WO2005111207A1 - Procede de purification de prourokinase - Google Patents

Procede de purification de prourokinase Download PDF

Info

Publication number
WO2005111207A1
WO2005111207A1 PCT/CN2005/000259 CN2005000259W WO2005111207A1 WO 2005111207 A1 WO2005111207 A1 WO 2005111207A1 CN 2005000259 W CN2005000259 W CN 2005000259W WO 2005111207 A1 WO2005111207 A1 WO 2005111207A1
Authority
WO
WIPO (PCT)
Prior art keywords
column
urokinase
phosphate buffer
detector
chromatography
Prior art date
Application number
PCT/CN2005/000259
Other languages
English (en)
Chinese (zh)
Inventor
Zhengguang Zhang
Shichong Li
Yan Jiang
Jian Liu
Original Assignee
Shanghai Tasly Pharmaceutical Co., Ltd.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Shanghai Tasly Pharmaceutical Co., Ltd. filed Critical Shanghai Tasly Pharmaceutical Co., Ltd.
Publication of WO2005111207A1 publication Critical patent/WO2005111207A1/fr

Links

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
    • 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
    • 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

Definitions

  • the present invention relates to a method for purifying urokinase, and more particularly to a method for purifying urokinase by chromatography. Background technique
  • Urokinase is a specific thrombolytic drug with good market prospects. Because the amount of proUK in natural materials is very small, it is difficult to prepare in large quantities, so people began to use genetic engineering methods to allow bacteria or mammalian cells to mass produce recombinant ProUK with therapeutic value. Urokinase produced by bacteria, because the product is an insoluble inclusion body, the urokinase produced is a non-glycosylated urokinase, and requires tedious denaturation and renaturation processes to obtain a pure product. At present, it has been possible to produce urokinase in a variety of genetically engineered cells such as E. coli, mammals, yeast, and insects through genetic recombination technology. Product purification technology is very important in the production of prourokinase, and it is a key step in determining product quality, affecting production costs and benefits.
  • CM-radial cation exchange chromatography carboxymethyl radial cation exchange chromatography
  • MPG microporous high silica glass beads
  • S-200 dextran polyacrylamide high performance gel Sephacryl S-200 HR
  • CM-radial cation exchange chromatography packing used in this purification method cannot be cleaned and disinfected online, has a short service life, has poor rigidity, has an irregular structure, is more troublesome to install a chromatography column, and is not conducive to scale-up.
  • the purification method has a low yield Only 50% is not ideal for large-scale industrial production. Summary of the invention
  • the invention aims to solve the problem of large-scale purification of urokinase expressed by engineered cells.
  • the present invention provides a method using Streamline SP expanded bed chromatography, Sephacryl S-200 gel chromatography, p-aminobenzidine Sepharose Fast Flow affinity chromatography, and DEAE-Sepharose Fast Flow anion exchange chromatography. Method for cultivating a large amount of purified genetic engineering products in a supernatant.
  • the present invention adopts the following technical solutions:
  • this method has a good effect for the purification of recombinant human urokinase.
  • the invention also provides preferred process conditions:
  • step A adjusting the collected cell culture supernatant to pH 5.5 to 6.8.
  • the chromatographic column was first rinsed with 0.005 ⁇ 0. 015mol / L phosphate buffer solution (pH 5. 5 ⁇ 6.8) and equilibrated 3 ⁇ 5 column volumes, and then the cell culture supernatant was added at a flow rate of 30 ⁇ 60ml / min 015mol / L phosphoric acid until the supernatant has passed through the column, and the column is rinsed with the same buffer, and the column effluent is monitored until the ultraviolet absorption value is 0.005 8 ⁇ 7. 8 and 7. 005 ⁇ 0.
  • step A The more preferred process conditions for step A are: the collected cell culture supernatant, and the pH is adjusted to 6.4.
  • the column was first rinsed with 0.005mol / L phosphate buffer (pH 6.4) and equilibrated with 3 to 5 column volumes, and then the cell culture supernatant was added at a flow rate of 40ml / min until the supernatant completely passed the column.
