CN110339828B - Chromatography medium with benzothiazolyl thiocarboxylic acid as functional ligand and preparation method thereof - Google Patents
Chromatography medium with benzothiazolyl thiocarboxylic acid as functional ligand and preparation method thereof Download PDFInfo
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- 239000003446 ligand Substances 0.000 title claims abstract description 58
- 239000012501 chromatography medium Substances 0.000 title claims abstract description 42
- JZEJEEZKZROKOZ-UHFFFAOYSA-N 1,3-benzothiazole-2-carbothioic s-acid Chemical compound C1=CC=C2SC(C(=O)S)=NC2=C1 JZEJEEZKZROKOZ-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 239000011159 matrix material Substances 0.000 claims abstract description 41
- 239000012504 chromatography matrix Substances 0.000 claims abstract description 37
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims abstract description 36
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 claims abstract description 24
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical compound NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000002156 mixing Methods 0.000 claims abstract description 21
- BHELZAPQIKSEDF-UHFFFAOYSA-N allyl bromide Chemical compound BrCC=C BHELZAPQIKSEDF-UHFFFAOYSA-N 0.000 claims abstract description 15
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 12
- 239000001632 sodium acetate Substances 0.000 claims abstract description 12
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 12
- 239000004005 microsphere Substances 0.000 claims abstract description 7
- 230000008878 coupling Effects 0.000 claims abstract description 5
- 238000010168 coupling process Methods 0.000 claims abstract description 5
- 238000005859 coupling reaction Methods 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 53
- 239000008367 deionised water Substances 0.000 claims description 44
- 229910021641 deionized water Inorganic materials 0.000 claims description 44
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 33
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 33
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 26
- 239000002609 medium Substances 0.000 claims description 26
- 238000005406 washing Methods 0.000 claims description 26
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 22
- DXSBAOMLHPFLMW-UHFFFAOYSA-N 3-(1,3-benzothiazol-2-ylsulfanyl)propanoic acid Chemical compound C1=CC=C2SC(SCCC(=O)O)=NC2=C1 DXSBAOMLHPFLMW-UHFFFAOYSA-N 0.000 claims description 13
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 13
- 238000000967 suction filtration Methods 0.000 claims description 13
- 230000003213 activating effect Effects 0.000 claims description 11
- 239000011543 agarose gel Substances 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 11
- -1 hexafluorophosphate Chemical compound 0.000 claims description 11
- 239000011259 mixed solution Substances 0.000 claims description 11
- ZZUQWNYNSKJLPI-UHFFFAOYSA-N 2-(1,3-benzothiazol-2-ylsulfanyl)acetic acid Chemical compound C1=CC=C2SC(SCC(=O)O)=NC2=C1 ZZUQWNYNSKJLPI-UHFFFAOYSA-N 0.000 claims description 10
- ZRMKAMAPVMHSFW-UHFFFAOYSA-N 2-(1,3-benzothiazol-2-ylsulfanyl)propanoic acid Chemical compound C1=CC=C2SC(SC(C)C(O)=O)=NC2=C1 ZRMKAMAPVMHSFW-UHFFFAOYSA-N 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- 150000003566 thiocarboxylic acids Chemical class 0.000 claims description 4
- 239000001913 cellulose Substances 0.000 claims description 2
- 229920002678 cellulose Polymers 0.000 claims description 2
- 238000000034 method Methods 0.000 claims description 2
- 238000005086 pumping Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 21
- 230000004913 activation Effects 0.000 abstract description 19
- 238000000926 separation method Methods 0.000 abstract description 16
- 238000001179 sorption measurement Methods 0.000 abstract description 13
