CN111647585A - Method for refining urokinase - Google Patents
Method for refining urokinase Download PDFInfo
- Publication number
- CN111647585A CN111647585A CN202010652909.0A CN202010652909A CN111647585A CN 111647585 A CN111647585 A CN 111647585A CN 202010652909 A CN202010652909 A CN 202010652909A CN 111647585 A CN111647585 A CN 111647585A
- Authority
- CN
- China
- Prior art keywords
- urokinase
- solution
- column
- buffer
- superdex
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 title claims abstract description 50
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 title claims abstract description 50
- 229960005356 urokinase Drugs 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000007670 refining Methods 0.000 title abstract description 6
- 239000000243 solution Substances 0.000 claims abstract description 33
- 238000005227 gel permeation chromatography Methods 0.000 claims abstract description 14
- 239000012505 Superdex™ Substances 0.000 claims abstract description 11
- 239000003480 eluent Substances 0.000 claims abstract description 8
- 239000012506 Sephacryl® Substances 0.000 claims abstract description 6
- 239000007853 buffer solution Substances 0.000 claims abstract description 5
- 239000000945 filler Substances 0.000 claims abstract description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 239000007979 citrate buffer Substances 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 239000006167 equilibration buffer Substances 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 239000008351 acetate buffer Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 9
- 230000008569 process Effects 0.000 abstract description 6
- 238000011084 recovery Methods 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 7
- 108010073863 saruplase Proteins 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 239000004005 microsphere Substances 0.000 description 3
- 239000008215 water for injection Substances 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 239000003527 fibrinolytic agent Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 229940012957 plasmin Drugs 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- PNKUSGQVOMIXLU-UHFFFAOYSA-N Formamidine Chemical compound NC=N PNKUSGQVOMIXLU-UHFFFAOYSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 208000001435 Thromboembolism Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000004926 tubular epithelial cell Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- 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
- C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
-
- 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)
- C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for refining urokinase, belonging to the technical field of urokinase purification. The method comprises the following steps: taking the urokinase intermediate solution, loading the urokinase intermediate solution on a gel chromatographic column, eluting the urokinase intermediate solution by using an equilibrium buffer solution, and collecting middle-section eluent to obtain a refined urokinase solution; wherein the filler of the gel chromatography comprises at least one of Superdex 75PG/GE, Superdex 200PG/GE, Sephacryl S-100HR/GE, Chromadex 75PG/BESTCHROM and Chromadex200 PG/BESTCHROM. The method is simple, safe, environment-friendly, high in purity, good in recovery rate and wide in application range, and can meet the requirement of mass production economic benefit by optimizing various process parameters and only needing to pass through a gel chromatographic column once.
Description
Technical Field
The invention belongs to the technical field of urokinase purification, and particularly relates to a urokinase refining method.
Background
Urokinase is a thrombolytic drug extracted from fresh human urine, is a serine protease produced by human renal tubular epithelial cells, is an alkaline protein, has an isoelectric point of about pH8.7, is white amorphous powder, and is easily soluble in water. The dilute solution is unstable in property, needs to be used fresh, and needs not to be diluted by an acid solution, and the freeze-dried state can be stable for years. Urokinase is a very specific proteolytic enzyme. The activity of the synthetic substrate is similar to that of trypsin and plasmin, and the synthetic substrate also has esterase activity, no antigenicity and no in vivo antibody production. The half-life period in vivo is (14 +/-6) min.
Urokinase activates plasminogen to active plasmin, which converts insoluble fibrin to soluble peptides, thereby dissolving the thrombus. Therefore, it is clinically used for treating thrombosis, thromboembolism and other diseases. When urokinase is combined with an anticancer agent, the urokinase can dissolve fibrin around cancer cells, so that the anticancer agent can penetrate into the cancer cells more effectively, thereby improving the capability of the anticancer agent in killing the cancer cells. Therefore, urokinase is also a good cancer adjuvant, and it has no problem of antigenicity and can be used for a long time.
D-160 adsorption resin is adopted in the past for refining urokinase, and the adsorption resin has the main problems of complicated treatment steps, large use of strong acid and strong base, high environmental protection pressure and great harm to working environment and people due to the use of large-volume strong acid.
Chinese patent application 200410018835.6 discloses a purification method of recombinant human prourokinase, comprising the following steps: A. recovering recombinant human prourokinase from the supernatant of the engineering cell culture by cation exchange Streamml ine-SP expanded bed chromatography; B. further purifying the crude urokinase product collected from A by Sephacryl S-200 gel chromatography; C. removing urokinase from the crude product by affinity chromatography using para aminobenzamidine-Sepharose Fast Flow; D. and (3) removing DNA of residual cells in the recombinant human prourokinase by using DEAE-Sepharose fast Flow anion exchange chromatography, and obtaining the recombinant human prourokinase meeting the SFDA requirement by adopting the method, wherein the total recovery rate is more than 70 percent, and the purity is more than 99 percent.