  • the column was rinsed with the same buffer solution, and the column effluent was monitored with a detector until the ultraviolet absorption value was 0.005, and then changed to 0.005mol / L phosphate buffer solution (pH6.4) and 0.005mol / L Phosphate buffer (containing 1.0 mol / L sodium chloride, pH 7.0) was subjected to gradient elution of urokinase adsorbed on the column, and the eluted protein absorption peak components were collected.
  • step B placing the Sephacryl S-200 gel chromatography column on 4 ° C and connected to a preparative high performance liquid chromatograph, with 0. 005 ⁇ 0. 015mol / L phosphate buffer (containing 0. 2 ⁇ 0. 6mol / L sodium chloride, pH 6. 8 ⁇ 7.8) Equilibrate 1 to 2 column volumes; A collect the urokinase sample through a sample tube, flow rate 10 ⁇ 40ml / min, UV detection wavelength 280nm, collect the protein absorption main peak effluent.
  • Step B is a more preferred process condition: the Sephacryl S-200 gel chromatography column is placed at 4 ° C and connected to a preparative high performance liquid chromatography, using 0. 005mol / L phosphate buffer (containing 0. 5mol / L sodium chloride, pH7.4. 4) Equilibrate 1 to 2 column volumes; A collected urokinase was loaded through a sample tube, the flow rate was 20ml / min, the UV detection wavelength was 280nm, and the main protein absorption peak effluent was collected.
  • step C preferred process conditions refer to: p-aminobenzidine-Sepharose Fast Flow affinity column inlet tube connected to a peristaltic pump, placed at 4 ° C, with 0. 005 ⁇ 0. 015mol / L phosphate Buffer (containing 0.2 to 0.6mol / L sodium chloride, pH 6. 8 to 7. 8) Equilibrate 3 to 5 column volumes, connect the column outlet to the UV detector, and pump the peristaltic pump into the liquid tube Insert into the B collection solution, monitor the effluent with a UV detector, and collect the effluent that passed through the protein absorption peak.
  • Step C more preferred process conditions are: p-aminobenzidine-Sepharose Fast Flow affinity column inlet tube connected to a peristaltic pump, placed at 4 ° C, with 0. 005mol / L phosphate buffer solution (containing 0 5mol / L sodium chloride, pH 7. 0) Equilibrate 3 to 5 column volumes, connect the column outlet to the UV detector, insert the peristaltic pump into the liquid tube into the B collection solution, and monitor the effluent with the UV detector Collect the effluent that passes through the protein absorption peak.
  • 005mol ⁇ Step D preferred process conditions refer to: DEAE- Sepharose Fast Flow anion exchange chromatography column at 4 ° C, the inlet tube is connected to the peristaltic pump, the outlet end is connected to the ultraviolet detector, with 0. 005 ⁇ 0. 010mol / L Tris- HC1 buffer solution to equilibrate 3 to 5 column volumes, adjust the pH of the passing solution collected in step C to 7. 5 ⁇ 8.5 with Tris solution, and load the column, monitor with a UV detector at a wavelength of 280 nm Protein absorption value, collect protein absorption through the liquid to obtain pure recombinant human urokinase.
  • step D Place the DEAE-Sepharose Fast Flow anion exchange column at 4 ° C, connect the inlet tube to the peristaltic pump, and connect the outlet end to the UV detector.
  • the preferred method is to include step E between steps C and D: Concentrate urokinase with a cation exchange Streamline-SP fixed bed chromatography column, which can be concentrated up to 10 to 15 times, which is greatly convenient.
  • step E Concentrate urokinase with a cation exchange Streamline-SP fixed bed chromatography column, which can be concentrated up to 10 to 15 times, which is greatly convenient.
  • step E placing the Streamline-SP fixed-bed chromatography column at 4 ° C,
  • the inlet tube is connected to a peristaltic pump, and the outlet end is connected to a UV detector, and the column is balanced with 3 to 5 column volumes by using 0. 005 ⁇ 0. 0.15mol / L phosphate buffer solution (pH 5. 5 ⁇ 6. 8)
  • the pH was adjusted to 5. 5 ⁇ 6.8 with NaOH, and then loaded on the column, followed by 0.005 ⁇ 0. 015mol / L phosphate buffer solution (containing 0.2 ⁇ 0. 6mol / L sodium chloride, pH 6.8 ⁇ 7.8), and collect the eluted protein absorption peak.
