CN107803187B - Affinity chromatography medium using tetrapeptide gastrin as functional ligand - Google Patents
Affinity chromatography medium using tetrapeptide gastrin as functional ligand Download PDFInfo
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- CN107803187B CN107803187B CN201711278656.XA CN201711278656A CN107803187B CN 107803187 B CN107803187 B CN 107803187B CN 201711278656 A CN201711278656 A CN 201711278656A CN 107803187 B CN107803187 B CN 107803187B
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- 102100021022 Gastrin Human genes 0.000 title claims abstract description 37
- 108010052343 Gastrins Proteins 0.000 title claims abstract description 37
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](C(=O)NCC(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 title claims abstract description 37
- 239000003446 ligand Substances 0.000 title claims abstract description 36
- 239000012501 chromatography medium Substances 0.000 title claims abstract description 23
- 238000001042 affinity chromatography Methods 0.000 title claims abstract description 13
- 239000012504 chromatography matrix Substances 0.000 claims abstract description 31
- HQSMEHLVLOGBCK-UHFFFAOYSA-N 1-ethenylsulfinylethene Chemical compound C=CS(=O)C=C HQSMEHLVLOGBCK-UHFFFAOYSA-N 0.000 claims abstract description 9
- 230000006698 induction Effects 0.000 claims abstract description 8
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 7
- 229920001661 Chitosan Polymers 0.000 claims abstract description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 20
- 239000008367 deionised water Substances 0.000 claims description 20
- 229910021641 deionized water Inorganic materials 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 15
- PAFZNILMFXTMIY-UHFFFAOYSA-N cyclohexylamine Chemical compound NC1CCCCC1 PAFZNILMFXTMIY-UHFFFAOYSA-N 0.000 claims description 14
- 230000008878 coupling Effects 0.000 claims description 13
- 238000010168 coupling process Methods 0.000 claims description 13
- 238000005859 coupling reaction Methods 0.000 claims description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000011159 matrix material Substances 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 9
- 238000000967 suction filtration Methods 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 6
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 claims description 5
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims 1
- 238000001179 sorption measurement Methods 0.000 abstract description 6
- 238000002360 preparation method Methods 0.000 abstract description 4
- 238000013375 chromatographic separation Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000004913 activation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
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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
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The affinity chromatography medium with the tetrapeptide gastrin as the functional ligand comprises a chromatography matrix and the ligand, wherein the chromatography matrix is chitosan, and the ligand is the tetrapeptide gastrin which is coupled after being activated by divinyl sulfoxide. The novel chromatographic medium developed by the invention takes the tetrapeptide gastrin as the functional ligand, has the characteristic of obvious hydrophobic charge induction chromatographic separation, has high adsorption capacity on the antibody, and can be used for large-scale preparation of the antibody.
Description
Technical Field
The invention relates to an affinity chromatography medium using tetrapeptide gastrin as a functional ligand, belonging to a protein chromatography separation technology in the field of biochemical engineering.
Background
The antibody is a biological missile, has the advantages of strong specificity, good curative effect, small toxic and side effect and the like, and is mainly used for treating malignant tumors, serious infectivity and immune system diseases. The global antibody sales is expected to break through $ 500 billion in 2011, which accounts for over 1/3 in the biotechnology drug market, and the antibody industry has become the competitive focus of biopharmaceuticals in various countries. However, the antibody industry in our country has just started.
The clinical dosage of the therapeutic antibody is large, the treatment period is long, the high treatment cost is forbidden by common patients, and the social demand of comprehensively reducing the price of the antibody is urgent.
Disclosure of Invention
Aiming at the technical problems in the prior art, the scheme provides an affinity chromatography medium taking tetrapeptide gastrin as a functional ligand.
In order to achieve the purpose, the scheme is achieved through the following technical scheme:
an affinity chromatography medium using tetrapeptide gastrin as a functional ligand comprises a chromatography matrix and a ligand, wherein the chromatography matrix is chitosan, and the ligand is tetrapeptide gastrin coupled after being activated by divinyl sulfoxide.