- 125000000524 functional group Chemical group 0.000 abstract description 2
- 230000015784 hyperosmotic salinity response Effects 0.000 abstract description 2
- 238000005576 amination reaction Methods 0.000 abstract 1
- 238000011084 recovery Methods 0.000 abstract 1
- 238000007789 sealing Methods 0.000 abstract 1
- 239000000758 substrate Substances 0.000 description 16
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- 102000004169 proteins and genes Human genes 0.000 description 11
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000010828 elution Methods 0.000 description 5
- 238000012434 mixed-mode chromatography Methods 0.000 description 5
- 229910003460 diamond Inorganic materials 0.000 description 4
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- 230000000694 effects Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- IOJUPLGTWVMSFF-UHFFFAOYSA-N benzothiazole Chemical class C1=CC=C2SC=NC2=C1 IOJUPLGTWVMSFF-UHFFFAOYSA-N 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
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- YHMYGUUIMTVXNW-UHFFFAOYSA-N 1,3-dihydrobenzimidazole-2-thione Chemical compound C1=CC=C2NC(S)=NC2=C1 YHMYGUUIMTVXNW-UHFFFAOYSA-N 0.000 description 1
- FQXRXTUXSODUFZ-UHFFFAOYSA-N 1h-imidazol-2-ylmethanethiol Chemical compound SCC1=NC=CN1 FQXRXTUXSODUFZ-UHFFFAOYSA-N 0.000 description 1
- DAPOMFMFTBUAKL-UHFFFAOYSA-N 2-ethylpyridine-3-thiol Chemical compound CCC1=NC=CC=C1S DAPOMFMFTBUAKL-UHFFFAOYSA-N 0.000 description 1
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- 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
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a chromatography medium with benzothiazolyl thiocarboxylic acid as functional ligand and a preparation method thereof. Taking hydrophilic porous microspheres as a chromatography matrix, and sequentially adding dimethyl sulfoxide and allyl bromide for activation; reacting the activated chromatography matrix with N-bromosuccinimide to perform bromoalcoholization; mixing the bromoalcoholized chromatography matrix with a hexamethylenediamine solution for amination; further coupling the aminated matrix with benzothiazolylthiocarboxylic acid ligand; and finally, sealing unreacted amino by adopting sodium acetate and acetic anhydride solution to obtain the chromatography medium taking benzothiazolyl thiocarboxylic acid as a functional group. The novel chromatographic medium has the characteristics of large antibody adsorption capacity, strong salt tolerance and the like, can realize the high-efficiency recovery of the antibody by adjusting the pH to weak acidity, has simple and convenient preparation process and low price, and can be used for antibody separation.
Description
Technical Field
The invention relates to a chromatography medium using benzothiazolyl thiocarboxylic acid as functional ligand and a preparation method thereof, belonging to the protein chromatography separation technology in the field of biochemical engineering.
Background
The antibody product often has high purity requirements while maintaining biological activity, and thus conventional separation processes are often difficult to meet. The protein A or protein G affinity chromatography medium can be specifically combined with an antibody, but the protein affinity ligand is easy to fall off, the repeated use times are low, the medium price is high, the use cost is extremely high, and the large-scale application is limited. Although the traditional methods such as ion exchange chromatography and hydrophobic interaction chromatography can combine antibodies, the specificity and selectivity are poor, the number of separation steps is large, and the purification effect is limited. Therefore, the development of non-protein ligands with antibody selectivity is the focus of research and development.
