CN1680550A - Purification of recommbined human urokinase zymogen - Google Patents
Purification of recommbined human urokinase zymogen Download PDFInfo
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- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 title claims abstract description 69
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 title claims abstract description 35
- 229960005356 urokinase Drugs 0.000 title claims abstract description 35
- 238000000746 purification Methods 0.000 title claims description 37
- 102000010911 Enzyme Precursors Human genes 0.000 title claims description 31
- 108010062466 Enzyme Precursors Proteins 0.000 title claims description 31
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 34
- 238000004113 cell culture Methods 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 20
- 229920002684 Sepharose Polymers 0.000 claims abstract description 16
- 239000012506 Sephacryl® Substances 0.000 claims abstract description 15
- 229920002271 DEAE-Sepharose Polymers 0.000 claims abstract description 14
- 239000000872 buffer Substances 0.000 claims description 74
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 64
- 239000002953 phosphate buffered saline Substances 0.000 claims description 64
- 239000007788 liquid Substances 0.000 claims description 63
- 238000010521 absorption reaction Methods 0.000 claims description 47
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 37
- 230000002572 peristaltic effect Effects 0.000 claims description 37
- 102000004169 proteins and genes Human genes 0.000 claims description 28
- 238000012546 transfer Methods 0.000 claims description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 239000012530 fluid Substances 0.000 claims description 20
- 239000000523 sample Substances 0.000 claims description 20
- 239000012228 culture supernatant Substances 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 11
- 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 10
- 239000007983 Tris buffer Substances 0.000 claims description 10
- 238000005349 anion exchange Methods 0.000 claims description 10
- 238000013016 damping Methods 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- 238000011010 flushing procedure Methods 0.000 claims description 10
- 238000012544 monitoring process Methods 0.000 claims description 10
- 238000002953 preparative HPLC Methods 0.000 claims description 10
- 238000001179 sorption measurement Methods 0.000 claims description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 10
- 238000000825 ultraviolet detection Methods 0.000 claims description 10
- 239000012470 diluted sample Substances 0.000 claims description 7
- 238000001042 affinity chromatography Methods 0.000 claims description 6
- 125000002091 cationic group Chemical group 0.000 claims description 4
- 238000005571 anion exchange chromatography Methods 0.000 claims description 3
- 238000005227 gel permeation chromatography Methods 0.000 claims description 3
- 230000002485 urinary effect Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 241000124008 Mammalia Species 0.000 abstract description 3
- 238000004587 chromatography analysis Methods 0.000 abstract description 3
- 238000011084 recovery Methods 0.000 abstract description 3
- 102100031358 Urokinase-type plasminogen activator Human genes 0.000 description 29
- 210000004027 cell Anatomy 0.000 description 16
- 238000012545 processing Methods 0.000 description 11
- 108010073863 saruplase Proteins 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 5
- WPANETAWYGDRLL-UHFFFAOYSA-N 4-aminobenzenecarboximidamide Chemical compound NC(=N)C1=CC=C(N)C=C1 WPANETAWYGDRLL-UHFFFAOYSA-N 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000005277 cation exchange chromatography Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 3
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 2
- 101150074155 DHFR gene Proteins 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 240000008791 Antiaris toxicaria Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 244000270834 Myristica fragrans Species 0.000 description 1
- 235000009421 Myristica fragrans Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 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
- 238000000975 co-precipitation Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000001702 nutmeg Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 229960000103 thrombolytic agent Drugs 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000003245 working effect Effects 0.000 description 1
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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
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- 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
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- 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
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Abstract
The invention is about a method to purify the recombined human urokinase by the chromatography. The invention relates to recovery the gene engineering production from the mammal cell culture using the Streamline-SP, Sephacryl S-200, Sepharose Fast Flow and DEAE-Sepharose Fast Flow method. The recombined human urokinase can meet the SFDA standard and the percent recovery is above 70%, the purity is above 99% by using the method.
Description
Technical field
The present invention relates to a kind of purification process of recombinant human urokinase zymogen, more specifically say so with chromatographic technique purification of Recombinant human pro-urokinase's method.
Background technology
UPA is a specific specificity thrombolytic agent, has good market outlook.At present, can be by gene recombination technology at cells produce uPAs such as several genes engineering cell such as intestinal bacteria, Mammals, yeast, insects.The purification technique of product is extremely important in the production process of uPA, is the committed step that determines quality product, influences production cost and benefit.
The purification technique of the uPA of open source literature report, as (1990) such as Avgennos G.C. with fast flow velocity sulfonic acid type sepharose pearl (S-Sepharose Fast Flow) cation-exchange chromatography, the p-Aminobenzamidine affinity chromatography, sulfonic acid type cation exchange fast protein liquid chromatogram (mono-S FPLC), p-Aminobenzamidine affinity chromatography four step rule second time purifying human pro-urokinase from CHO genetically engineered cell culture, it is more loaded down with trivial details that but this four-step method is operated in actual applications, prolonged the production operation time, and the uPA overall yield of purifying only is 40%.According to said method amplify suitability for industrialized production, certainly will be restricted.