Preferably, the tetrapeptide gastrin is used as the functionAffinity chromatography medium of ligand, wherein, the density of the ligand of the chromatography medium is 30-120 mu mol/m3。
Preferably, the affinity chromatography medium using tetrapeptide gastrin as a functional ligand comprises the following steps:
1) after the chromatography matrix is dried by suction, adding 25 percent (v/v) of dimethyl sulfoxide 0.1-0.6 time of the mass of the chromatography matrix, divinyl sulfoxide 0.5-1.5 times of the mass of the chromatography matrix and sodium hydroxide 0.3-0.6 time of the mass of the chromatography matrix, activating for 5-20 hours in a shaking table at 200rpm at 25 ℃, performing suction filtration, and washing with deionized water to obtain an activated chromatography matrix;
2) mixing the activated chromatography matrix with 1-3 times of 3-mercaptopropionic acid and 0.1-0.3 time of ammonium persulfate by mass of the chromatography matrix for alcoholization, reacting in a shaking table at 200rpm at 50 ℃ for 3-7 hours, performing suction filtration, and washing with deionized water to obtain an alcoholized matrix;
3) mixing an alcoholized matrix, tetrapeptide gastrin with the mass of 0.1-0.3 time of that of a chromatography matrix and 0.5-1M sodium carbonate buffer solution, and reacting in a shaking table at the temperature of 30 ℃ and the rpm of 200 for 5-20 hours to obtain a medium after tetrapeptide gastrin coupling;
4) and (3) filtering the medium after the tetrapeptide gastrin coupling, washing with deionized water, adding into cyclohexylamine, reacting for 4-10 hours in a shaking table at 200rpm at 30 ℃, and washing with deionized water to obtain the hydrophobic charge induction chromatography medium taking the tetrapeptide gastrin as a functional ligand.
Preferably, the preparation method of the affinity chromatography medium using the tetrapeptide gastrin as the functional ligand is characterized in that the mass ratio of the cyclohexylamine to the chromatography matrix is 1-5: 1.
Preferably, the preparation method of the affinity chromatography medium using the tetrapeptide gastrin as the functional ligand is characterized in that the pH of the sodium carbonate buffer solution is 10-12.
The invention has the beneficial effects that:
(1) the antibody has large adsorption capacity, the saturated adsorption capacity can reach 150mg/ml wet medium, and the processing capacity is strong;
(2) the adsorption process is not influenced by salt, the adsorption capacity is basically kept unchanged within a wide conductivity range (0-100mS/cm), the feed liquid is not required to be diluted or treated by adding salt, the protein can be directly captured, and the operation steps are reduced;
(3) the ligand density is high, the charge induction effect is strong, the coupling efficiency is high by coupling the ligand through the primary amino site on the ligand, and the amino site can also be protonated, so that the auxiliary electrostatic repulsion effect can be provided;
(4) the medium has stable property, the divinyl sulfoxide is adopted for activation and coupling with the ligand, the ligand of the obtained medium is stable, and the cleaning and regeneration are convenient;
(5) the novel chromatographic medium developed by the invention takes the tetrapeptide gastrin as the functional ligand, has the characteristic of obvious hydrophobic charge induction chromatographic separation, has high adsorption capacity on the antibody, and can be used for large-scale preparation of the antibody.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
Example 1
Taking 10g of chitosan, adding 2g of 25% (v/v) dimethyl sulfoxide, 5g of divinyl sulfoxide and 3g of sodium hydroxide, activating for 10 hours in a shaking table at the temperature of 25 ℃ and at the speed of 200rpm, performing suction filtration, and washing with deionized water to obtain an activated chromatography matrix; mixing the activated chromatography matrix with 10g of 3-mercaptopropionic acid and 2g of ammonium persulfate for alcoholization, reacting for 3 hours in a shaking table at the temperature of 50 ℃ and the rpm of 200, performing suction filtration, and washing with deionized water to obtain an alcoholized matrix; mixing an alcoholized matrix with 1g of tetrapeptide gastrin and 0.5M sodium carbonate buffer solution, wherein the pH value of the sodium carbonate buffer solution is 10, and reacting in a shaker at 30 ℃ and 200rpm for 10 hours to obtain a medium after tetrapeptide gastrin coupling; and (2) filtering the medium after the tetrapeptide gastrin coupling, washing with deionized water, adding into 50g of cyclohexylamine, reacting for 5 hours in a shaking table at the temperature of 30 ℃ and at 200rpm, and washing with deionized water to obtain the hydrophobic charge induction chromatography medium taking the tetrapeptide gastrin as a functional ligand. The ligand density was 50. mu. mol/m3。
Example 2