The non-protein ligands for antibody separation mainly include bionic affinity ligands, polypeptide ligands and the like, and the ligands usually pay more attention to specificity and affinity, so that the antibody elution condition is harsh or the yield is low. Mixed mode chromatography is a novel biological separation technology, and the ligand has multiple functional groups and can generate multiple interactions such as hydrophobicity, static electricity and the like with target protein. The ligand density of the mixed-mode medium is generally higher, the adsorption capacity is large, the mixed-mode medium has salt-tolerant adsorption characteristics, the elution condition is mild, and the mixed-mode medium is particularly suitable for large-scale separation and purification and has been applied to the separation and purification of proteins such as antibodies and the like. The key to mixed mode chromatography is the specially designed combination of multiple interacting functional ligands. Patents CN101402671B, CN101899110B and CN101948535A report that mixed mode chromatography media using mercaptoethylpyridine, mercaptomethylimidazole and mercaptobenzimidazole as ligands separate immunoglobulin from livestock and poultry blood, and have the characteristics of simple steps, high separation efficiency and low cost. Patent EP2459308A1 reports the preparation of mixed mode chromatography media with benzothiazole compounds as ligands to achieve the separation and purification of various proteins. Patents US6919436B2 and CN1972961B report that benzothiazole compounds are used as separation ligands of chromatography media, coupled to agarose matrix, and used for separation and purification of proteins such as serum or plasma. Wang et al (J.Chromatogr. B,2013,936:33) separated IgG from mock serum using mixed mode media Bestarose Diamond MMA, Bestarose Diamond MMC, MEP HyperCel and PPA HyperCel media, where Bestarose Diamond MMA media adsorbed IgG and HSA simultaneously with poor adsorption selectivity; bestarose Diamond MMC and PPARHyperCel media have difficulty in antibody elution and are not beneficial to practical separation and application; MEP HyperCel media adsorb mainly IgG, but still bind small amounts of HSA, with modest IgG selectivity. Therefore, the development of mixed mode media with better performance is of great significance to the large-scale separation and purification of antibodies.
Aiming at antibody separation, small molecular compound ligand libraries such as pyridine compounds, imidazole compounds, thiazole compounds, polyaromatic ring compounds and the like are screened, affinity characterization of ligands and IgG is carried out, and benzothiazolylthiocarboxylic acid compounds are found to have high affinity and strong salt tolerance to IgG and are potential novel mixed-mode ligands for antibody separation.
Disclosure of Invention
The invention aims to provide a chromatography medium taking benzothiazolylthiocarboxylic acid as a functional ligand and a preparation method thereof.
The chromatography medium with benzothiazolyl thiocarboxylic acid as functional ligand comprises a chromatography matrix and the ligand, wherein the chromatography matrix is hydrophilic porous microspheres with hydroxyl, the ligand is benzothiazolyl thiocarboxylic acid which is coupled after being activated by allyl bromide, and the benzothiazolyl thiocarboxylic acid is one of 3- (2-benzothiazolylthio) propionic acid, 2- (benzothiazolylthio) acetic acid or 2- (2-benzothiazolylthio) propionic acid.
When the benzothiazolylthiocarboxylic acid is 3- (2-benzothiazolylthio) propionic acid, the chromatographic medium structurally consists of:
when the benzothiazolylthiocarboxylic acid is 2- (benzothiazolylthio) acetic acid, the chromatographic medium structurally consists of:
when the benzothiazolylthiocarboxylic acid is 2- (2-benzothiazolylthio) propionic acid, the chromatographic medium structurally comprises:
the structure composition of the chromatography medium only shows a chromatography matrix and a ligand molecule structure, and is only an example for illustration, and the surface and the inner pore channel surface of the chromatography matrix are provided with a large number of ligand molecules.
The chromatography matrix is hydrophilic microspheres with a porous structure and surface hydroxyl groups, and the structural formula is as follows:
the structural formula is given only one-OH, and is merely illustrative, and the surface thereof has a large number of-OH.
The chromatography matrix is agarose gel or cellulose microspheres.
The density of the ligand of the chromatography medium is 40-140 mu mol/ml.
