CN115386563A - Method for rapidly preparing urokinase raw material - Google Patents
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- CN115386563A CN115386563A CN202211143608.0A CN202211143608A CN115386563A CN 115386563 A CN115386563 A CN 115386563A CN 202211143608 A CN202211143608 A CN 202211143608A CN 115386563 A CN115386563 A CN 115386563A
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- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 title claims abstract description 61
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 title claims abstract description 61
- 229960005356 urokinase Drugs 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000002994 raw material Substances 0.000 title claims abstract description 18
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 46
- 239000003480 eluent Substances 0.000 claims abstract description 44
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical class O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 38
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 29
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 29
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 29
- 238000001179 sorption measurement Methods 0.000 claims abstract description 24
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 23
- 238000001914 filtration Methods 0.000 claims abstract description 21
- 239000000047 product Substances 0.000 claims abstract description 17
- 210000002700 urine Anatomy 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 238000005406 washing Methods 0.000 claims abstract description 9
- 238000004108 freeze drying Methods 0.000 claims abstract description 7
- 238000011068 loading method Methods 0.000 claims abstract description 6
- 239000002244 precipitate Substances 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 37
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 10
- 239000001488 sodium phosphate Substances 0.000 claims description 8
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 8
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 230000001502 supplementing effect Effects 0.000 claims description 5
- 238000005349 anion exchange Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 3
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 229910000077 silane Inorganic materials 0.000 claims description 3
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 238000003889 chemical engineering Methods 0.000 abstract description 2
- 239000006087 Silane Coupling Agent Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- -1 amino modified silica gel Chemical class 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 208000007536 Thrombosis Diseases 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- 206010008088 Cerebral artery embolism Diseases 0.000 description 1
- 206010008132 Cerebral thrombosis Diseases 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 1
- 102000001399 Kallikrein Human genes 0.000 description 1
- 108060005987 Kallikrein Proteins 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 102000001938 Plasminogen Activators Human genes 0.000 description 1
- 108010001014 Plasminogen Activators Proteins 0.000 description 1
- 208000010378 Pulmonary Embolism Diseases 0.000 description 1
- 206010038908 Retinal vein thrombosis Diseases 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 206010000891 acute myocardial infarction Diseases 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 201000010849 intracranial embolism Diseases 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 229940039088 kininogenase Drugs 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 229940127126 plasminogen activator Drugs 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001957 retinal vein Anatomy 0.000 description 1
- 208000004644 retinal vein occlusion Diseases 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001732 thrombotic effect Effects 0.000 description 1
- HQYALQRYBUJWDH-UHFFFAOYSA-N trimethoxy(propyl)silane Chemical compound CCC[Si](OC)(OC)OC HQYALQRYBUJWDH-UHFFFAOYSA-N 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21031—Urokinase (3.4.21.31)
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method for quickly preparing a urokinase raw material, belonging to the field of biological engineering and chemical engineering. The preparation method of the urokinase raw material comprises the following steps: (1) adding modified silica gel into urine for adsorption; (2) Washing the modified silica gel with water, eluting with an eluent I, and collecting the eluent; (3) Adding ammonium sulfate into the eluent, and collecting the precipitate to obtain a crude urokinase product; (4) Dissolving crude urokinase in phosphoric acid, filtering, and concentrating to obtain concentrated solution; (5) Balancing the adsorption column with an eluent II, loading the concentrated solution obtained in the step (4) on the adsorption column, eluting the adsorption column with the eluent II again, and collecting the eluent; (6) And (5) filtering the eluent obtained in the step (5), and freeze-drying to obtain a urokinase raw material. The preparation method is simple, low in production cost and high in yield, and can quickly prepare high-quality urokinase raw materials.
Description
Technical Field
The invention belongs to the field of biological engineering and chemical engineering, and relates to a method for quickly preparing a urokinase raw material.