Chinese patent application 201711260186.4 discloses a four-step purification process of a new thrombolytic drug, recombinant human prourokinase, which comprises four steps of carboxymethyl radial ion exchange chromatography (CM-RIEC), microporous glass bead adsorption chromatography (MPG), dextran-polyacrylamide high performance gel chromatography (Sephacryl S-200HR) and formamidine affinity chromatography (PABZ), and is characterized in that the CM-radial ion exchange chromatography is matched with the combination of other three methods. After purification, the specific activity of prourokinase is increased by 280 times, the impurity protein can be removed by more than 98%, and the total recovery rate is up to more than 50%.
However, the methods all need the combination of gel chromatography and other chromatographic columns, and different chromatographic columns are used for multiple times, so that the complexity of the purification process is greatly improved.
Chinese patent application 201710311962.2 discloses a method for separating and purifying urokinase, which comprises the following steps: 1) preparing a crude urokinase product; 2) purifying urokinase; the step 2) comprises the following steps: purifying the clear liquid obtained by dissolving the crude urokinase prepared in the step 1) by a dextran microsphere column and/or an agarose microsphere column. The separation and purification method provided by the application can simultaneously improve the specific activity of the urokinase to more than 2 ten thousand IU/mg protein, the yield reaches 75-87%, and the proportion of the large and small molecular urokinase is more than 85%. The crude product prepared by the method is prepared by sequentially passing through a diatomite column and a CM-C column, the purity is improved to a certain extent, and the same effect cannot be obtained when the crude product is directly used for purifying a finished urokinase product in a purification step.
In view of the above, the invention provides a simple, safe and environment-friendly urokinase refining method, which has high purity, good recovery rate and wide application range, and meets the economic benefit requirement of mass production by optimizing various process parameters and only needing to pass through a gel chromatographic column once.
Disclosure of Invention
The invention aims to simplify the purification process of urokinase by adopting high-resolution gel chromatography and combining the optimization of other process conditions on the premise of ensuring the purity and recovery rate of urokinase, meet the requirement of mass production on economic benefit, simultaneously do not need strong acid, and are safe and environment-friendly.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for purifying urokinase, comprising the following steps:
and (3) taking the urokinase intermediate solution, loading the urokinase intermediate solution on a gel chromatographic column, eluting the urokinase intermediate solution by using an equilibrium buffer solution, and collecting middle-section eluent to obtain a refined urokinase solution.
Wherein, the filler of the gel chromatographic column comprises at least one of Superdex 75PG/GE, Superdex 200PG/GE, Sephacryl S-100HR/GE, Chromadex 75PG/BESTCHROM and Chromadex200PG/BESTCHROM, preferably Superdex 75 PG.
Preferably, the urokinase intermediate solution is a human urokinase intermediate solution, and the purity of the urokinase intermediate solution is preferably 65-85%, and more preferably 70%.
Preferably, the protein concentration of the upper gel chromatographic column is 2-20mg/mL, and more preferably 10 mg/mL.
Preferably, the upper column volume of the upper gel chromatography column is 2 to 8%, more preferably 5% of the column volume.
Preferably, the equilibrium buffer is at least one of an acetate buffer, a citrate buffer, a phosphate buffer and a Tris-HCl buffer, and preferably is a citrate buffer; the buffer solution contains sodium chloride, and the content of the sodium chloride in the buffer solution is 150-500 mM.
Preferably, the pH of the equilibration buffer is 4.5-8.0, more preferably 6.
Preferably, the elution is carried out at a conductivity value of 15 to 90ms/cm, more preferably 50 ms/cm.
Preferably, the mid-stream eluent is a liquid with a retention time of 40-70min, and more preferably 55 min.
Preferably, after obtaining the refined urokinase solution, the method further comprises a step of washing; the cleaning step is preferably: washing the column with a washing solution, and then washing with injection water to neutrality; further preferably, the cleaning solution is 0.1-2M NaOH solution, and the cleaning time is 0.5-2 hours.
Compared with the prior art, the invention has the beneficial effects that:
under the condition of ensuring good activity yield, the gel chromatography process is used, so that the method is simple and convenient to operate, high in purity, good in recovery rate and wide in application range; strong acid is not used, the environmental protection pressure is low, the working environment and the personnel safety are guaranteed, and the economic benefit meets the requirement of the mass production of enterprises.