The preparation method of the chromatography medium with benzothiazolyl thiocarboxylic acid as functional ligand comprises the following steps:
1) after the chromatography matrix is dried by pumping, adding 20 percent (v/v) of dimethyl sulfoxide 0.2-1 time of the mass of the chromatography matrix, allyl bromide 0.1-1 time of the mass of the chromatography matrix and sodium hydroxide 0.1-0.5 time of the mass of the chromatography matrix, activating for 8-48 hours in a shaking table at the temperature of 25 ℃ and at the speed of 150rpm, performing suction filtration, and washing with deionized water to obtain an activated chromatography matrix;
2) mixing the activated chromatography matrix and N-bromosuccinimide with the mass of 0.1-0.5 time of that of the chromatography matrix for bromoalcoholization, reacting in a shaking table at 150rpm at 25 ℃ for 1-5 hours, performing suction filtration, and washing with deionized water to obtain a bromoalcoholized matrix;
3) mixing bromoalcoholized matrix, hexamethylenediamine 0.1-1 times of chromatography matrix mass and 0.5-1M sodium carbonate buffer solution 0.1-1 times of chromatography matrix mass, wherein the pH of the sodium carbonate buffer solution is 10-12, and reacting in a shaking table at 25 ℃ and 150rpm for 8-48 hours to obtain aminated matrix;
4) mixing an aminated matrix, benzothiazyl thiocarboxylic acid with the mass of 0.1-0.3 time of that of a chromatography matrix, 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate with the mass of 0.4-2 times of that of the chromatography matrix, and N, N-dimethylformamide solution of N, N-diisopropylethylamine with the mass of 0.8-4 times of that of the chromatography matrix, and reacting in a shaking table with 150rpm at 25 ℃ for 8-48 hours to obtain a medium after coupling of the benzothiazyl thiocarboxylic acid;
5) and (2) filtering the medium after coupling the benzothiazolyl thiocarboxylic acid, washing with deionized water, adding the filtered medium into a mixed solution of sodium acetate and acetic anhydride, reacting for 2-8 hours in a shaking table at the temperature of 25 ℃ and at the speed of 150rpm, and washing with deionized water to obtain the chromatography medium taking the benzothiazolyl thiocarboxylic acid as a functional ligand.
The chromatographic medium which is developed by the invention and takes benzothiazolylthiocarboxylic acid as functional ligand can be used for mixed mode chromatographic separation, and has the following advantages: (1) the antibody has large adsorption capacity and strong processing capacity, and the static adsorption capacity of hIgG can reach 140mg/ml wet medium; (2) the salt-tolerant adsorption property is strong, and the adsorption capacity is basically kept unchanged within a wider conductivity range (0-100 mS/cm); (3) the elution is convenient, the pH of the eluent is about 3.5, the complete elution can be realized, and the adverse effects of peracid, over-alkali or high salt and the like on the structure and the activity of the protein are avoided; (4) the medium has stable property, the ligand of the obtained medium is stable and is convenient to clean and regenerate by adopting allyl bromide to activate and couple the ligand. The novel mixed-mode chromatography medium developed by the invention takes benzothiazolylthiocarboxylic acid as a functional ligand, has the characteristics of obvious mixed-mode protein adsorption and chromatographic separation, has good adsorption capacity and selectivity on an antibody, and can be used for large-scale preparation of the antibody.
Drawings
FIG. 1 is a graph of the binding capacity of IgG adsorbed on a chromatography medium coupled with 3- (2-benzothiazolylthio) propionic acid in example 2.
Detailed Description
The invention is further described by way of examples below:
example 1
Taking 10g of the drained agarose gel, adding 2g of 20% (v/v) dimethyl sulfoxide, 1g of allyl bromide and 1g of sodium hydroxide, activating in a shaking table at the temperature of 25 ℃ and the speed of 150rpm for 48 hours, carrying out suction filtration, and washing with deionized water to obtain an activated matrix; then mixing the activated substrate and 1g N-bromosuccinimide for bromoalcoholization, reacting for 2 hours in a shaking table with 150rpm at 25 ℃, filtering, and washing with deionized water; then, mixing the brominated matrix with 2g of hexamethylenediamine and 1M sodium carbonate buffer (pH12), and reacting for 48 hours in a shaker at the temperature of 25 ℃ and at the speed of 150rpm to obtain an amino activated matrix; taking 1g of amino activation matrix, adding the amino activation matrix into 2mL of N, N-dimethylformamide containing 3g of 3- (2-benzothiazolylthio) propionic acid, 60mg of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and 37.5 mu L N, N-diisopropylethylamine, and reacting in a water bath shaking table at 25 ℃ for 8 hours; finally, the medium is filtered, washed by deionized water, added into the mixed solution of sodium acetate and acetic anhydride, reacted for 12 hours in a shaking table with 150rpm at 25 ℃, and washed by deionized water to obtain the chromatography medium taking 3- (2-benzothiazolylthio) propionic acid as the ligand, wherein the density of the ligand is 40 mu mol/ml.