Background
Urokinase, UK for short, is a plasminogen activator, produced by the kidney and secreted into the urine, a serine protease that specifically recognizes plasminogen and catalyzes its conversion to plasmin. Urokinase breaks down insoluble fibrin into soluble small peptides, thereby dissolving the thrombus. Therefore, UK is commonly used clinically for the treatment of acute myocardial infarction, acute cerebral thrombosis, cerebral embolism, thrombotic occlusive disease, pulmonary embolism and central retinal vein thrombosis. The human urokinase mainly comprises two types of natural high molecular weight urokinase (54000 Dal) and low molecular weight urokinase (33000 Dal), the clinical effect of dissolving thrombus of the high molecular weight urokinase is about 3 times higher than that of the low molecular weight urokinase, and the relative molecular weight of the urokinase is one of important indexes of quality standard internationally.
Chinese patent application 201510549397.4 discloses a method for preparing urokinase, which is a method for enriching urokinase, wherein urine protein in urine is directly adsorbed in a urinal or a urinal by using a filter cloth bag filled with modified silica gel, and the filter cloth bag adsorbing the urine protein is transported to a processing point for subsequent treatment. The method utilizes the isoelectric point property of specific urine protein, and directly and effectively adsorbs urine protein such as urine trypsin inhibitor, human urine kininogenase, urokinase and the like by using modified silica gel, macroporous resin, chitin, ion resin and the like, thereby avoiding the step of collecting urine. The method has no obvious influence on the sanitary condition of the toilet, and greatly reduces the cost of urine transportation and a series of environmental problems. However, the preparation method of the modified silica gel used in the method is complex and high in cost, and the obtained urokinase product has low activity.
Chinese patent application 201510549397.4 discloses a preparation method of urokinase and freeze-dried powder thereof, which specifically comprises the following steps: (1) preparing a crude urokinase product; (2) preparing a fine urokinase product; and (3) purifying. Dissolving the urokinase refined product obtained in the step (2) in the step (3), and adding the urokinase refined product solution into a cation exchange chromatography column for purification; and (4) freeze-drying the purified urokinase to obtain the urokinase freeze-dried powder. However, the method uses a common silica gel column to prepare the crude urokinase, has poor elution effect, and still needs more complicated filtering and purifying steps in the subsequent process, so that the production process is more complicated and the production cost is relatively high.
Therefore, there is a need to develop a method for preparing a high-quality urokinase raw material with simple preparation method, low production cost and high yield.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the method for quickly preparing the urokinase raw material, which is simple, low in production cost and high in yield.
In order to realize the purpose, the technical scheme adopted by the invention is as follows:
firstly, a preparation method of a urokinase raw material is provided, which comprises the following steps:
(1) Adding modified silica gel into urine for adsorption;
(2) Washing the modified silica gel with water, eluting with an eluent I, and collecting the eluent;
(3) Adding ammonium sulfate solution into the eluent, stirring, and taking supernatant; supplementing ammonium sulfate, stirring to dissolve, filtering, and collecting precipitate to obtain crude urokinase product;
(4) Adding phosphoric acid into the crude urokinase product for dissolving, filtering and concentrating to obtain a concentrated solution;
(5) Balancing the adsorption column with an eluent II, loading the concentrated solution obtained in the step (4) on the adsorption column, eluting the adsorption column with the eluent II again, and collecting the eluent;
(6) And (5) filtering the eluent obtained in the step (5), and freeze-drying to obtain a urokinase raw material.
Further, the modified silica gel in the step (1) is one or two of silane coupling agent modified silica gel and amino modified silica gel.
Further, the preparation method of the silane coupling agent modified silica gel comprises the following steps: 50g of spherical silica gel and 100g of gamma- (2, 3-glycidoxy) propyltrimethoxysilane were dispersed in 250mL of a toluene solution and reacted at 80 ℃ for 2 hours to obtain 53g of a silane coupling agent-modified silica gel.
Further, the weight ratio of the silane coupling agent modified silica gel to the amino modified silica gel is 2-2.5:1-1.5.