  • Step E more preferred process conditions are: the Streamline-SP fixed-bed chromatography column is placed at 4 ° C, the inlet pipe is connected to a peristaltic pump, the outlet end is connected to the ultraviolet detector, with 0. 005mol / L phosphate buffer solution ( pH 6. 4) Equilibrate the column with 3 to 5 column volumes. After the sample from step C is diluted, adjust the pH to 6.4 with NaOH and load it on the column. Then use 0.005mol / L phosphate buffer solution ( Contains 0.5 mol / L sodium chloride ', pH 7.0. Elution, and collect the eluted protein absorption peak.
  • the purification method used in the present invention allows the supernatant of the culture solution to be directly applied to the column without centrifugation or filtration, and the purification method has a large processing capacity and a fast flow rate, which can save operating steps, save costs, increase yield, and is suitable for large Large-scale production;
  • the separation medium used is convenient for packing chromatographic columns, and is easy to operate and easy to clean and disinfect.
  • the original urokinase product separated and purified by the above method can meet the requirements of SF A for biological products, and the recovery rate is more than 70%, which is superior to the previous purification methods of the original urokinase product. detailed description
  • the full-length human urokinase cDNA gene was constructed and cloned into the pUC19 plasmid.
  • the PMM-UK recombinant plasmid was obtained and confirmed by sequencing.
  • the 5 'end of the gene contains Hind III and EcoR l single digestion sites, and the 3, the end contains Sai l, Xba l, Sma l, KPn l and Sac I five digestion sites, which is convenient for cloning of urokinesis genes.
  • the plasmid was double digested with pVU I and EcoR V and Klenow F enzyme was used to fill the ends, and a 3.8Kb fragment (containing the full-length prourokinase cDNA) was recovered by electrophoresis, and the fragment was inserted into the Bgl II digestion and Klenow F enzyme end-filled PMTSV-dhfr intermediate vector to obtain a 12Kb vector pMTs V- du that can express urokinase.
  • pMTSV-du plasmid DNA was transfected into CHO-dhfr-cells by calcium phosphate co-precipitation method.
  • the cells were first selected with HAT selection medium, and then changed to selection medium containing 1-3 ⁇ 10 ′ 8 ⁇ ⁇ Double screening of dhfr and MTX was performed, and positive transformed cells expressing urokinase activity were repeatedly subcloned and MTX-pressurized to amplify genes, induced by zinc ions, and finally a cell line capable of efficiently expressing urokinase was selected.
  • CHO engineering cells are made from square bottles (single-layer adherent culture)-spinner bottles (single-layer adherent culture)-stirred bottles (porous microcarrier culture)-5L Celligen reactor (porous microcarrier culture)-30L Biostat
  • the UC reactor (porous microcarrier culture) was scaled-up. Because cells can be automatically transferred between the cell-filled carrier and the empty carrier, in each stage of the expansion culture, an appropriate amount of medium and a processed porous microcarrier are first added in a larger reactor in advance, and the cell-filled porous The microcarriers are piped directly into the next reactor.
  • Control pH is 7.0 ⁇ 0.5
  • DO is 7% -40%
  • temperature It is 37.0 ⁇ 0.1 ° C
  • the stirring speed is 70r / min-90r / min.
  • the concentration of the porous microcarrier is 2g L medium.
  • the batch-type liquid exchange continuous culture method is adopted, and the liquid is exchanged through the cell retention system every day for 1-1.2 working volumes. The microcarriers are trapped in the reactor, the product-containing supernatant is harvested and fresh medium is added.
  • the high-density culture of CHO engineering cells is perfusion culture in a reactor controlled by a perfusion culture device using solid polyethylene microcarrier Biosilon or SH-2 and porous cellulose microcarrier Cytopore.
  • the column was first rinsed with 0.005mol / L phosphate buffer ( PH 6. 4) and equilibrated 3 to 5 column volumes, and then the cell culture supernatant was added at a flow rate of 40ml / min until the supernatant completely passed the column. Then, rinse the column with the same buffer solution, and monitor the column effluent with a detector until the UV absorption value is 0.005, and then use 0.005mol / L phosphate buffer solution (pH 6. 4) and 0.005mol. / L phosphate buffer