Detailed Description
The present invention will be further explained with reference to specific embodiments in order to make the technical means, the original characteristics, the achieved objects and the effects of the present invention easy to understand, but the following embodiments are only preferred embodiments of the present invention, and not all embodiments are possible. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.
The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The urokinase intermediate solutions used in the following examples were made by the company under lots 20190426, 20190518.
Example 1
20190426 batches of high-purity human urokinase intermediate solution with the purity of 70 percent are taken, and the elution is carried out under the condition of the conductance value of 15ms/cm according to the protein concentration of 2mg/mL and the sample loading volume of 2 percent of the column volume by using a high-resolution gel chromatography column Superdex 200pg and washing the column by using acetate buffer solution containing 150mM sodium chloride with the pH value of 4.5. Collecting and reserving middle-section eluent for 40min to obtain refined urokinase solution.
After completion, the column was washed with 0.1M NaOH solution for 2 hours and washed to neutrality with water for injection.
Example 2
20190518 batches of high-purity human urokinase intermediate solution with the purity of 70 percent are taken, and the high-resolution gel chromatographic column Sephacryl S-100HR is adopted according to the protein concentration of 20mg/mL and the sample loading volume of 8 percent of the column volume, the pH value is 8.0, phosphate buffer solution containing 500mM sodium chloride is used for washing the column, and elution is carried out under the condition of the electric conductivity value of 90 ms/cm. Collecting and reserving the middle-section eluent for 70min to obtain the refined urokinase solution.
After completion, the column was washed with 2M NaOH solution for 0.5 hour and washed to neutrality with water for injection.
Example 3
20190426 batches of high-purity human urokinase intermediate solution with the purity of 70 percent are taken, the Superdex 75pg of high-resolution gel chromatographic column is added according to the protein concentration of 10mg/mL and the sample loading volume of 5 percent of the column volume, the column is washed by citrate buffer solution with the pH value of 6 and 150mM sodium chloride, and the elution is carried out under the condition of the electric conductivity value of 50 ms/cm. Collecting and reserving the middle section eluent of 55min to obtain the refined urokinase solution.
After completion, the column was washed with 1M NaOH solution for 1 hour and washed with water for injection to neutrality.
Example 4
In contrast to example 3, a high resolution gel chromatography column Chromadex 75PG was used, the rest being identical.
Example 5
The difference from example 3 is that Tris-HCl buffer is used as the equilibration buffer, and the rest is the same.
Example 6
In contrast to example 1, a high resolution gel chromatography column Superdex 75pg was used, the rest being identical.
Example 7
Unlike example 1, the protein concentration on the gel column was 10mg/mL, and the rest was the same.
Comparative example 1
The difference from example 3 is that CM-dextran microsphere column was used, and the rest was the same.
Comparative example 2
The same as in example 3, except that a CM-Sepharose column was used.
Comparative example 3
Unlike example 3, the protein concentration on the gel column was 30mg/mL, and the rest was the same.
Test example
The purity and the activity yield of the products of examples and comparative examples were measured and calculated according to the second part of the chinese pharmacopoeia, and the results are shown in table 1.
Table 1.
| Batch number | Purity of | Yield of activity |
| Example 1 | 95％ | 76％ |
| Example 2 | 95％ | 72％ |
| Example 3 | 99％ | 83％ |
| Example 4 | 97％ | 79％ |
| Example 5 | 96％ | 78％ |
| Example 6 | 95％ | 74％ |
| Example 7 | 96％ | 76％ |
| Comparative example 1 | 88％ | 68％ |
| Comparative example 2 | 89％ | 67％ |
| Comparative example 3 | 91％ | 71％ |
As can be seen from Table 1, the purity of the product obtained in the examples of the present invention is not less than 95%, the activity yield is not less than 70%, and example 3 is the best example, and the purity thereof is 99%. The activity yield was 83%, demonstrating that the process parameters under each aspect of this example are optimal. However, it is found from examples 6 and 7 that a single factor change does not significantly improve the final effect, demonstrating that the process parameter combination of the present scheme improves purity and yield.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A method for purifying urokinase, which is characterized by comprising the following steps:
taking the urokinase intermediate solution, loading the urokinase intermediate solution on a gel chromatographic column, eluting the urokinase intermediate solution by using an equilibrium buffer solution, and collecting middle-section eluent to obtain a refined urokinase solution;
wherein the filler of the gel chromatographic column comprises at least one of Superdex 75PG/GE, Superdex 200PG/GE, Sephacryl S-100HR/GE, Chromadex 75PG/BESTCHROM and Chromadex200 PG/BESTCHROM.