Example 2
Taking 10g of the drained agarose gel, adding 10g of 20% (v/v) dimethyl sulfoxide, 10g of allyl bromide and 5g of sodium hydroxide, activating in a shaking table at the temperature of 25 ℃ and the speed of 150rpm for 48 hours, carrying out suction filtration, and washing with deionized water to obtain an activated matrix; then mixing the activated substrate and 5g N-bromosuccinimide for bromoalcoholization, reacting in a shaking table at 150rpm at 25 ℃ for 3 hours, filtering, and washing with deionized water; then, the bromo-substrate was mixed with 3g of hexamethylenediamine and 1M sodium carbonate buffer (pH12) and reacted in a shaker at 150rpm at 25 ℃ for 24 hours; taking 1g of amino activation matrix, adding the amino activation matrix into 2mL of N, N-dimethylformamide containing 10g of 3- (2-benzothiazolylthio) propionic acid, 100mg of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and 64 mu L N, N-diisopropylethylamine, and reacting for 8 hours in a water bath shaking table at 25 ℃; finally, the medium is filtered, washed by deionized water, added into the mixed solution of sodium acetate and acetic anhydride, reacted for 48 hours in a shaking table with 150rpm at 25 ℃, and washed by deionized water to obtain the chromatography medium taking 3- (2-benzothiazolylthio) propionic acid as the ligand, wherein the density of the ligand is 140 mu mol/ml. Under different pH values and NaCl concentrations, the binding capacity of the medium to human immunoglobulin IgG is measured, a loading capacity distribution diagram is shown in figure 1, the abscissa is pH, the ordinate is NaCl concentration, the color and the numerical value in the diagram represent the binding capacity, and the loading capacity unit is mg/ml medium, so that the IgG binding capacity is larger than 100mg/ml medium and can reach 140mg/ml medium at most in the range of pH 5.0-9.0 and 0-500 mM NaCl concentration, which indicates that the adsorption capacity is large, the influence of the salt concentration on the adsorption is small, and the salt-tolerant adsorption characteristic is obvious.
Example 3
Taking 10g of the drained agarose gel, adding 6g of 20% (v/v) dimethyl sulfoxide, 6g of allyl bromide and 4g of sodium hydroxide, activating for 25 hours in a shaking table at the temperature of 25 ℃ and the speed of 150rpm, carrying out suction filtration, and washing with deionized water to obtain an activated matrix; then mixing the activated substrate and 3g N-bromosuccinimide for bromoalcoholization, reacting for 3 hours in a shaking table with 150rpm at 25 ℃, filtering, and washing with deionized water; the brominated substrate was then mixed with 1.8g of hexamethylenediamine and 0.5M sodium carbonate buffer (pH 11) and reacted in a shaker at 150rpm for 16 hours at 25 ℃; taking 1g of amino activation matrix, adding the amino activation matrix into 2mL of N, N-dimethylformamide containing 6g of 3- (2-benzothiazolylthio) propionic acid, 60mg of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and 38 mu L N, N-diisopropylethylamine, and reacting for 15 hours in a water bath shaking table at 25 ℃; finally, the medium is filtered, washed by deionized water, added into the mixed solution of sodium acetate and acetic anhydride, reacted for 7 hours in a shaking table with 150rpm at 25 ℃, and washed by deionized water to obtain the chromatography medium taking 3- (2-benzothiazolylthio) propionic acid as the ligand, wherein the density of the ligand is 60 mu mol/ml.