  • the DEAE-Sepharose Fast Flow anion exchange column was placed at 4 ° C, the inlet tube was connected to a peristaltic pump, the outlet end was connected to a UV detector, and 3 to 5 were equilibrated with 0.005mol / L Tris-HCl buffer solution. Column volume, Tris solution was used to adjust the pH of the passing solution collected in step C to 8.2, and then loaded onto the column, the protein absorption value was monitored at 280 nm with a UV detector, and the protein was collected and passed through the solution to obtain pure recombinant human Prourokinase.
  • Tris solution was used to adjust the pH of the passing solution collected in step C to 8.2, and then loaded onto the column, the protein absorption value was monitored at 280 nm with a UV detector, and the protein was collected and passed through the solution to obtain pure recombinant human Prourokinase.
  • the chromatographic column was first rinsed and equilibrated with 3 to 5 column volumes with 0.008 mol / L phosphate buffer solution (pH 6.0), and then the cell culture supernatant was added at a flow rate of 40 ml / minute until the supernatant passed through the column.
  • the column was rinsed with the same buffer solution, and the column effluent was monitored with a detector until the ultraviolet absorption value was 0.005, and then changed to 0.008mol / L phosphate buffer solution (pH 6.0) and 0.008mol.
  • the DEAE-Sepharose Fast Flow anion exchange column was placed at 4 ° C, the inlet tube was connected to a peristaltic pump, the outlet end was connected to a UV detector, and 3 to 5 were equilibrated with 0.008mol / L Tris-HCl buffer solution. Column volume, Tris solution was used to adjust the pH of the passing solution collected in step C to 8.2, and then loaded onto the column, the protein absorption value was monitored at 280 nm with a UV detector, and the protein was collected and passed through the solution to obtain pure recombinant human Prourokinase.
  • the DEAE-Sepharose Fast Flow anion exchange column was placed at 4 ° C, the inlet tube was connected to a peristaltic pump, the outlet end was connected to a UV detector, and 3 to 5 were equilibrated with 0.010mol / L Tris-HC1 buffer. Column volume, Tris solution was used to adjust the pH of the passing solution collected in step C to 7.5, and then loaded onto the column. The protein absorption value was monitored at 280 nm with a UV detector, and the protein was collected and passed through the solution to obtain pure recombinant human. Prourokinase.
  • 0. 012mol / L phosphate buffer solution ( PH 6. 4) Rinse and equilibrate 3 to 5 column volumes, and then add the cell culture supernatant at a flow rate of 50 ml / min until the supernatant completely passes through the column.
  • kind of buffer solution to flush the column, and monitor the column effluent with a detector until the UV absorption value is 0.005, and then use 0.012mol / L phosphate buffer (pH6.4) and 0.012mol / L phosphate
  • the buffer solution (containing 1.2 mol / L sodium chloride, pH 7.8) was subjected to gradient elution of urokinase adsorbed on the column, and the eluted protein absorption peak components were collected.
  • the DEAE-Sepharose Fast Flow anion exchange column was placed at 4 ° C, the inlet tube was connected to a peristaltic pump, the outlet end was connected to a UV detector, and 3 to 5 were equilibrated with 0. Olmol / L Tris-HCl buffer 0 ⁇ After the column volume, the pH of the permeate collected in step C was adjusted to 8.0 with Tris solution Load the column, monitor the protein absorption value with a UV detector at a wavelength of 280 nm, and collect the protein absorbed through the liquid to obtain pure recombinant human urokinase.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne un procédé de purification de prourokinase, plus particulièrement, un procédé de purification de prourokinase par chromatographie. Cette invention concerne un procédé de purification de produits de recombinaison provenant de cellules cultivées, notamment: une chromatographie à lit étendu par ligne de courant SP, une chromatographie sur gel séphacryl S-200, une chromatographie d'affinité à écoulement rapide de p-aminobenzamidine sépharose, ainsi qu'une chromatographie d'échange d'anions à écoulement rapide de DEAE-sépharose. Une étape E peut être ajoutée entre les étapes C et D: la concentration de prourokinase par chromatographie à lit fixe par ligne de courant SP d'échange de cations.
PCT/CN2005/000259 2004-04-05 2005-03-03 Procede de purification de prourokinase WO2005111207A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN200410018835.6 2004-04-05
CNB2004100188356A CN100432222C (zh) 2004-04-05 2004-04-05 一种重组人尿激酶原的纯化方法