In addition as patent application " purifying process of thrombus dissolving new drug---recombinant human urokinase zymogen " (applicant country: China, publication number: CN1164536A, open day on November 12nd, 1997) disclose a kind of four step purifying process to recombinant human urokinase zymogen in the CHO genetically engineered cell nutrient solution, comprise: the first step carboxymethyl is cation-exchange chromatography (CM-is cation-exchange chromatography radially) radially, second step micro pore high silicon granulated glass sphere (MPG) adsorption chromatography, the 3rd step S-200 type dextran polyacrylamide efficient gel (Sephacryl S-200HR) chromatography and the 4th step p-Aminobenzamidine affinity chromatography.The CM-that this purification process adopts radially cation-exchange chromatography filler can not on-line cleaning sterilize, and work-ing life is short, and poor rigidity, irregular structure, and the filling chromatographic column is cumbersome, is unfavorable for amplifying; In addition, the yield of this purification process is lower, only has 50%, and it is not ideal enough to be used for large-scale commercial production.
Summary of the invention
The present invention is intended to solve the large scale purification problem of the uPA of animal cell expression.
For this reason, the invention provides a kind of with Streamline-SP expanding bed chromatogram, Sephacryl S-200 gel chromatography, p-Aminobenzamidine Sepharose Fast Flow affinity chromatography and DEAE-Sepharose Fast Flow anion-exchange chromatography etc., the method for large-scale purification gene engineering product from Mammals engineering cell culture supernatant.
For achieving the above object, the present invention is by the following technical solutions:
A. from the engineering cell culture supernatant, reclaim recombinant human urokinase zymogen with cationic exchange Streamline-SP expanding bed chromatogram;
B. be further purified the thick product of uPA that above-mentioned A collects with Sephacryl S-200 gel chromatography;
C. remove the urokinase in the thick product with p-Aminobenzamidine-Sepharose Fast Flow affinity chromatography;
D. remove residual cell DNA in the recombinant human urokinase zymogen with DEAE-Sepharose Fast Flow anion-exchange chromatography.
The present invention also provides preferred processing condition:
Wherein the preferred processing condition of steps A are meant: with the cells and supernatant of collecting, transfer pH to 5.5～6.8.Chromatographic column is earlier with 0.005～0.015mol/L phosphate buffered saline buffer (pH5.5～6.8) flushing and 3～5 column volumes of balance, add the cell culture supernatant then, flow velocity 30～60ml/ minute, all pass through chromatographic column until supernatant liquor, wash chromatographic column with same buffer again, and with detector monitors chromatographic column effluent liquid, until ultraviolet absorption value≤0.005, use 0.005～0.015mol/L phosphate buffered saline buffer (pH5.5～6.8) and 0.005～0.015mol/L phosphate buffered saline buffer then instead and (contain 0.8～1.2mol/L sodium-chlor, pH6.8～7.8) carry out gradient elution and be adsorbed on uPA on the chromatographic column, collect eluted protein absorption peak component.
The preferred processing condition of steps A are: the cells and supernatant of collection, transfer pH to 6.4.Chromatographic column is earlier with 0.005mol/L phosphate buffered saline buffer (pH6.4) flushing and 3～5 column volumes of balance, add the cell culture supernatant then, flow velocity 40ml/ minute, all pass through chromatographic column until supernatant liquor, wash chromatographic column with same buffer again, and with detector monitors chromatographic column effluent liquid, until ultraviolet absorption value≤0.005, use 0.005mol/L phosphate buffered saline buffer (pH6.4) and 0.005mol/L phosphate buffered saline buffer then instead and (contain 1.0mol/L sodium-chlor, pH7.0) carry out gradient elution and be adsorbed on uPA on the chromatographic column, collect eluted protein absorption peak component.
Wherein the preferred processing condition of step B are meant: Sephacryl S-200 gel chromatographic columns is placed 4 ℃ and link to each other with a preparative high performance liquid chromatography instrument, with 1～2 column volume of 0.005～0.015mol/L phosphate buffered saline buffer (containing 0.2～0.6mol/L sodium-chlor, pH6.8～7.8) balance; The uPA that A collects is by sample on the sample introduction pipe, and flow velocity 10～40ml/ minute, ultraviolet detection wavelength 280nm collected albumen and absorbs the main peak effluent liquid.
The preferred processing condition of step B are: Sephacryl S-200 gel chromatographic columns is placed 4 ℃ and link to each other with a preparative high performance liquid chromatography instrument, (contain 0.5mol/L sodium-chlor, pH7.4) 1～2 column volume of balance with the 0.005mol/L phosphate buffered saline buffer; The uPA that A collects is by sample on the sample introduction pipe, and flow velocity 20ml/ minute, ultraviolet detection wavelength 280nm collected albumen and absorbs the main peak effluent liquid.
Wherein the preferred processing condition of step C are meant: p-Aminobenzamidine-Sepharose Fast Flow affinity chromatographic column liquid-inlet pipe is linked to each other with peristaltic pump, place 4 ℃, (contain 0.2～0.6mol/L sodium-chlor with 0.005～0.015mol/L phosphate buffered saline buffer, pH6.8～7.8) 3～5 column volumes of balance, the chromatographic column outlet is connected with UV-detector, the peristaltic pump liquid-inlet pipe is inserted B collect in the liquid,, collect and pass albumen absorption peak effluent liquid with UV-detector monitoring stream fluid.
The preferred processing condition of step C are: p-Aminobenzamidine-Sepharose Fast Flow affinity chromatographic column liquid-inlet pipe is linked to each other with peristaltic pump, place 4 ℃, (contain 0.5mol/L sodium-chlor with the 0.005mol/L phosphate buffered saline buffer, pH7.0) 3～5 column volumes of balance, the chromatographic column outlet is connected with UV-detector, the peristaltic pump liquid-inlet pipe is inserted B collect in the liquid,, collect and pass albumen absorption peak effluent liquid with UV-detector monitoring stream fluid.
Wherein the preferred processing condition of step D are meant: DEAE-Sepharose Fast Flow anion-exchange column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.005～0.010mol/L Tris-HCl damping fluid balance, the pH that passes liquid that step C is collected with Tris solution transfers to 7.5～8.5 back upper props, under the 280nm wavelength, monitor the albumen absorption value with UV-detector, collect protein adsorption and pass liquid, obtain pure recombinant human urokinase zymogen.
The preferred processing condition of step D are: DEAE-Sepharose Fast Flow anion-exchange column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.005mol/L Tris-HCl damping fluid balance, the pH that passes liquid that step C is collected with 1mol/L Tris solution transfers to 8.2 back upper props, under the 280nm wavelength, monitor the albumen absorption value with UV-detector, collect protein adsorption and pass liquid, obtain pure recombinant human urokinase zymogen.
In order to reach better experiment effect, preferable methods is also to comprise step e between step C and D: with cationic exchange Streamline-SP fixed bed chromatographic column urinary concentration prokinase, concentrated can reaching about 10～15 times greatly facilitates subsequent operations.
The preferred processing condition of step e are: Streamline-SP fixed bed chromatographic column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.005～0.015mol/L phosphate buffered saline buffer (pH5.5～6.8) balance chromatographic column, after C goes on foot the diluted sample that gets off, behind NaOH accent pH to 5.5～6.8, upper prop, use 0.005～0.015mol/L phosphate buffered saline buffer (to contain 0.2～0.6mol/L sodium-chlor subsequently, pH6.8～7.8) wash-out, and collect the eluted protein absorption peak.
The preferred processing condition of step e are: Streamline-SP fixed bed chromatographic column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.005mol/L phosphate buffered saline buffer (pH6.4) balance chromatographic column, after C goes on foot the diluted sample that gets off, transfer pH to 6.4 with NaOH after, upper prop, use the 0.005mol/L phosphate buffered saline buffer (to contain 0.5mol/L sodium-chlor, pH7.0) wash-out, and collection eluted protein absorption peak subsequently.
Purification process of the present invention, nutrient solution supernatant do not need centrifugal or filter, and get final product direct upper prop, and this purification process treatment capacity are big, and flow velocity is fast, can save operation steps, save cost, improve yield, are suitable for scale operation; In addition, the separating medium that is adopted is convenient to load chromatographic column, and easy and simple to handle, is easy to cleaning and sterilizing.UPA product through the aforesaid method separation and purification can reach the requirement of SFDA to biological products, and the rate of recovery reaches more than 70%, is better than uPA purifying products method in the past.
Embodiment
In order to further specify the present invention, below enumerate the enforcement specific embodiments of the invention.
Embodiment one
The clone of uPA gene and the structure of expression vector
(referring to Chinese patent: the preparation method of reorganized human saccharified urokinasen, notification number CN1062016C, the day for announcing: February 14 calendar year 2001)
Adopt nutmeg ester (PMA) to induce Detroit 562 cells, extract cell total rna, the construction cDNA library, obtained to contain the positive clone strain of coding urokinase gene fragment by screening, obtain human pro-urokinase's full-length cDNA gene and be cloned into the pUC19 plasmid, obtained the PMM-UK recombinant plasmid;
From the pMM-UK plasmid DNA, separate pro-UK cDNA and insert intermediate carrier 1pSV
2Obtain to express the recombinant plasmid pSV of uPA among the-pro-UK
2-pro-UK;
From pSV
2Obtain including SV40 enhanser and dihydrofolate reductase gene in-the dhfr carrier, this fragment is inserted in the pXMT carrier, thereby obtain intermediate carrier pMTSV-dhfr;
From pSV
2Obtain containing SV40 enhanser and total length pro-urokinase cDNA among-the pro-UK, and this fragment is inserted in the PMTSV-dhfr intermediate carrier, can obtain to express the carrier pMTsv-du of uPA.
Embodiment two
The CHO engineering cell that transfection and screening efficiently express
20-40 μ g pMTSV-du plasmid DNA by coprecipitation of calcium phosphate method transfection CHO-dhfr-cell, is selected the substratum screening with HAT earlier, and 10 change into after big and contain 1-3 * 10
-8The selection substratum of M MTX carries out dhfr and the dual screening of MTX, and expression is had the active positive transformant of uPA through repeatedly subclone and MTX pressurization amplification gene, and zine ion is induced, and finishing screen is chosen the cell strain that can efficiently express uPA.
Embodiment three
The cultivation of CHO engineering cell and amplification
The CHO engineering cell is by square vase (monolayer adherence cultivation) rolling bottle (monolayer adherence cultivation) blender jar (porous microcarrier cultivation) 5LCelligen reactor (porous microcarrier cultivation) 30L Biostat UC reactor (porous microcarrier cultivation) amplification culture step by step.Because cell can shift between carrier that covers with cell and empty carrier automatically, during every grade of amplification culture, earlier add an amount of substratum and treated porous microcarrier in advance in more massive reactor, the porous microcarrier that covers with cell is by in the direct person of modern times's next stage of the pipeline reactor.Control pH is 7.0 ± 0.5, and DO is 7%-40%, and temperature is 37.0 ± 0.1 ℃, and mixing speed is that the concentration of 70r/min-90r/min. porous microcarrier is the 2g/L-g/L substratum.Adopt batch formula to change liquid cultured continuously mode, change liquid 1-1.2 working volume by the cell retention system every day, and microcarrier is trapped in the reactor, and results contain the supernatant of product and add fresh culture.
Embodiment four
With chromatogram purification method purification of Recombinant human pro-urokinase
A. collect 50 liters of engineering cell culture supernatant, transfer pH to 6.4 with HCl.Chromatographic column is earlier with 0.005mol/L phosphate buffered saline buffer (pH6.4) flushing and 3～5 column volumes of balance, add the cell culture supernatant then, flow velocity 40ml/ minute, all pass through chromatographic column until supernatant liquor, wash chromatographic column with same buffer again, and with detector monitors chromatographic column effluent liquid, until ultraviolet absorption value≤0.005, use 0.005mol/L phosphate buffered saline buffer (pH6.4) and 0.005mol/L phosphate buffered saline buffer then instead and (contain 1.0mol/L sodium-chlor, pH7.0) carry out gradient elution and be adsorbed on uPA on the chromatographic column, collect eluted protein absorption peak component.
B. Sephacryl S-200 gel chromatographic columns is placed 4 ℃ and link to each other, (contain 0.5mol/L sodium-chlor, pH7.0) 1～2 column volume of balance with the 0.005mol/L phosphate buffered saline buffer with a preparative high performance liquid chromatography instrument; The uPA that A collects is by sample on the sample introduction pipe, and flow velocity 20ml/ minute, ultraviolet detection wavelength 280nm collected albumen and absorbs the main peak effluent liquid.
C. p-Aminobenzamidine-Sepharose Fast Flow affinity chromatographic column liquid-inlet pipe is linked to each other with peristaltic pump, place 4 ℃, (contain 0.5mol/L sodium-chlor with the 0.005mol/L phosphate buffered saline buffer, pH7.0) 3～5 column volumes of balance, the chromatographic column outlet is connected with UV-detector, the peristaltic pump liquid-inlet pipe is inserted B collect in the liquid,, collect and pass albumen absorption peak effluent liquid with UV-detector monitoring stream fluid.
D. DEAE-Sepharose Fast Flow anion-exchange column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.005mol/L Tris-HCl damping fluid balance, the pH that passes liquid that step C is collected with Tris solution transfers to 8.2 back upper props, under the 280nm wavelength, monitor the albumen absorption value with UV-detector, collect protein adsorption and pass liquid, obtain pure recombinant human urokinase zymogen.
Embodiment five
With chromatogram purification method purification of Recombinant human pro-urokinase
A. collect 100 liters of engineering cell culture supernatant, transfer pH to 6.0 with HCl.Chromatographic column is earlier with 0.008mol/L phosphate buffered saline buffer (pH6.0) flushing and 3～5 column volumes of balance, add the cell culture supernatant then, flow velocity 40ml/ minute, all pass through chromatographic column until supernatant liquor, wash chromatographic column with same buffer again, and with detector monitors chromatographic column effluent liquid, until ultraviolet absorption value≤0.005, use 0.008mol/L phosphate buffered saline buffer (pH6.0) and 0.008mol/L phosphate buffered saline buffer then instead and (contain 1.2mol/L sodium-chlor, pH7.6) carry out gradient elution and be adsorbed on uPA on the chromatographic column, collect eluted protein absorption peak component.
B. Sephacryl S-200 gel chromatographic columns is placed 4 ℃ and link to each other, (contain 0.2mol/L sodium-chlor, pH7.6) 1～2 column volume of balance with the 0.008mol/L phosphate buffered saline buffer with a preparative high performance liquid chromatography instrument; The uPA that A collects is by sample on the sample introduction pipe, and flow velocity 20ml/ minute, ultraviolet detection wavelength 280nm collected albumen and absorbs the main peak effluent liquid.
C. p-Aminobenzamidine-Sepharose Fast Flow affinity chromatographic column liquid-inlet pipe is linked to each other with peristaltic pump, place 4 ℃, (contain 0.2mol/L sodium-chlor with the 0.008mol/L phosphate buffered saline buffer, pH7.6) 3～5 column volumes of balance, the chromatographic column outlet is connected with UV-detector, the peristaltic pump liquid-inlet pipe is inserted B collect in the liquid,, collect and pass albumen absorption peak effluent liquid with UV-detector monitoring stream fluid.
D. DEAE-Sepharose Fast Flow anion-exchange column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.01mol/L Tris-HCl damping fluid balance, the pH that passes liquid that step C is collected with Tris solution transfers to 7.8 back upper props, under the 280nm wavelength, monitor the albumen absorption value with UV-detector, collect protein adsorption and pass liquid, obtain pure recombinant human urokinase zymogen.
Embodiment six
With chromatogram purification method purification of Recombinant human pro-urokinase
A. collect 200 liters of engineering cell culture supernatant, transfer pH to 5.8 with HCl.Chromatographic column is earlier with 0.012mol/L phosphate buffered saline buffer (pH5.8) flushing and 3～5 column volumes of balance, add the cell culture supernatant then, flow velocity 60ml/ minute, all pass through chromatographic column until supernatant liquor, wash chromatographic column with same buffer again, and with detector monitors chromatographic column effluent liquid, until ultraviolet absorption value≤0.005, use 0.012mol/L phosphate buffered saline buffer (pH5.8) and 0.012mol/L phosphate buffered saline buffer then instead and (contain 0.8mol/L sodium-chlor, pH6.8) carry out gradient elution and be adsorbed on uPA on the chromatographic column, collect eluted protein absorption peak component.
B. Sephacryl S-200 gel chromatographic columns is placed 4 ℃ and link to each other, (contain 0.4mol/L sodium-chlor, pH6.8) 1～2 column volume of balance with the 0.012mol/L phosphate buffered saline buffer with a preparative high performance liquid chromatography instrument; The uPA that A collects is by sample on the sample introduction pipe, and flow velocity 40ml/ minute, ultraviolet detection wavelength 280nm collected albumen and absorbs the main peak effluent liquid.