2. The method of claim 1, wherein the packing of the gel chromatography column is Superdex 75 pg/GE.
3. The method of claim 1, wherein the protein concentration of the upper gel chromatography column is 2-20 mg/mL.
4. The method of claim 1, wherein the protein concentration of the upper gel chromatography column is 10 mg/mL.
5. The method of claim 1, wherein the upper gel chromatography column has an upper column volume of 2-8% of the column volume.
6. The method of claim 1, wherein the equilibration buffer is at least one of an acetate buffer, a citrate buffer, a phosphate buffer, and a Tris-HCl buffer.
7. The method of claim 1, wherein the equilibration buffer is a citrate buffer.
8. The method of claim 1, wherein the equilibration buffer has a ph of 4.5-8.0.
9. The method according to claim 1, wherein the elution is carried out at a conductance value of 15 to 90 ms/cm.
10. The method of claim 1, wherein the mid-stream eluent is a liquid with a retention time of 40-70 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010652909.0A CN111647585A (en) | 2020-07-08 | 2020-07-08 | Method for refining urokinase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010652909.0A CN111647585A (en) | 2020-07-08 | 2020-07-08 | Method for refining urokinase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111647585A true CN111647585A (en) | 2020-09-11 |
Family
ID=72343268
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010652909.0A Pending CN111647585A (en) | 2020-07-08 | 2020-07-08 | Method for refining urokinase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111647585A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB802326A (en) * | 1955-07-01 | 1958-10-01 | Knud Abildgaard | Method of recovering urokinase from urine |
| CN101024827A (en) * | 2006-02-22 | 2007-08-29 | 中国科学院福建物质结构研究所 | Expression, purification and crystallization of urokinase catalyst structure domain mutant |
-
2020
- 2020-07-08 CN CN202010652909.0A patent/CN111647585A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB802326A (en) * | 1955-07-01 | 1958-10-01 | Knud Abildgaard | Method of recovering urokinase from urine |
| CN101024827A (en) * | 2006-02-22 | 2007-08-29 | 中国科学院福建物质结构研究所 | Expression, purification and crystallization of urokinase catalyst structure domain mutant |
Non-Patent Citations (2)
| Title |
|---|
| 祝一锋等: "尿激酶精制新工艺", 《浙江工业大学学报》 * |
| 陈于红等: "重组人尿激酶原的工业化制备与性质研究", 《南京大学学报(自然科学版)》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110894495B (en) | Preparation method of urokinase and freeze-dried powder thereof | |
| Magdolen et al. | Systematic mutational analysis of the receptor‐binding region of the human urokinase‐type plasminogen activator | |
| EP0405285A1 (en) | Novel plasminogen activator | |
| EP0363126A2 (en) | Method for the purification of vitamin K-dependent proteins | |
| FI86746C (en) | FOERFARANDE FOERFARANDE AV MAENSKLIG VAEVNADS PLASMINOGENAKTIVATOR (TPA) MED HJAELP AV SACCHAROMYCES CEREVISIAE -JEST OCH I FOERFARANDET ANVAENDA HYBRIDVEKTORER | |
| CN109498815B (en) | Chemical modifier of recombinant human kallikrein and application thereof | |
| US4381346A (en) | Isolation of plasminogen activators useful as therapeutic and diagnostic agents | |
| CN111662896A (en) | Method for purifying crude urokinase | |
| CN111647585A (en) | Method for refining urokinase | |
| US4066506A (en) | Method of separating and purifying two active forms of urokinase using affinity chromatography | |
| CN1167808C (en) | Reversed-phase HPLC assay for plasminogen activators | |
| CN101291951B (en) | Method for manufacturing high purified factor IX | |
| Nevaldine et al. | Bovine pepsinogen and pepsin. IV. A new method of purification of the pepsin | |
| CN100432222C (en) | Purification of recommbined human urokinase zymogen | |
| WO1987001389A1 (en) | Methods for the recovery of tissue plasminogen activator | |
| CN111647587A (en) | Method for purifying urokinase intermediate | |
| Bansal et al. | Production and purification of urokinase: a comprehensive review | |
| CN101880655B (en) | Method of purifying CHO cell expressing prourokinase | |
| KR100508043B1 (en) | Method for the production of rDSPAα1 | |
| KR20130136883A (en) | A novel method for purifying long-acting human growth hormone | |
| WO1981001417A1 (en) | Isolation of plasminogen activators useful as therapeutic and diagnostic agents | |
| US20130330808A1 (en) | Novel process for the purification of tissue plasminogen activator | |
| CN111748545B (en) | Method for adsorbing urokinase in urine by using agarose gel 4B | |
| EP0477277B1 (en) | Chymosin recovery and purification | |
| EP0196226A2 (en) | Method for preparing a monoclonal antibody against plasminogen-activating factor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200911 |
|
| RJ01 | Rejection of invention patent application after publication |