Further, in the step (2), the eluent I is a mixed solution containing sodium chloride and phosphoric acid, and the elution process comprises the following steps: eluting with a mixture containing 0.1-0.2mol/L sodium chloride and 0.1-0.15mol/L phosphoric acid, and eluting with a mixture containing 0.3-0.4mol/L sodium chloride and 0.2-0.25mol/L phosphoric acid.
Further, the dosage ratio of the eluent, the ammonium sulfate solution and the ammonium sulfate in the step (3) is 80-100mL:80-90mL:1g.
Further, the concentration of the ammonium sulfate solution is 1.0-1.5mol/L.
Further, the dosage ratio of the crude urokinase to the phosphoric acid in the step (4) is 1g:4-6mL.
Further, the eluent II in the step (5) is 0.5-0.6mol/L sodium phosphate solution.
Further, the adsorption column in the step (5) is a weak anion exchange column, and the filler of the weak anion exchange column comprises one or more of D201, D301, D311 and DEAE.
Further, the filtration in the step (6) is carried out by using a filter element with the diameter of 20 nm.
In some embodiments, a method for preparing a urokinase starting material comprises the steps of:
(1) Adding silane coupling agent modified silica gel and amino modified silica gel compounded modified silica gel into urine for adsorption;
(2) Washing the modified silica gel with water, eluting the obtained washing liquid with a mixed liquid containing 0.1-0.2mol/L sodium chloride and 0.1-0.15mol/L phosphoric acid, eluting with a mixed liquid containing 0.3-0.4mol/L sodium chloride and 0.2-0.25mol/L phosphoric acid, controlling the flow rate at 100mL/min, and collecting the eluent;
(3) Adding 1.0mol/L ammonium sulfate solution into the eluent, stirring, and taking the supernatant; supplementing ammonium sulfate, stirring to dissolve, filtering, and collecting precipitate to obtain crude urokinase product;
(4) Taking a crude urokinase product, adding 0.1mol/L phosphoric acid for dissolving, filtering and concentrating to obtain a concentrated solution; wherein the dosage ratio of the urokinase crude product to the phosphoric acid is 1g:5mL;
(5) Balancing an adsorption column by using 0.5mol/L sodium phosphate solution, then loading the concentrated solution obtained in the step (4) onto the adsorption column (D301), eluting the adsorption column by using 0.5mol/L sodium phosphate solution again, controlling the flow rate at 100mL/min, and collecting eluent;
(6) And (5) filtering the eluent obtained in the step (5) by using a filter element with the diameter of 20nm, and freeze-drying to obtain a urokinase raw material.
Compared with the prior art, the invention has the following beneficial effects:
(1) The purity of the urokinase prepared by the method is up to 99 percent, and the relative content of the high-molecular urokinase is up to 95 percent; the activity yield of the urokinase reaches 92 percent, and the specific activity reaches 3200 IU/g.pr;
(2) The purification method provided by the invention has the advantages of simple preparation method and low production cost, and can be used for quickly preparing the urokinase raw material.
Detailed Description
It should be noted that the raw materials used in the present invention are all common commercial products, and the sources thereof are not particularly limited.
The following reagent sources are illustrative:
amino-modified silica gel available from Nilappa Kjeldahl high tech materials Co., ltd, fuji Rich, japanScotch-shaped high-purity silica gel filler NH 2 Amino-linked modified silica gel 100A 20/45 μm.
