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Purification of recommbined human urokinase zymogen

A technology of prourokinase and purification method, which is applied in peptide preparation methods, chemical instruments and methods, biochemical equipment and methods, etc. It can solve the problems of short service life, unfavorable amplification, irregular structure, etc., and achieve saving operation steps, easy to clean and disinfect, cost-saving effect

Active Publication Date: 2005-10-12
TASLY BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The CM-radial cation exchange chromatographic filler used in this purification method cannot be cleaned and sterilized on-line, has a short service life, and has poor rigidity and irregular structure. It is troublesome to fill the chromatographic column, which is not conducive to amplification; , only 50%, not ideal for large-scale industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Cloning of Prourokinase Gene and Construction of Expression Vector

[0028] (See Chinese patent: preparation method of recombinant human glycosylated pro-urokinase, announcement number CN1062016C, announcement date: February 14, 2001)

[0029] Detroit 562 cells were induced by myristyl ester (PMA), the total RNA was extracted, and a cDNA library was constructed. A positive clone containing a gene fragment encoding urokinase was obtained by screening, and the full-length cDNA gene of human prourokinase was obtained and cloned into pUC19 Plasmid, obtained the PMM-UK recombinant plasmid;

[0030] From the pMM-UK plasmid DNA, the pro-UK cDNA was isolated and inserted into the intermediate vector 1pSV 2 -A recombinant plasmid pSV capable of expressing prourokinase was obtained in pro-UK 2 -pro-UK;

[0031] from pSV2 - The dhfr vector contains the SV40 enhancer and dihydrofolate reductase gene, and this fragment is inserted into the pXMT vector to obtain the intermediate v...

Embodiment 2

[0034] Transfection and screening of highly expressed CHO engineered cells

[0035] Transfect 20-40μg pMTSV-du plasmid DNA into CHO-dhfr-cells by calcium phosphate co-precipitation method, select with HAT selection medium first, and replace with 1-3×10 -8 The selection medium of M MTX is double-screened by dhfr and MTX, and the positively transformed cells expressing pro-urokinase activity are subjected to multiple subcloning and MTX pressurized gene amplification, zinc ion induction, and finally screened to highly express urokinase original cell line.

Embodiment 3

[0037] Cultivation and scale-up of CHO engineered cells

[0038] CHO engineered cells consist of square flask (monolayer adherent culture)-spinner bottle (monolayer adherent culture)-stirred flask (porous microcarrier culture)-5LCelligen reactor (porous microcarrier culture)-30L Biostat UC reactor (porous microcarrier culture) Microcarrier culture) step-by-step scale-up culture. Since the cells can be automatically transferred between the carrier full of cells and the empty carrier, when each stage of scale-up culture is carried out, an appropriate amount of medium and treated porous microcarriers are pre-added in a larger-scale reactor, and the porous microcarriers full of cells The microcarriers are directly approached to the next-stage reactor through the pipeline. The control pH is 7.0±0.5, DO is 7%-40%, temperature is 37.0±0.1°C, stirring speed is 70r / min-90r / min. The concentration of porous microcarrier is 2g / L-g / L culture medium. Batch-type medium-changing continuous ...

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PUM

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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 [0001] The invention relates to a method for purifying recombinant human pro-urokinase, more specifically a method for purifying recombinant human pro-urokinase by chromatographic technology. Background technique [0002] Prourokinase is a specific thrombolytic drug with good market prospects. At present, it has been possible to produce prourokinase in a variety of genetically engineered cells such as Escherichia coli, mammals, yeast, insects and other cells by gene recombination technology. Product purification technology is very important in the production process of prourokinase, and it is a key step that determines product quality and affects production cost and benefit. [0003] The purification technology of pro-urokinase reported in open literature, such as Avgennos G.C. etc. (1990) with fast flow rate sulfonic acid type agarose gel beads (S-Sepharose Fast Flow) cation exchange chromatography, p-aminobenzamidine affinity chromatography, sulfonic acid ...

Claims

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Application Information

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IPC IPC(8): C07K1/14C07K1/16C12N9/72
CPCC12N9/6456C07K1/16C12Y304/21073
Inventor 肖成祖张正光姜燕胡显文胥照平李世崇刘健高丽华
Owner TASLY BIOPHARMACEUTICALS CO LTD
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