C. p-Aminobenzamidine-Sepharose Fast Flow affinity chromatographic column liquid-inlet pipe is linked to each other with peristaltic pump, place 4 ℃, (contain 0.4mol/L sodium-chlor with the 0.012mol/L phosphate buffered saline buffer, pH6.8) 3～5 column volumes of balance, the chromatographic column outlet is connected with UV-detector, the peristaltic pump liquid-inlet pipe is inserted B collect in the liquid,, collect and pass albumen absorption peak effluent liquid with UV-detector monitoring stream fluid.
D. DEAE-Sepharose Fast Flow anion-exchange column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.008mol/L Tris-HCl damping fluid balance, the pH that passes liquid that step C is collected with Tris solution transfers to 8.2 back upper props, under the 280nm wavelength, monitor the albumen absorption value with UV-detector, collect protein adsorption and pass liquid, obtain pure recombinant human urokinase zymogen.
Embodiment seven
With chromatogram purification method purification of Recombinant human pro-urokinase
A. collect 200 liters of engineering cell culture supernatant, transfer pH to 5.5 with HCl.Chromatographic column is earlier with 0.01mol/L phosphate buffered saline buffer (pH5.5) flushing and 3～5 column volumes of balance, add the cell culture supernatant then, flow velocity 45ml/ minute, all pass through chromatographic column until supernatant liquor, wash chromatographic column with same buffer again, and with detector monitors chromatographic column effluent liquid, until ultraviolet absorption value≤0.005, use 0.01mol/L phosphate buffered saline buffer (pH5.5) and 0.01mol/L phosphate buffered saline buffer then instead and (contain 0.8mol/L sodium-chlor, pH7.5) carry out gradient elution and be adsorbed on uPA on the chromatographic column, collect eluted protein absorption peak component.
B. Sephacryl S-200 gel chromatographic columns is placed 4 ℃ and link to each other, (contain 0.3mol/L sodium-chlor, pH7.5) 1～2 column volume of balance with the 0.01mol/L phosphate buffered saline buffer with a preparative high performance liquid chromatography instrument; The uPA that A collects is by sample on the sample introduction pipe, and flow velocity 30ml/ minute, ultraviolet detection wavelength 280nm collected albumen and absorbs the main peak effluent liquid.
C. p-Aminobenzamidine-Sepharose Fast Flow affinity chromatographic column liquid-inlet pipe is linked to each other with peristaltic pump, place 4 ℃, (contain 0.3mol/L sodium-chlor with the 0.01mol/L phosphate buffered saline buffer, pH7.5) 3～5 column volumes of balance, the chromatographic column outlet is connected with UV-detector, the peristaltic pump liquid-inlet pipe is inserted B collect in the liquid,, collect and pass albumen absorption peak effluent liquid with UV-detector monitoring stream fluid.
E. Streamline-SP fixed bed chromatographic column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.01mol/L phosphate buffered saline buffer (pH5.5) balance chromatographic column, after C goes on foot the diluted sample that gets off, transfer pH to 5.5 with NaOH after, upper prop, use the 0.01mol/L phosphate buffered saline buffer (to contain 0.3mol/L sodium-chlor, pH7.5) wash-out, and collection eluted protein absorption peak subsequently.
D. DEAE-Sepharose Fast Flow anion-exchange column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.010mol/L Tris-HCl damping fluid balance, the pH that passes liquid that step C is collected with Tris solution transfers to 7.5 back upper props, under the 280nm wavelength, monitor the albumen absorption value with UV-detector, collect protein adsorption and pass liquid, obtain pure recombinant human urokinase zymogen.
Embodiment eight
With chromatogram purification method purification of Recombinant human pro-urokinase
A. collect 150 liters of engineering cell culture supernatant, transfer pH to 6.4 with HCl.Chromatographic column is earlier with 0.012mol/L phosphate buffered saline buffer (pH6.4) flushing and 3～5 column volumes of balance, add the cell culture supernatant then, flow velocity 50ml/ minute, all pass through chromatographic column until supernatant liquor, wash chromatographic column with same buffer again, and with detector monitors chromatographic column effluent liquid, until ultraviolet absorption value≤0.005, use 0.012mol/L phosphate buffered saline buffer (pH6.4) and 0.012mol/L phosphate buffered saline buffer then instead and (contain 1.2mol/L sodium-chlor, pH7.8) carry out gradient elution and be adsorbed on uPA on the chromatographic column, collect eluted protein absorption peak component.
B. Sephacryl S-200 gel chromatographic columns is placed 4 ℃ and link to each other, (contain 0.2mol/L sodium-chlor, pH7.8) 1～2 column volume of balance with the 0.012mol/L phosphate buffered saline buffer with a preparative high performance liquid chromatography instrument; The uPA that A collects is by sample on the sample introduction pipe, and flow velocity 40ml/ minute, ultraviolet detection wavelength 280nm collected albumen and absorbs the main peak effluent liquid.
C. p-Aminobenzamidine-Sepharose Fast Flow affinity chromatographic column liquid-inlet pipe is linked to each other with peristaltic pump, place 4 ℃, (contain 0.2mol/L sodium-chlor with the 0.012mol/L phosphate buffered saline buffer, pH7.8) 3～5 column volumes of balance, the chromatographic column outlet is connected with UV-detector, the peristaltic pump liquid-inlet pipe is inserted B collect in the liquid,, collect and pass albumen absorption peak effluent liquid with UV-detector monitoring stream fluid.