Publications (1)

Publication Number Publication Date
WO2005111207A1 true WO2005111207A1 (fr) 2005-11-24

Family

ID=35067308

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2005/000259 WO2005111207A1 (fr) 2004-04-05 2005-03-03 Procede de purification de prourokinase

Country Status (2)

Country Link
CN (1) CN100432222C (fr)
WO (1) WO2005111207A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101880655B (zh) * 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 一种纯化cho细胞表达尿激酶原的方法
SG10201603931TA (en) * 2011-06-08 2016-07-28 Agency Science Tech & Res Purification Of Biological Products By Constrained Cohydration Chromatography
CN103159849B (zh) * 2011-12-08 2015-05-13 鲁南新时代生物技术有限公司 一种制备重组人胰岛素原的方法
CN103789291B (zh) * 2014-02-24 2016-08-17 东北制药集团股份有限公司 一种重组大肠杆菌发酵液中分离纯化重组人尿激酶原的制备工艺
CN106867984B (zh) * 2015-12-11 2021-11-12 天士力生物医药股份有限公司 一种重组人尿激酶原的纯化方法
CN106867983A (zh) * 2015-12-11 2017-06-20 上海天士力药业有限公司 一种重组人尿激酶原的病毒去除方法
CN106867985A (zh) * 2015-12-11 2017-06-20 上海天士力药业有限公司 一种重组人尿激酶原的纯化及去除病毒方法
CN108226363B (zh) * 2017-12-04 2019-01-18 赵欣雨 一种利用hplc检测尿激酶分子组分比的方法
CN114191385B (zh) * 2021-12-22 2023-06-30 天士力生物医药股份有限公司 一种重组人尿激酶原注射液及其制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03164172A (ja) * 1989-11-21 1991-07-16 Tosoh Corp ウロキナーゼ前駆体様蛋白質の精製方法
JPH0471488A (ja) * 1990-07-13 1992-03-06 Tosoh Corp ウロキナーゼ前駆体の精製法
CN1164536A (zh) * 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 溶血栓新药-重组人尿激酶原的纯化工艺
KR20020094255A (ko) * 2001-06-08 2002-12-18 씨제이 주식회사 활성형 재조합 인체 프로유로키나제의 정제 방법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03164172A (ja) * 1989-11-21 1991-07-16 Tosoh Corp ウロキナーゼ前駆体様蛋白質の精製方法
JPH0471488A (ja) * 1990-07-13 1992-03-06 Tosoh Corp ウロキナーゼ前駆体の精製法
CN1164536A (zh) * 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 溶血栓新药-重组人尿激酶原的纯化工艺
KR20020094255A (ko) * 2001-06-08 2002-12-18 씨제이 주식회사 활성형 재조합 인체 프로유로키나제의 정제 방법

Also Published As

Publication number Publication date
CN1680550A (zh) 2005-10-12
CN100432222C (zh) 2008-11-12

Similar Documents

Publication Publication Date Title
WO2005111207A1 (fr) Procede de purification de prourokinase
AU635222B2 (en) Method for the purification of proteins
US6399333B1 (en) Process for producing erythropoietin containing no animal proteins
JPH11504217A (ja) 抗肥満タンパク質を製造する方法
WO1996040883A1 (fr) Nouveaux procedes de purification du facteur ix
JPH0134596B2 (fr)
CN109627322A (zh) 一种从Cohn组分Ⅴ上清中分离纯化人血清白蛋白的方法
CN114249817B (zh) 一种分离纯化人抗凝血酶ⅲ的方法
CN102121013B (zh) 可高效表达的成熟鸡α干扰素多肽的制备方法
US5731186A (en) Method for the production of rDSPA α1
WO1993003061A1 (fr) Multiplicateur de cellules souches hematopoietiques
CN101880655B (zh) 一种纯化cho细胞表达尿激酶原的方法
CN1062016C (zh) 重组人糖基化尿激酶原的制备方法
CN103555760B (zh) 一种重组人血小板生成素的制备方法及其制剂
WO1992004450A1 (fr) Activateurs de plasminogene hybrides
CN1164536A (zh) 溶血栓新药-重组人尿激酶原的纯化工艺
CN113121638B (zh) 一种纯化重组蛋白的方法
CN102040659A (zh) 一种重组人促红素分离纯化方法
RU2683950C1 (ru) Способ очистки рекомбинантного тканевого активатора плазминогена для медицинского применения
JP2008520220A (ja) 組換え型ヒトｘｉｉｉ因子の精製
CN106867985A (zh) 一种重组人尿激酶原的纯化及去除病毒方法
CN1190130A (zh) 重组人红细胞生成素制剂的制备生产方法
CN1096541A (zh) 家蚕基因工程生产重组人巨噬细胞集落刺激因子
CN118126991A (zh) 多片段插入提高胰蛋白酶热稳定性的方法
CN1277206A (zh) 含s(主蛋白)和前s1(大蛋白)乙肝表面基因工程融合抗原的纯化工艺

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase