-methyl-N-propanesulfonic acid monomer being from (1:90) to (99:1).
When the grafting comonomer contains 4-acryloylmorpholine neutral monomer and 2-acrylamido-2-methyl-N-propane sulfonic Acid Monomer (AMPSA), and the dosage of the grafting comonomer and the monomer is regulated, the synthesized ion exchange chromatography medium can provide salt-resistant high-load adsorption, and can also show high adsorption load to antibody protein (immunoglobin) under the condition of high conductivity.
The high electric conductivity referred to in the present invention means electric conductivity higher than 8ms/cm, particularly higher than 10ms/cm.
The mass ratio of the 4-acryloylmorpholine monomer to the 2-acrylamido-2-methyl-N-propanesulfonic acid monomer is (1:90) to (99:1), for example 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:5, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, etc., preferably (1:90) to (99:10). If the mass ratio is too large, the loading will be too low, and if the mass ratio is too small, the chromatography medium will be poorly resistant to high salts.
The mass ratio of the matrix to the 4-acryloylmorpholine monomer is (0.1:1) to (100:1), preferably (5:1) to (50:1), for example 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1, etc.). If the mass of 4-acryloylmorpholine monomer is too much, the loading will be reduced, and if the mass of 4-acryloylmorpholine monomer is too little, the high salt tolerance will be reduced.
The matrix is microsphere.
The microsphere is one of polyacrylate microsphere, polystyrene-divinylbenzene microsphere, agarose microsphere, dextran microsphere or cellulose microsphere.
The second object of the present invention is to provide a method for preparing a cation exchange chromatography medium according to one of the objects, comprising the steps of:
mixing the substrate with deionized water according to a proportion, adding 4-acryloylmorpholine monomer for dissolution, adding an initiator for graft polymerization reaction, and carrying out suction filtration and cleaning on the substrate to obtain the cation exchange chromatography medium.
The graft polymerization reaction temperature is 30-50deg.C, such as 30deg.C, 31deg.C, 32deg.C, 33deg.C, 35deg.C, 37deg.C, 38deg.C, 39deg.C, 40deg.C, 41 deg.C, 42 deg.C, 43deg.C, 45deg.C, 46 deg.C, 47 deg.C, 48deg.C, 49deg.C, 50deg.C, etc.; the time is 5-8h, for example 5h, 6h, 7h or 8h, etc.
Preferably, the initiator is cerium sulfate or ammonium cerium nitrate;
preferably, the mass ratio of the monomer to initiator is from (10:1) to (100:1), for example 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1, etc.
Preferably, the preparation method of the cation exchange chromatography medium comprises the following steps:
adding the substrate and deionized water into a reaction kettle according to the proportion, adding 4-acryloylmorpholine and 2-acrylamido-2-methyl-N-propane sulfonic acid monomer for dissolution, adding an initiator at 30-50 ℃ for graft polymerization for 5-8h, and carrying out suction filtration and cleaning on the substrate to obtain the cation exchange chromatography medium.
Compared with the prior art, the invention has the beneficial effects that:
the cation exchange chromatography medium has good salt tolerance and high adsorption load, and can also show high adsorption load to antibody protein under the high-conductivity condition of more than 8 ms/cm. Specifically, the cation exchange chromatography medium with ion exchange capacity has the ion exchange capacity of 0.09-0.13mmol/mL, the dynamic capacity of the antibody under the 4.1ms/cm conductivity condition of 108-143mg/mL and the dynamic capacity of the antibody under the 10.2ms/cm conductivity condition of 122-161mg/mL.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments.
The various starting materials of the present invention are commercially available, or may be prepared according to methods conventional in the art, unless specifically indicated.
Example 1
The cation exchange chromatography media of this example were prepared by the following method:
80g of polyacrylate microspheres and 200g of deionized water are added into a 500mL three-neck flask, the system temperature is controlled at 40 ℃, 7.7g of 4-acryloylmorpholine and 4.1g of 2-acrylamido-2-methyl-N-propanesulfonic acid are added into the flask and stirred for 10 minutes, then 0.5g of cerium sulfate is added into the flask, the system is stirred for 6 hours, after the time, the microspheres are filtered to remove mother liquor by suction, and then are washed with 300mL of deionized water, 300mL of ethanol and 500mL of deionized water in sequence, so as to obtain the cation exchange chromatography medium.