E. Streamline-SP fixed bed chromatographic column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.012mol/L phosphate buffered saline buffer (pH6.4) balance chromatographic column, after C goes on foot the diluted sample that gets off, transfer pH to 6.4 with NaOH after, upper prop, use the 0.012mol/L phosphate buffered saline buffer (to contain 0.2mol/L sodium-chlor, pH7.8) wash-out, and collection eluted protein absorption peak subsequently.
D. DEAE-Sepharose Fast Flow anion-exchange column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.01mol/L Tris-HCl damping fluid balance, the pH that passes liquid that step C is collected with Tris solution transfers to 8.0 back upper props, under the 280nm wavelength, monitor the albumen absorption value with UV-detector, collect protein adsorption and pass liquid, obtain pure recombinant human urokinase zymogen.
Embodiment nine
With chromatogram purification method purification of Recombinant human pro-urokinase
A. collect 150 liters of engineering cell culture supernatant, transfer pH to 6.8 with HCl.Chromatographic column is earlier with 0.005mol/L phosphate buffered saline buffer (pH6.8) flushing and 3～5 column volumes of balance, add the cell culture supernatant then, flow velocity 50ml/ minute, all pass through chromatographic column until supernatant liquor, wash chromatographic column with same buffer again, and with detector monitors chromatographic column effluent liquid, until ultraviolet absorption value≤0.005, use 0.005mol/L phosphate buffered saline buffer (pH6.8) and 0.005mol/L phosphate buffered saline buffer then instead and (contain 1.2mol/L sodium-chlor, pH7.0) carry out gradient elution and be adsorbed on uPA on the chromatographic column, collect eluted protein absorption peak component.
B. Sephacryl S-200 gel chromatographic columns is placed 4 ℃ and link to each other, (contain 0.5mol/L sodium-chlor, pH7.0) 1～2 column volume of balance with the 0.005mol/L phosphate buffered saline buffer with a preparative high performance liquid chromatography instrument; The uPA that A collects is by sample on the sample introduction pipe, and flow velocity 35ml/ minute, ultraviolet detection wavelength 280nm collected albumen and absorbs the main peak effluent liquid.
C. p-Aminobenzamidine-Sepharose Fast Flow affinity chromatographic column liquid-inlet pipe is linked to each other with peristaltic pump, place 4 ℃, (contain 0.5mol/L sodium-chlor with the 0.005mol/L phosphate buffered saline buffer, pH7.0) 3～5 column volumes of balance, the chromatographic column outlet is connected with UV-detector, the peristaltic pump liquid-inlet pipe is inserted B collect in the liquid,, collect and pass albumen absorption peak effluent liquid with UV-detector monitoring stream fluid.
E. Streamline-SP fixed bed chromatographic column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.005mol/L phosphate buffered saline buffer (pH6.8) balance chromatographic column, after C goes on foot the diluted sample that gets off, transfer pH to 6.8 with NaOH after, upper prop, use the 0.005mol/L phosphate buffered saline buffer (to contain 0.5mol/L sodium-chlor, pH7.0) wash-out, and collection eluted protein absorption peak subsequently.
D. DEAE-Sepharose Fast Flow anion-exchange column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.005mol/L Tris-HCl damping fluid balance, the pH that passes liquid that step C is collected with Tris solution transfers to 8.2 back upper props, under the 280nm wavelength, monitor the albumen absorption value with UV-detector, collect protein adsorption and pass liquid, obtain pure recombinant human urokinase zymogen.
Claims (12)
1, a kind of purification process of recombinant human urokinase zymogen is characterized in that this method comprises the steps:
A. from the engineering cell culture supernatant, reclaim recombinant human urokinase zymogen with cationic exchange Streamline-SP expanding bed chromatogram;
B. be further purified the thick product of uPA that above-mentioned A collects with Sephacryl S-200 gel chromatography;
C. remove the urokinase in the thick product with p-Aminobenzamidine-Sepharose Fast Flow affinity chromatography;
D. remove the DNA of residual cell in the recombinant human urokinase zymogen with DEAE-Sepharose Fast Flow anion-exchange chromatography.
2,, it is characterized in that between step C and D, also comprising step e according to the purification process of the described recombinant human urokinase zymogen of claim 1:
With cationic exchange Streamline-SP fixed bed chromatogram urinary concentration prokinase.
3, purification process according to the described recombinant human urokinase zymogen of claim 2, it is characterized in that: described step e is meant Streamline-SP fixed bed chromatographic column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.005～0.015mol/L phosphate buffered saline buffer (pH5.5～6.8) balance chromatographic column, after C goes on foot the diluted sample that gets off, behind NaOH accent pH to 5.5～6.8, upper prop, use 0.005～0.015mol/L phosphate buffered saline buffer (to contain 0.2～0.6mol/L sodium-chlor subsequently, pH6.8～7.8) wash-out, and collect the eluted protein absorption peak.
4, according to the purification process of the described recombinant human urokinase zymogen of claim 3, it is characterized in that: described step e is meant Streamline-SP fixed bed chromatographic column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.005mol/L phosphate buffered saline buffer (pH6.4) balance chromatographic column, after C goes on foot the diluted sample that gets off, behind NaOH accent pH to 6.4, upper prop, use the 0.01mol/L phosphate buffered saline buffer (to contain 0.5mol/L sodium-chlor subsequently, pH7.0) wash-out, and collect the eluted protein absorption peak.