Example 1
(1) Adding modified silica gel compounded by silane coupling agent modified silica gel and amino modified silica gel (weight ratio is 2.5;
(2) Washing the modified silica gel with water, eluting the obtained washing liquid with a mixed solution containing 0.2mol/L sodium chloride and 0.15mol/L phosphoric acid, eluting with a mixed solution containing 0.3mol/L sodium chloride and 0.2mol/L phosphoric acid, controlling the flow rate at 100mL/min, and collecting the eluent;
(3) Adding 1.0mol/L ammonium sulfate solution into the eluent, stirring, and taking the supernatant; supplementing ammonium sulfate, stirring to dissolve, filtering, and collecting precipitate to obtain crude urokinase product; wherein the dosage ratio of the eluent, the ammonium sulfate solution and the ammonium sulfate is 90mL:80mL of: 1g;
(4) Taking a crude urokinase product, adding 0.1mol/L phosphoric acid for dissolving, filtering and concentrating to obtain a concentrated solution; wherein the dosage ratio of the urokinase crude product to the phosphoric acid is 1g:5mL;
(5) Balancing an adsorption column by using 0.5mol/L sodium phosphate solution, then loading the concentrated solution obtained in the step (4) onto the adsorption column (D301), eluting the adsorption column by using 0.5mol/L sodium phosphate solution again, controlling the flow rate at 100mL/min, and collecting eluent;
(6) And (5) filtering the eluent obtained in the step (5) by using a filter element with the diameter of 20nm, and freeze-drying to obtain a urokinase raw material.
Example 2
(1) Adding modified silica gel compounded by silane coupling agent modified silica gel and amino modified silica gel (weight ratio is 2;
(2) Washing the modified silica gel with water, eluting the obtained washing liquid with a mixed liquid containing 0.1mol/L sodium chloride and 0.1mol/L phosphoric acid, eluting with a mixed liquid containing 0.4mol/L sodium chloride and 0.25mol/L phosphoric acid, controlling the flow rate at 100mL/min, and collecting the eluent;
(3) Adding 1.0mol/L ammonium sulfate solution into the eluent, stirring, and taking the supernatant; supplementing ammonium sulfate, stirring to dissolve, filtering, and collecting precipitate to obtain crude urokinase product; wherein the dosage ratio of the eluent, the ammonium sulfate solution and the ammonium sulfate is 100mL:90mL:1g of a compound;
(4) Taking a crude urokinase product, adding 0.1mol/L phosphoric acid for dissolving, filtering and concentrating to obtain a concentrated solution; wherein the dosage ratio of the urokinase crude product to the phosphoric acid is 1g:5mL;
(5) Balancing an adsorption column by using 0.5mol/L sodium phosphate solution, loading the concentrated solution obtained in the step (4) onto the adsorption column (D301), eluting the adsorption column by using 0.5mol/L sodium phosphate solution again, controlling the flow rate at 100mL/min, and collecting eluent;
(6) And (5) filtering the eluent obtained in the step (5) by using a filter element with the diameter of 20nm, and freeze-drying to obtain a urokinase raw material.
Example 3
The difference from example 1 is that the modified silica gel in step (1) is modified with only the silane coupling agent (ensuring that the total weight of the modified silica gel is unchanged), and the other steps are the same as in example 1.
Example 4
The difference from example 1 is that the modified silica gel in step (1) is modified with only amino groups (ensuring that the total weight of the modified silica gel is unchanged), and the other steps are the same as example 1.
Example 5
The difference from the example 1 is that the weight ratio of the silane coupling agent modified silica gel to the amino modified silica gel added in the step (1) is 1.5:2, the other steps are the same as in example 1.
Example 6
The difference from example 1 is that the amount ratio of the eluent, the ammonium sulfate solution and the ammonium sulfate in step (3) is 75mL:95mL of: 1g, the other steps are the same as in example 1.
Example 7
The difference from example 1 is that no ammonium sulfate solid is added in step (3), and the ratio of the eluent to the ammonium sulfate solution is 90mL:87.6mL (ensuring that the molar amount of ammonium sulfate added is unchanged), and the other steps are the same as in example 1.
Comparative example 1
The difference from example 1 is that no ammonium sulfate solution is added in step (3), and the ratio of the eluent to the ammonium sulfate is 90mL:11.56g (ensuring that the molar amount of ammonium sulfate added is unchanged), and the other steps are the same as in example 1.