Example 4
Taking 10g of the drained agarose gel, adding 3g of 20% (v/v) dimethyl sulfoxide, 2g of allyl bromide and 1g of sodium hydroxide, activating for 8 hours in a shaking table at the temperature of 25 ℃ and the speed of 150rpm, carrying out suction filtration, and washing with deionized water to obtain an activated matrix; then mixing the activated substrate and 1g N-bromosuccinimide for bromoalcoholization, reacting for 2 hours in a shaking table with 150rpm at 25 ℃, filtering, and washing with deionized water; then mixing the brominated substrate with 1g of hexamethylenediamine and 1M sodium carbonate buffer (pH12), and reacting in a shaker at 150rpm and 25 ℃ for 8 hours; taking 1g of amino activation matrix, adding the amino activation matrix into 2mL of N, N-dimethylformamide containing 2g of 3- (2-benzothiazolylthio) propionic acid, 20mg of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and 12 mu L N, N-diisopropylethylamine, and reacting for 8 hours in a water bath shaking table at 25 ℃; finally, the medium is filtered, washed by deionized water, added into the mixed solution of sodium acetate and acetic anhydride, reacted for 5 hours in a shaking table with 150rpm at 25 ℃, and washed by deionized water to obtain the chromatography medium taking 3- (2-benzothiazolylthio) propionic acid as the ligand, wherein the density of the ligand is 45 mu mol/ml.
Example 5
Taking 10g of the drained agarose gel, adding 10g of 20% (v/v) dimethyl sulfoxide, 10g of allyl bromide and 5g of sodium hydroxide, activating in a shaking table at the temperature of 25 ℃ and the speed of 150rpm for 48 hours, carrying out suction filtration, and washing with deionized water to obtain an activated matrix; then mixing the activated substrate and 5g N-bromosuccinimide for bromoalcoholization, reacting in a shaking table at 150rpm at 25 ℃ for 3 hours, filtering, and washing with deionized water; then, the bromo-substrate was mixed with 3g of hexamethylenediamine and 1M sodium carbonate buffer (pH12) and reacted in a shaker at 150rpm at 25 ℃ for 24 hours; taking 1g of amino activation matrix, adding the amino activation matrix into 2mL of N, N-dimethylformamide containing 10g of 2- (benzothiazolylthio) acetic acid, 100mg of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and 64 mu L N, N-diisopropylethylamine, and reacting for 8 hours in a water bath shaking table at 25 ℃; finally, the medium is filtered, washed by deionized water, added into the mixed solution of sodium acetate and acetic anhydride, reacted for 48 hours in a shaking table with 150rpm at 25 ℃, and washed by deionized water to obtain the chromatography medium taking 2- (benzothiazolylthio) acetic acid as the ligand, wherein the density of the ligand is 140 mu mol/ml.
Example 6
Taking 10g of the drained agarose gel, adding 2g of 20% (v/v) dimethyl sulfoxide, 1g of allyl bromide and 1g of sodium hydroxide, activating in a shaking table at the temperature of 25 ℃ and the speed of 150rpm for 48 hours, carrying out suction filtration, and washing with deionized water to obtain an activated matrix; then mixing the activated substrate and 1g N-bromosuccinimide for bromoalcoholization, reacting for 2 hours in a shaking table with 150rpm at 25 ℃, filtering, and washing with deionized water; then, mixing the brominated matrix with 2g of hexamethylenediamine and 1M sodium carbonate buffer (pH12), and reacting for 48 hours in a shaker at the temperature of 25 ℃ and at the speed of 150rpm to obtain an amino activated matrix; taking 1g of amino activation matrix, adding the amino activation matrix into 2mL of N, N-dimethylformamide containing 1g of 2- (benzothiazolylthio) acetic acid, 60mg of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and 37.5 mu L N, N-diisopropylethylamine, and reacting for 8 hours in a water bath shaking table at 25 ℃; finally, the medium is filtered, washed by deionized water, added into the mixed solution of sodium acetate and acetic anhydride, reacted for 12 hours in a shaking table with 150rpm at 25 ℃, and washed by deionized water to obtain the chromatography medium taking 2- (benzothiazolylthio) acetic acid as the ligand, wherein the density of the ligand is 40 mu mol/ml.