5, according to the purification process of claim 1,2 or 3 described recombinant human urokinase zymogens, it is characterized in that: described steps A is meant the cells and supernatant of will collect, and transfers pH to 5.5～6.8.Chromatographic column is earlier with 0.005～0.015mol/L phosphate buffered saline buffer (pH5.5～6.8) flushing and 3～5 column volumes of balance, add the cell culture supernatant then, flow velocity 30～60ml/ minute, all pass through chromatographic column until supernatant liquor, wash chromatographic column with same buffer again, and with detector monitors chromatographic column effluent liquid, until ultraviolet absorption value≤0.005, use 0.005～0.015mol/L phosphate buffered saline buffer (pH5.5～6.8) and 0.005～0.015mol/L phosphate buffered saline buffer then instead and (contain 0.8～1.2mol/L sodium-chlor, pH6.8～7.8) carry out gradient elution and be adsorbed on uPA on the chromatographic column, collect eluted protein absorption peak component.
6, according to the purification process of the described recombinant human urokinase zymogen of claim 5, it is characterized in that: described steps A is meant the cells and supernatant of will collect, and transfers pH to 6.4.Chromatographic column is earlier with 0.005mol/L phosphate buffered saline buffer (pH6.4) flushing and 3～5 column volumes of balance, add the cell culture supernatant then, flow velocity 40ml/ minute, all pass through chromatographic column until supernatant liquor, wash chromatographic column with same buffer again, and with detector monitors chromatographic column effluent liquid, until ultraviolet absorption value≤0.005, use 0.005mol/L phosphate buffered saline buffer (pH6.4) and 0.005mol/L phosphate buffered saline buffer then instead and (contain 1.0mol/L sodium-chlor, pH7.0) carry out gradient elution and be adsorbed on uPA on the chromatographic column, collect eluted protein absorption peak component.
7, according to the purification process of claim 1,2 or 3 described recombinant human urokinase zymogens, it is characterized in that: described step B is meant Sephacryl S-200 gel chromatographic columns is placed 4 ℃ and link to each other with a preparative high performance liquid chromatography instrument, with 1～2 column volume of 0.005～0.015mol/L phosphate buffered saline buffer (containing 0.2～0.6mol/L sodium-chlor, pH6.8～7.8) balance; The uPA that A collects is by sample on the sample introduction pipe, and flow velocity 10～40ml/ minute, ultraviolet detection wavelength 280nm collected albumen and absorbs the main peak effluent liquid.
8, according to the purification process of the described recombinant human urokinase zymogen of claim 7, it is characterized in that: described step B is meant Sephacryl S-200 gel chromatographic columns is placed 4 ℃ and link to each other with a preparative high performance liquid chromatography instrument, (contain 0.5mol/L sodium-chlor, pH7.0) 1～2 column volume of balance with the 0.005mol/L phosphate buffered saline buffer; The uPA that A collects is by sample on the sample introduction pipe, and flow velocity 20ml/ minute, ultraviolet detection wavelength 280nm collected albumen and absorbs the main peak effluent liquid.
9, according to the purification process of claim 1,2 or 3 described recombinant human urokinase zymogens, it is characterized in that: described step C is meant p-Aminobenzamidine-Sepharose Fast Flow affinity chromatographic column liquid-inlet pipe is linked to each other with peristaltic pump, place 4 ℃, (contain 0.2～0.6mol/L sodium-chlor with 0.005～0.015mol/L phosphate buffered saline buffer, pH6.8～7.8) 3～5 column volumes of balance, the chromatographic column outlet is connected with UV-detector, the peristaltic pump liquid-inlet pipe is inserted B to be collected in the liquid, with UV-detector monitoring stream fluid, collect and pass albumen absorption peak effluent liquid.
10, according to the purification process of the described recombinant human urokinase zymogen of claim 9, it is characterized in that: described step C is meant p-Aminobenzamidine-Sepharose Fast Flow affinity chromatographic column liquid-inlet pipe is linked to each other with peristaltic pump, place 4 ℃, (contain 0.5mol/L sodium-chlor with the 0.005mol/L phosphate buffered saline buffer, pH7.0) 3～5 column volumes of balance, the chromatographic column outlet is connected with UV-detector, the peristaltic pump liquid-inlet pipe is inserted B to be collected in the liquid, with UV-detector monitoring stream fluid, collect and pass albumen absorption peak effluent liquid.
11, according to claim 1, the purification process of 2 or 3 described recombinant human urokinase zymogens, it is characterized in that: described step D is meant DEAE-Sepharose Fast Flow anion-exchange column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.005～0.010mol/L Tris-HCl damping fluid balance, pass liquid pH to 7.5～8.5 back upper props with what the rapid C of Tris solution pacing collected, under the 280nm wavelength, monitor the albumen absorption value with UV-detector, collect protein adsorption and pass liquid, obtain pure recombinant human urokinase zymogen.
