Pharmaceutical composition containing recombinant tissue plasminogen activator

A composition and drug technology, applied in the directions of drug combination, recombinant DNA technology, medical preparations containing active ingredients, etc., can solve the problems of increased downstream protein purification work, low yield of expressed cell lines, poor stability of tPA protein, etc.

Active Publication Date: 2014-03-12
AMPSOURCE BIOPHARMA (SHANGHAI) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, continuous perfusion culture needs to continuously add fresh medium while harvesting the culture medium, which is very costly and makes downstream protein purification more difficult
At present, the published tPA recombinant products all use continuous perfusion culture technology, mainly due to the poor stability of tPA protein itself and the low expression level of cell lines, so perfusion culture technology is a more suitable choice
[0014] In summary, there are still some limitations in the clinical application of TNK-tPA products, such as relatively short half-life, and the treatment time window is not wide enough.
In addition, the yield of the constructed expression cell line is low, and the perfusion production process adopted increases the production cost and makes downstream protein purification more complicated and difficult

Method used

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  • Pharmaceutical composition containing recombinant tissue plasminogen activator
  • Pharmaceutical composition containing recombinant tissue plasminogen activator
  • Pharmaceutical composition containing recombinant tissue plasminogen activator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1. Construction of the gene encoding the TNK-tPA-L-vFcγ fusion protein

[0079] The gene sequence encoding TNK-tPA leading peptide and mature protein is artificially optimized CHO cell preferred codons and obtained by chemical synthesis. In order to facilitate the insertion of the target fragment into the specific site of the expression vector, there is a restriction enzyme endonuclease site at the 5' and 3' ends of the synthesized fragment, respectively SpeI and BamHI. The full-length 1705bp DNA fragment is inserted between the EcoRV restriction enzyme sites in the transfer vector such as pUC57 to obtain an intermediate plasmid whose TNK-tPA gene sequence is verified by DNA sequencing.

[0080] Flexible peptide linker and human IgG Fc region Fc γ2 variant vFc γ2 (Pro331Ser mutation), Fc γ4 variant vFc γ4 (Ser228Pro and Leu235Ala mutations), Fc γ1 variant vFc γ1 (Leu234Val, Leu235Ala, and Pro331SSer mutations) fusion genes are also artificially optimized C...

Embodiment 2

[0082] Example 2. Expression of Fusion Proteins in Transfected Cell Lines

[0083] Transfection of recombinant expression vector plasmids into mammalian host cell lines to express TNK-tPA-L-vFc γ fusion protein. For stable high-level expression, a preferred host cell line is a DHFR enzyme-deficient CHO-cell (US Patent No. 4,818,679). A preferred method of transfection is electroporation, although other methods including calcium phosphate co-sedimentation, lipofection, and protoplast fusion can also be used. In electroporation, use a Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA) set to a 210V electric field and a 1050 μFd capacitance, in a cuvette of 2 to 3 × 1O 7 Add 20 μg of the plasmid linearized with PvuI to each cell. Two days after transfection, the medium was changed to growth medium containing 0.6 mg / mL G418. Transfectants were screened for resistance to the selective drug using an anti-human IgG Fc ELISA assay. The expression of fusion protein ca...

Embodiment 3

[0085] Example 3. Production of fusion proteins

[0086] The high-yield cell line preferably obtained in Example 2 is first subjected to serum-free acclimation culture in a petri dish, and then transferred to a shake flask for suspension acclimatization culture. After the cells are adapted to these culture conditions, they are then cultured in a 300ml shake flask with feed supplementation or simulated perfusion culture by changing the medium every day. The above-mentioned CHO-derived cell lines were fed-cultured in 100ml shake flasks for 13 days, and the cumulative yield of the expressed recombinant fusion protein was 1.90g / L ( Image 6 ). Between days 6 and 10 of cell culture, the viable cell density can reach a maximum of 22 × 10 6 A / mL. In order to obtain more TNK-tPA-L-vFc recombinant protein, 2000ml shake flask culture can also be selected. In another culture method, the above-mentioned CHO-derived cell line is replaced with a medium in a 100ml volume shake flask every...

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Abstract

The invention discloses a pharmaceutical composition containing a recombinant tissue plasminogen activator. The pharmaceutical composition consists of a pharmaceutically acceptable carrier, excipient or thinner, as well as an effective amount of recombinant TNK-tPA-L-Fc fusion protein; from N terminal to C terminal, the protein sequentially includes TNK-tPA, a flexible peptide joint and human IgG natural or variant Fc. The fusion protein is similar to human TNK-tPA in both in vitro and in vivo bioactivities, and can greatly prolong plasma half-life.

Description

technical field [0001] The present invention relates to the field of molecular medicine, more specifically, the present invention relates to an Fc fusion protein of recombinant long-acting tissue-type plasminogen activator mutant TNK-tPA, and on the other hand relates to the preparation process of the fusion protein and its Medical applications, especially in the treatment of acute myocardial infarction, acute pulmonary embolism, acute ischemic stroke and other diseases that require rapid dissolution of thrombus, as well as its use in medical devices or biomedicine that come into contact with human blood or tissue New uses for coating the surface of materials in medicine. Background technique [0002] Tissue plasminogen activator (tissue plasminogen activator, tPA) is a kind of plasminogen activator, which is mainly synthesized and released by vascular endothelial cells. It is a serine protease mainly present in mammalian plasma. tPA efficiently and specifically binds to fi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/49A61K47/48A61P7/02A61P9/10C12N15/62C07K19/00C12N15/63C12N5/10A61L27/54A61L27/34A61L29/16A61L29/08A61L31/16A61L31/10
Inventor 李强孙乃超周若芸刘瑞贤金宜慧
Owner AMPSOURCE BIOPHARMA (SHANGHAI) INC
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