Example 7
Taking 10g of the drained agarose gel, adding 3g of 20% (v/v) dimethyl sulfoxide, 2g of allyl bromide and 1g of sodium hydroxide, activating for 8 hours in a shaking table at the temperature of 25 ℃ and the speed of 150rpm, carrying out suction filtration, and washing with deionized water to obtain an activated matrix; then mixing the activated substrate and 1g N-bromosuccinimide for bromoalcoholization, reacting for 2 hours in a shaking table with 150rpm at 25 ℃, filtering, and washing with deionized water; then mixing the brominated substrate with 1g of hexamethylenediamine and 1M sodium carbonate buffer (pH12), and reacting in a shaker at 150rpm and 25 ℃ for 8 hours; taking 1g of amino activation matrix, adding the amino activation matrix into 2mL of N, N-dimethylformamide containing 2g of 2- (benzothiazolylthio) acetic acid, 20mg of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and 12 mu L N, N-diisopropylethylamine, and reacting for 8 hours in a water bath shaking table at 25 ℃; finally, the medium is filtered, washed by deionized water, added into the mixed solution of sodium acetate and acetic anhydride, reacted for 5 hours in a shaking table with 150rpm at 25 ℃, and washed by deionized water to obtain the chromatography medium taking 2- (benzothiazolylthio) acetic acid as the ligand, wherein the density of the ligand is 45 mu mol/ml.
Example 8
Taking 10g of the drained agarose gel, adding 10g of 20% (v/v) dimethyl sulfoxide, 10g of allyl bromide and 5g of sodium hydroxide, activating in a shaking table at the temperature of 25 ℃ and the speed of 150rpm for 48 hours, carrying out suction filtration, and washing with deionized water to obtain an activated matrix; then mixing the activated substrate and 5g N-bromosuccinimide for bromoalcoholization, reacting in a shaking table at 150rpm at 25 ℃ for 3 hours, filtering, and washing with deionized water; then the bromo-substrate was mixed with 3g hexamethylenediamine and 1M sodium carbonate buffer (pH12) and reacted in a shaker at 150rpm at 25 ℃ for 24 hours; taking 1g of amino activation matrix, adding the amino activation matrix into 2mL of N, N-dimethylformamide containing 10g of 2- (2-benzothiazolylthio) propionic acid, 100mg of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and 64 mu L N, N-diisopropylethylamine, and reacting for 8 hours in a water bath shaking table at 25 ℃; finally, the medium is filtered, washed by deionized water, added into the mixed solution of sodium acetate and acetic anhydride, reacted for 48 hours in a shaking table with 150rpm at 25 ℃, and washed by deionized water to obtain the chromatography medium taking 2- (2-benzothiazolylthio) propionic acid as the ligand, wherein the density of the ligand is 140 mu mol/ml.
Example 9
Taking 10g of the drained agarose gel, adding 3g of 20% (v/v) dimethyl sulfoxide, 2g of allyl bromide and 1g of sodium hydroxide, activating for 8 hours in a shaking table at the temperature of 25 ℃ and the speed of 150rpm, carrying out suction filtration, and washing with deionized water to obtain an activated matrix; then mixing the activated substrate and 1g N-bromosuccinimide for bromoalcoholization, reacting for 2 hours in a shaking table with 150rpm at 25 ℃, filtering, and washing with deionized water; then mixing the brominated substrate with 1g of hexamethylenediamine and 1M sodium carbonate buffer (pH12), and reacting in a shaker at 150rpm and 25 ℃ for 8 hours; taking 1g of amino activation matrix, adding the amino activation matrix into 2mL of N, N-dimethylformamide containing 2g of 2- (2-benzothiazolylthio) propionic acid, 20mg of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and 12 mu L N, N-diisopropylethylamine, and reacting for 8 hours in a water bath shaking table at 25 ℃; finally, the medium is filtered, washed by deionized water, added into the mixed solution of sodium acetate and acetic anhydride, reacted for 5 hours in a shaking table with 150rpm at 25 ℃, and washed by deionized water to obtain the chromatography medium taking 2- (2-benzothiazolylthio) propionic acid as the ligand, wherein the density of the ligand is 45 mu mol/ml.