12, purification process according to the described recombinant human urokinase zymogen of claim 11, it is characterized in that: described step D is meant DEAE-Sepharose Fast Flow anion-exchange column is placed 4 ℃, liquid-inlet pipe links to each other with peristaltic pump, exit end links to each other with UV-detector, with 3～5 column volumes of 0.005mol/L Tris-HCl damping fluid balance, pass liquid pH to 8.2 back upper prop with what the rapid C of 1mol/L Tris solution pacing collected, under the 280nm wavelength, monitor the albumen absorption value with UV-detector, collect protein adsorption and pass liquid, obtain pure recombinant human urokinase zymogen.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100188356A CN100432222C (en) | 2004-04-05 | 2004-04-05 | Purification of recommbined human urokinase zymogen |
| PCT/CN2005/000259 WO2005111207A1 (en) | 2004-04-05 | 2005-03-03 | A method of purifying prourokinase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100188356A CN100432222C (en) | 2004-04-05 | 2004-04-05 | Purification of recommbined human urokinase zymogen |
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| CN100432222C CN100432222C (en) | 2008-11-12 |
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| WO (1) | WO2005111207A1 (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880655A (en) * | 2009-05-06 | 2010-11-10 | 中国人民解放军军事医学科学院生物工程研究所 | Method of purifying CHO cell expressing prourokinase |
| CN103159849A (en) * | 2011-12-08 | 2013-06-19 | 鲁南新时代生物技术有限公司 | Recombinant human proinsulin preparation method |
| CN103717284A (en) * | 2011-06-08 | 2014-04-09 | 新加坡科技研究局 | Purification of biological products by constrained cohydration chromatography |
| CN103789291A (en) * | 2014-02-24 | 2014-05-14 | 东北制药集团股份有限公司 | Preparation process of separating and purifying recombinant human pro-urokinase in recombinant E. colifermentation broth |
| CN106867985A (en) * | 2015-12-11 | 2017-06-20 | 上海天士力药业有限公司 | A kind of purifying of recombinant human urokinase zymogen and removal viral methods |
| CN106867984A (en) * | 2015-12-11 | 2017-06-20 | 上海天士力药业有限公司 | A kind of purification process of recombinant human urokinase zymogen |
| CN106867983A (en) * | 2015-12-11 | 2017-06-20 | 上海天士力药业有限公司 | A kind of virus removal method of recombinant human urokinase zymogen |
| CN108226363A (en) * | 2017-12-04 | 2018-06-29 | 赵欣雨 | It is a kind of using HPLC detect urokinase molecular components than method |
| CN114191385A (en) * | 2021-12-22 | 2022-03-18 | 天士力生物医药股份有限公司 | Recombinant human prourokinase injection and preparation method thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03164172A (en) * | 1989-11-21 | 1991-07-16 | Tosoh Corp | Purification of urokinase precursor-like protein |
| JPH0471488A (en) * | 1990-07-13 | 1992-03-06 | Tosoh Corp | Method for purifying urokinase precursor |
| CN1164536A (en) * | 1996-05-03 | 1997-11-12 | 中国人民解放军军事医学科学院生物工程研究所 | Purifying process of new thrombolytic drug-recombinant human urokinase zymogen |
| KR100454891B1 (en) * | 2001-06-08 | 2004-11-09 | 씨제이 주식회사 | A method for purifying the active recombinant human prourokinase |
-
2004
- 2004-04-05 CN CNB2004100188356A patent/CN100432222C/en not_active Expired - Lifetime
-
2005
- 2005-03-03 WO PCT/CN2005/000259 patent/WO2005111207A1/en active Application Filing
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880655B (en) * | 2009-05-06 | 2013-12-04 | 中国人民解放军军事医学科学院生物工程研究所 | Method of purifying CHO cell expressing prourokinase |
| CN101880655A (en) * | 2009-05-06 | 2010-11-10 | 中国人民解放军军事医学科学院生物工程研究所 | Method of purifying CHO cell expressing prourokinase |
| CN103717284B (en) * | 2011-06-08 | 2016-02-24 | 新加坡科技研究局 | By retraining common hydration chromatogram purification biological products |
| CN103717284A (en) * | 2011-06-08 | 2014-04-09 | 新加坡科技研究局 | Purification of biological products by constrained cohydration chromatography |
| US9809639B2 (en) | 2011-06-08 | 2017-11-07 | Agency For Science, Technology And Research | Purification of biological products by constrained cohydration chromatography |
| CN103159849B (en) * | 2011-12-08 | 2015-05-13 | 鲁南新时代生物技术有限公司 | Recombinant human proinsulin preparation method |
| CN103159849A (en) * | 2011-12-08 | 2013-06-19 | 鲁南新时代生物技术有限公司 | Recombinant human proinsulin preparation method |
| CN103789291A (en) * | 2014-02-24 | 2014-05-14 | 东北制药集团股份有限公司 | Preparation process of separating and purifying recombinant human pro-urokinase in recombinant E. colifermentation broth |
| CN103789291B (en) * | 2014-02-24 | 2016-08-17 | 东北制药集团股份有限公司 | The preparation technology of isolated and purified recombinant human urokinase zymogen in a kind of Recombinant E. coli Fermentation Broth |
| CN106867985A (en) * | 2015-12-11 | 2017-06-20 | 上海天士力药业有限公司 | A kind of purifying of recombinant human urokinase zymogen and removal viral methods |
| CN106867984A (en) * | 2015-12-11 | 2017-06-20 | 上海天士力药业有限公司 | A kind of purification process of recombinant human urokinase zymogen |
| CN106867983A (en) * | 2015-12-11 | 2017-06-20 | 上海天士力药业有限公司 | A kind of virus removal method of recombinant human urokinase zymogen |
| CN106867984B (en) * | 2015-12-11 | 2021-11-12 | 天士力生物医药股份有限公司 | Purification method of recombinant human prourokinase |
| CN108226363A (en) * | 2017-12-04 | 2018-06-29 | 赵欣雨 | It is a kind of using HPLC detect urokinase molecular components than method |
| CN114191385A (en) * | 2021-12-22 | 2022-03-18 | 天士力生物医药股份有限公司 | Recombinant human prourokinase injection and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2005111207A1 (en) | 2005-11-24 |
| CN100432222C (en) | 2008-11-12 |
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