Example 2
80g of polyacrylate microspheres and 200g of deionized water are added into a 500mL three-neck flask, the temperature of the system is controlled at 40 ℃, 3.8g of 4-acryloylmorpholine and 8.3g of 2-acrylamido-2-methyl-N-propanesulfonic acid are added into the flask and stirred for 10 minutes, then 0.5g of cerium sulfate is added into the flask, the system is stirred for 6 hours, after the time, the microspheres are filtered to remove mother liquor by suction, and then are washed with 300mL of deionized water, 300mL of ethanol and 500mL of deionized water in sequence, so as to obtain the cation exchange chromatography medium.
Example 3
80g of polystyrene-divinylbenzene microspheres and 200g of deionized water are added into a 500-mL three-neck flask, the temperature of the system is controlled at 40 ℃, 7.7g of 4-acryloylmorpholine and 4.1g of 2-acrylamido-2-methyl-N-propanesulfonic acid are added into the flask and stirred for 10 minutes, then 0.5g of cerium sulfate is added into the flask, the system is stirred for 6 hours, after the time is up, the microspheres are pumped and filtered to remove mother liquor, and then are washed with 300mL of deionized water, 300mL of ethanol and 500mL of deionized water in sequence, so that the cation exchange chromatography medium is obtained.
Example 4
This example differs from example 1 in that the matrix was replaced with agarose microspheres, and the other is the same as example 1.
Comparative example 1
The cation exchange chromatography medium of this comparative example was prepared by the following method:
80g of polyacrylate microspheres and 200g of deionized water are added into a 500mL three-neck flask, the system temperature is controlled at 40 ℃, 12.4g of 2-acrylamido-2-methyl-N-propane sulfonic acid is added into the flask and stirred for 10 minutes, then 0.5g of cerium sulfate is added into the flask, the system is continuously stirred for 6 hours, after the time is up, the microspheres are pumped and filtered to remove mother liquor, and then are washed with 300mL of deionized water, 300mL of ethanol and 500mL of deionized water in sequence, so that the cation exchange chromatography medium is obtained.
Comparative example 2
The cation exchange chromatography medium of this comparative example was prepared by the following method:
80g of polystyrene-divinylbenzene microspheres and 200g of deionized water are added into a 500mL three-necked flask, the system temperature is controlled at 40 ℃, 12.4g of 2-acrylamido-2-methyl-N-propane sulfonic acid is added into the flask and stirred for 10 minutes, then 0.5g of cerium sulfate is added into the flask, the system is stirred for 6 hours, after the time is up, the microspheres are pumped and filtered to remove mother liquor, and then are washed with 300mL of deionized water, 300mL of ethanol and 500mL of deionized water in sequence, so as to obtain the cation exchange chromatography medium.
Comparative example 3
The cation exchange chromatography medium of this comparative example was prepared by the following method:
80g of polyacrylate microspheres and 200g of deionized water are added into a 500mL three-neck flask, the temperature of the system is controlled at 40 ℃, 7.7g of 4-acryloylmorpholine is added into the flask and stirred for 10 minutes, then 0.5g of cerium sulfate is added into the flask, the system is stirred for 6 hours, after the time is up, the microspheres are pumped and filtered to remove mother liquor, and then are washed with 300mL of deionized water, 300mL of ethanol and 500mL of deionized water in sequence, so that the cation exchange chromatography medium is obtained.
Comparative example 4
This comparative example differs from example 1 in that the amount of 4-acryloylmorpholine added was too much, specifically 10g of 4-acryloylmorpholine and 0.1g of 2-acrylamido-2-methyl-N-propanesulfonic acid, and the other was the same as in example 1.
Comparative example 5
This comparative example differs from example 1 in that the amount of 4-acryloylmorpholine added was too small, specifically 0.1g of 4-acryloylmorpholine and 10g of 2-acrylamido-2-methyl-N-propanesulfonic acid, and the other was the same as in example 1.
Comparative example 6
The monomers used in this comparative example are conventional monomers in the prior art, and the specific preparation method is as follows:
80g of polyacrylate microspheres and 200g of deionized water are added into a 500mL three-neck flask, the system temperature is controlled at 40 ℃, 6.3g N-acryloylethanolamine and 4.1g of 2-acrylamido-2-methyl-N-propane sulfonic acid are added into the flask and stirred for 10 minutes, then 0.5g of cerium sulfate is added into the flask, the system is stirred for 6 hours, after the time is up, the microspheres are filtered to remove mother liquor by suction, and then are washed with 300mL of deionized water, 300mL of ethanol and 500mL of deionized water in sequence, so that the cation exchange chromatography medium is obtained.
