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Process for cultivating recombinant human tectotype fibrinolytic enzyme activator TNK mutant with micro-carrier

A technology of microcarrier culture and plasminogen, applied in the direction of recombinant DNA technology, cells modified by introducing foreign genetic material, etc., can solve the problems of complicated process operation and high cost, and achieve high cell density, low cost and high expression product higher than live effect

Inactive Publication Date: 2008-01-02
冯来坤
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AI Technical Summary

Problems solved by technology

[0004] The present invention provides a process for culturing and producing recombinant human tissue plasminogen activator TNK mutants with microcarriers, which overcomes the deficiencies of the existing fermenter suspension and batch culture methods, such as high cost and complicated process operation, etc. Suitable for large-scale cultivation and industrial production

Method used

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Embodiment

[0017] 1. Materials

[0018] 1.1 Porous microcarriers

[0019] The Cytopore microcarrier is a product of Pharmacia, the matrix is ​​cellulose, the diameter is 200 μm, the pore size is 30 μm, and the swelling volume is 40 mL / g.

[0020] After soaking in 0.1M, pH7.0 phosphate buffer solution (PBS) for 4 hours before use, the PBS was poured off, and then washed three times with PBS. After autoclaving at 121°C for 30 minutes, the PBS was aspirated and washed twice with DMEN medium.

[0021] 1.2 cells

[0022] The engineered cells producing rhTNK-tPA are CHO cell lines containing TNK-tPA cDNA and dhfr gene.

[0023] 1.3 Medium

[0024] The medium was DMEN containing 5% fetal bovine serum (FBS). The culture medium and FBS are both products of GIBCO Company,

[0025] 1.4 Reagents

[0026] CHO-S-S II, a product of GIBCO

[0027] MTX, product of SIGAN company

[0028] Gentamicin, domestic pharmaceutical grade

[0029] 0.25% trypsin, domestic analytically pure product

[0030...

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PUM

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Abstract

The invention discloses a technology for producing and recombining a human tectotype profibrinolytic activator TNK mutant with Cytopore porous microcarrier culture method, which comprises the following steps: suspending TNK-tPA engineering project CHO cell strain with the culture medium; culturing by transferring to the cell culture bottle; forming dense monolayer cell; culturing by transferring the cell to Wheaton agitator; transferring to the Celligen reactor (the culture temperature is 37 Deg. C, PH is 7.1+-0.1, the dissolved oxygen is 30-60%, the original rotating speed is 20rpm, the rotating speed increases progressively after 4 hours and reaches 40rpm when inoculating for 6 hours); culturing 20 days by replenishing the culture liquid and the fresh microcarrier; collecting when the cell concentration reaches the finite level. TNK-tPA with the invention has the big cell density, the low cost, the high expression level and the high expression product specific activity.

Description

technical field [0001] The invention relates to a new production process of recombinant human tissue-type plasminogen activator TNK mutant (rhTNK-tPA), especially discloses a production method of recombinant human tissue-type plasminogen activator TNK by adopting Cytopore porous microcarrier culture method The technology of the mutant to produce rhTNK-tPA belongs to the technical field of cell culture production technology. Background technique [0002] The rhTNK-tPA involved in the present invention can be used clinically for rescue and treatment of thrombotic diseases such as myocardial infarction and cerebral thrombosis. And easy to rescue and many other advantages. Since rhTNK-tPA is used in a large amount clinically, generally 30-40 mg / dose, if the large-scale culture technology of engineered cell lines cannot efficiently express and produce rhTNK-tPA, the industrialization of rhTNK-tPA will not be carried out, making rhTNK It is impossible for an excellent drug like ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/09
Inventor 冯来坤
Owner 冯来坤
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