10g of chitosan was added with 5g of 25% (v/v) dimethyl sulfoxide, 10g of divinyl sulfoxide and 5g ofActivating with sodium hydroxide in a shaker at 200rpm at 25 deg.C for 12 hr, vacuum filtering, and washing with deionized water to obtain activated chromatographic matrix; mixing the activated chromatography matrix with 10g of 3-mercaptopropionic acid and 2g of ammonium persulfate for alcoholization, reacting for 3 hours in a shaking table at the temperature of 50 ℃ and the rpm of 200, performing suction filtration, and washing with deionized water to obtain an alcoholized matrix; mixing an alcoholized matrix with 1g of tetrapeptide gastrin and 0.5M sodium carbonate buffer solution, wherein the pH value of the sodium carbonate buffer solution is 10, and reacting in a shaker at 30 ℃ and 200rpm for 8 hours to obtain a medium after tetrapeptide gastrin coupling; and (2) filtering the medium after the tetrapeptide gastrin coupling, washing with deionized water, adding the washed medium into 10g of cyclohexylamine, reacting for 7 hours in a shaking table at the temperature of 30 ℃ and at the speed of 200rpm, and washing with deionized water to obtain the hydrophobic charge induction chromatography medium taking the tetrapeptide gastrin as a functional ligand. The ligand density was 100. mu. mol/m3。
Example 3
Taking 10g of chitosan, adding 2g of 25% (v/v) dimethyl sulfoxide, 10g of divinyl sulfoxide and 5g of sodium hydroxide, activating for 10 hours in a shaking table at the temperature of 25 ℃ and at the speed of 200rpm, performing suction filtration, and washing with deionized water to obtain an activated chromatography matrix; mixing the activated chromatography matrix with 10g of 3-mercaptopropionic acid and 3g of ammonium persulfate for alcoholization, reacting for 6 hours in a shaking table at the temperature of 50 ℃ and the rpm of 200, performing suction filtration, and washing with deionized water to obtain an alcoholized matrix; mixing an alcoholized matrix with 2g of tetrapeptide gastrin and 0.5M sodium carbonate buffer solution, wherein the pH value of the sodium carbonate buffer solution is 10, and reacting in a shaker at 30 ℃ and 200rpm for 20 hours to obtain a medium after tetrapeptide gastrin coupling; and (2) filtering the medium after the tetrapeptide gastrin coupling, washing with deionized water, adding the washed medium into 10g of cyclohexylamine, reacting for 10 hours in a shaking table at the temperature of 30 ℃ and at the speed of 200rpm, and washing with deionized water to obtain the hydrophobic charge induction chromatography medium taking the tetrapeptide gastrin as a functional ligand. The ligand density was 120. mu. mol/m3。
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (5)
1. An affinity chromatography medium using tetrapeptide gastrin as a functional ligand is characterized by comprising a chromatography matrix and a ligand, wherein the chromatography matrix is chitosan, and the ligand is tetrapeptide gastrin; the chromatography matrix is activated by divinyl sulfoxide and then coupled with ligand to form a chromatography medium.
2. The affinity chromatography media of claim 1, wherein said affinity chromatography media has a ligand density of 30-120 μmol/m3。
3. A method of preparing an affinity chromatography medium as claimed in claim 1 having a tetrapeptide gastrin as a functional ligand, comprising the steps of:
1) after the chromatography matrix is dried by suction, adding 25 percent (v/v) of dimethyl sulfoxide 0.1-0.6 time of the mass of the chromatography matrix, divinyl sulfoxide 0.5-1.5 times of the mass of the chromatography matrix and sodium hydroxide 0.3-0.6 time of the mass of the chromatography matrix, activating for 5-20 hours in a shaking table at 200rpm at 25 ℃, performing suction filtration, and washing with deionized water to obtain an activated chromatography matrix;
2) mixing the activated chromatography matrix with 1-3 times of 3-mercaptopropionic acid and 0.1-0.3 time of ammonium persulfate by mass of the chromatography matrix for alcoholization, reacting in a shaking table at 200rpm at 50 ℃ for 3-7 hours, performing suction filtration, and washing with deionized water to obtain an alcoholized matrix;
3) mixing an alcoholized matrix, tetrapeptide gastrin with the mass of 0.1-0.3 time of that of a chromatography matrix and 0.5-1M sodium carbonate buffer solution, and reacting in a shaking table at the temperature of 30 ℃ and the rpm of 200 for 5-20 hours to obtain a medium after tetrapeptide gastrin coupling;
4) and (3) filtering the medium after the tetrapeptide gastrin coupling, washing with deionized water, adding into cyclohexylamine, reacting for 4-10 hours in a shaking table at 200rpm at 30 ℃, and washing with deionized water to obtain the hydrophobic charge induction chromatography medium taking the tetrapeptide gastrin as a functional ligand.
4. The method of claim 3, wherein the ratio of cyclohexylamine mass to chromatography matrix mass is from 1 to 5: 1.
5. The method of claim 3, wherein the sodium carbonate buffer has a pH of from about 10 to about 12.
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| CN201711278656.XA CN107803187B (en) | 2017-12-06 | 2017-12-06 | Affinity chromatography medium using tetrapeptide gastrin as functional ligand |
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| CN201711278656.XA CN107803187B (en) | 2017-12-06 | 2017-12-06 | Affinity chromatography medium using tetrapeptide gastrin as functional ligand |
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| CN107803187B true CN107803187B (en) | 2020-07-14 |
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| CN104117345B (en) * | 2014-05-13 | 2016-09-14 | 浙江大学 | Hydrophobic charge-induction chromatography medium with bifunctional group and preparation method thereof |
| CN104096544B (en) * | 2014-05-13 | 2016-06-01 | 浙江大学 | Taking amino benzoglyoxaline as the chromatography media of functional ligand and its preparation method |
| CN104645949B (en) * | 2015-02-04 | 2017-01-25 | 浙江大学 | Affinity chromatography medium employing tetrapeptide as functional ligand and preparation method of affinity chromatography medium |
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