The cation exchange chromatography media prepared in examples 1 to 4 and comparative examples 1 to 6 were subjected to ion exchange amounts and dynamic loads of antibodies under different conductivities, and experimental results are shown in table 1.
The test of the ion exchange capacity is referred to national standard GB/T8144-2008, and the test method of the dynamic loading of the antibody under different conductivities is as follows:
and (3) after balancing the chromatographic column by using the 5CV balancing solution, switching to a sample loading solution, continuing sample injection until UV 10% is penetrated, wherein the sample quantity corresponding to the penetrating volume is the dynamic loading capacity of the chromatographic column, and the specific parameters are as follows:
7mm I.D.x 25mm;
equilibration solution in 20mM morpholinoethanesulfonic acid buffer (pH 4.8);
low salt loading (4.1 ms/cm) antibody protein IgG (2 mg/mL) was dissolved in 20mM morpholinoethanesulfonic acid buffer (pH 4.8) +40mM sodium chloride;
high salt loading (10.2 ms/cm) antibody protein IgG (2 mg/mL) was dissolved in 20mM morpholinoethanesulfonic acid buffer (pH 4.8) +100mM sodium chloride;
the flow rate is 0.5mL/min;
a detector: sieve SCG-030 (UV@280 nm).
TABLE 1
As can be seen from the data in Table 1, the cation exchange chromatography media of the present invention have good salt tolerance, high adsorption capacity, and also exhibit high adsorption capacity to antibody proteins under high conductance (10.2 ms/cm) conditions. Specifically, the cation exchange chromatography medium with ion exchange capacity has the ion exchange capacity of 0.09-0.13mmol/mL, the dynamic capacity of the antibody under the 4.1ms/cm conductivity condition of 108-143mg/mL and the dynamic capacity of the antibody under the 10.2ms/cm conductivity condition of 122-161mg/mL.
Comparative data for example 1 and comparative example 1 (polyacrylate microsphere matrix), example 3 and comparative example 2 (polystyrene-divinylbenzene microsphere matrix), example 1 and comparative example 6 (conventional monomer N-acryloylethanolamine in the prior art) it can be seen that the cation exchange chromatography media synthesized using 4-acryloylmorpholine as a spacer monomer in the present invention has significant salt tolerance.
It can be seen from examples 1, 2 and comparative example 1 that the higher the ratio of 4-acryloylmorpholine used, the higher the salt tolerance, while the dynamic loading can be significantly improved by using 4-acryloylmorpholine.
Example 1 and comparative example 3 show that the use of two monomers in example 1 gives a significant increase in loading relative to comparative example 3, which uses only one monomer, 4-acryloylmorpholine.
As can be seen from examples 1, 1 and 2, the use of only one monomer, 2-acrylamido-2-methyl-N-propanesulfonic acid, for example 1, results in a significant increase in loading and an increase in salt tolerance.
Too much 4-acryloylmorpholine in comparative example 4 added results in a significant reduction in the loading.
Too little of 4-acryloylmorpholine in comparative example 5 was added to significantly lower the high salt tolerance.
The detailed process equipment and process flow of the present invention are described by the above embodiments, but the present invention is not limited to, i.e., it does not mean that the present invention must be practiced depending on the detailed process equipment and process flow. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (5)
1. A cation exchange chromatography medium comprising a matrix, the surface of the matrix having grafted thereto monomers comprising 4-acryloylmorpholine monomer and 2-acrylamido-2-methyl-N-propanesulfonic acid monomer, the mass ratio of 4-acryloylmorpholine monomer to 2-acrylamido-2-methyl-N-propanesulfonic acid monomer being from (1:5) to (5:1);
the mass ratio of the matrix to the 4-acryloylmorpholine monomer is (10:1) to (50:1);
the matrix is a microsphere, and the microsphere is a polyacrylate microsphere or a polystyrene-divinylbenzene microsphere.
2. A method of preparing a cation exchange chromatography medium according to claim 1, comprising the steps of:
mixing the substrate with deionized water, adding 4-acryloylmorpholine monomer and 2-acrylamido-2-methyl-N-propane sulfonic acid monomer for dissolution, adding initiator for graft polymerization reaction, and filtering and cleaning the substrate to obtain the cation exchange chromatography medium.
3. The process according to claim 2, wherein the graft polymerization is carried out at a temperature of 30 to 50℃for a period of 5 to 8 hours.
4. The method of claim 2, wherein the initiator is cerium sulfate or ammonium cerium nitrate.
5. The method of claim 2, wherein the mass ratio of monomer to initiator is from (10:1) to (100:1).
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