Claims (5)
1. A chromatography medium with benzothiazolyl thiocarboxylic acid group as functional ligand is characterized by comprising a chromatography matrix and ligand, wherein the chromatography matrix is a hydrophilic porous microsphere with hydroxyl, the ligand is benzothiazolyl thiocarboxylic acid which is coupled after being activated by allyl bromide, and the benzothiazolyl thiocarboxylic acid is one of 3- (2-benzothiazolylthio) propionic acid, 2- (benzothiazolylthio) acetic acid or 2- (2-benzothiazolylthio) propionic acid;
when the benzothiazolylthiocarboxylic acid is 3- (2-benzothiazolylthio) propionic acid, the chromatographic medium structurally consists of:
when the benzothiazolylthiocarboxylic acid is 2- (benzothiazolylthio) acetic acid, the chromatographic medium structurally consists of:
when the benzothiazolylthiocarboxylic acid is 2- (2-benzothiazolylthio) propionic acid, the chromatographic medium structurally comprises:
2. a chromatography medium according to claim 1, wherein said chromatography matrix is hydrophilic microspheres with porous structure and surface hydroxyl groups.
3. A chromatographic medium with benzothiazolylthiocarboxylic acid as functional ligand according to claim 1, wherein the chromatographic matrix is agarose gel or cellulose microsphere.
4. A chromatography medium according to claim 1 wherein said chromatography medium comprises a ligand density of 40-140 μmol/ml.
5. A process for preparing a chromatography medium according to claim 1, wherein said chromatography medium comprises benzothiazolylthiocarboxylic acid as a functional ligand, said process comprising the steps of:
1) after the chromatography matrix is dried by pumping, adding dimethyl sulfoxide with the volume percentage content of 20 percent which is 0.2 to 1 time of the mass of the chromatography matrix, allyl bromide with the mass of 0.1 to 1 time of the chromatography matrix and sodium hydroxide with the mass of 0.1 to 0.5 time of the chromatography matrix, activating for 8 to 48 hours in a shaking table with 150rpm at the temperature of 25 ℃, performing suction filtration, and washing with deionized water to obtain an activated chromatography matrix;
2) mixing the activated chromatography matrix and N-bromosuccinimide with the mass of 0.1-0.5 time of that of the chromatography matrix for bromoalcoholization, reacting in a shaking table at 150rpm at 25 ℃ for 1-5 hours, performing suction filtration, and washing with deionized water to obtain a bromoalcoholized matrix;
3) mixing bromoalcoholized matrix, hexamethylenediamine 0.1-1 times of chromatography matrix mass and 0.5-1M sodium carbonate buffer solution 0.1-1 times of chromatography matrix mass, wherein the pH of the sodium carbonate buffer solution is 10-12, and reacting in a shaking table at 25 ℃ and 150rpm for 8-48 hours to obtain aminated matrix;
4) mixing an aminated matrix, benzothiazyl thiocarboxylic acid with the mass of 0.1-0.3 time of that of a chromatography matrix, 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate with the mass of 0.4-2 times of that of the chromatography matrix, and N, N-dimethylformamide solution of N, N-diisopropylethylamine with the mass of 0.8-4 times of that of the chromatography matrix, and reacting in a shaking table with 150rpm at 25 ℃ for 8-48 hours to obtain a medium after coupling of the benzothiazyl thiocarboxylic acid;
5) and (2) filtering the medium after coupling the benzothiazolyl thiocarboxylic acid, washing with deionized water, adding the filtered medium into a mixed solution of sodium acetate and acetic anhydride, reacting for 2-8 hours in a shaking table at the temperature of 25 ℃ and at the speed of 150rpm, and washing with deionized water to obtain the chromatography medium taking the benzothiazolyl thiocarboxylic acid as a functional ligand.
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