Examples of the experiments
The relative contents of high molecular urokinase, the purity, the activity yield and the specific activity of the urokinase obtained in examples 1 to 7 and comparative example 1 were calculated according to the method described in the first part of the text variety of the second edition of Chinese pharmacopoeia 2020. The test results are shown in Table 1.
TABLE 1
| Examples of the invention | Purity/%) | Relative content of Polymer urokinase (%) | Activity yield (%) | Specific activity (IU/g pr) |
| Example 1 | 99 | 95 | 92 | 3200 |
| Example 2 | 99 | 94 | 91 | 3200 |
| Example 3 | 92 | 90 | 86 | 2900 |
| Example 4 | 93 | 91 | 86 | 2800 |
| Example 5 | 95 | 93 | 89 | 3100 |
| Example 6 | 94 | 91 | 87 | 3000 |
| Example 7 | 93 | 91 | 85 | 2700 |
| Comparative example 1 | 88 | 79 | 71 | 2200 |
Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and do not limit the protection scope of the present invention, and those skilled in the art can make simple modifications or equivalent substitutions on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A preparation method of a urokinase raw material is characterized by comprising the following steps:
(1) Adding modified silica gel into urine for adsorption;
(2) Washing the modified silica gel with water, eluting with an eluent I, and collecting the eluent;
(3) Adding ammonium sulfate solution into the eluent, stirring, and taking supernatant; supplementing ammonium sulfate, stirring to dissolve, filtering, and collecting precipitate to obtain crude urokinase product;
(4) Adding phosphoric acid into the crude urokinase product for dissolving, filtering and concentrating to obtain a concentrated solution;
(5) Balancing the adsorption column with an eluent II, loading the concentrated solution obtained in the step (4) on the adsorption column, eluting the adsorption column with the eluent II again, and collecting the eluent;
(6) And (5) filtering the eluent obtained in the step (5), and freeze-drying to obtain a urokinase raw material.
2. The method according to claim 1, wherein the modified silica gel in step (1) is one or both of a silane coupling agent-modified silica gel and an amino-modified silica gel.
3. The method according to claim 2, wherein the weight ratio of the silane coupling agent-modified silica gel to the amino-modified silica gel is 2-2.5:1-1.5.
4. The preparation method according to claim 1, wherein the eluent I in the step (2) is a mixed solution containing sodium chloride and phosphoric acid, and the elution process comprises: eluting with a mixture containing 0.1-0.2mol/L sodium chloride and 0.1-0.15mol/L phosphoric acid, and eluting with a mixture containing 0.3-0.4mol/L sodium chloride and 0.2-0.25mol/L phosphoric acid.
5. The preparation method according to claim 1, wherein the eluent, the ammonium sulfate solution and the ammonium sulfate in the step (3) are used in a ratio of 80-100mL:80-90mL:1g of the total weight of the composition.
6. The method according to claim 5, wherein the concentration of the ammonium sulfate solution is 1.0 to 1.5mol/L.
7. The method according to claim 1, wherein the crude urokinase and the phosphoric acid in the step (4) are used in a ratio of 1g:4-6mL.
8. The process according to claim 1, wherein the eluent II in the step (5) is 0.5 to 0.6mol/L sodium phosphate solution.
9. The preparation method according to claim 1, wherein the adsorption column in step (5) is a weak anion exchange column, and the packing of the weak anion exchange column comprises one or more of D201, D301, D311 and DEAE.
10. The method according to claim 1, wherein the filtration in the step (6) is a filtration with a 20nm filter cartridge.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| US2983647A (en) * | 1955-07-01 | 1961-05-09 | Leo Pharm Prod Ltd | Urokinase and method of recovering the same |
| CN101863974A (en) * | 2009-07-13 | 2010-10-20 | 广东天普生化医药股份有限公司 | Method for enriching urine protein directly |
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| CN111662896A (en) * | 2020-07-10 | 2020-09-15 | 江苏尤里卡生物科技有限公司 | Method for purifying crude urokinase |
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