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Purification method for recombined human tissue type plasminogen exciter TNK mutant rhTNK-tPA

A technology of plasminogen and purification method, applied in the direction of hydrolase and the like, can solve the problems of high price, cannot be cleaned with alkali, t-PA is not ideal, etc., and achieves the effects of simple operation, good repeatability and low price

Active Publication Date: 2012-07-04
石药集团明复乐药业(广州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Benzamidine is used to purify serine protease, and some people also use it to purify t-PA or separate single-chain and double-chain t-PA, but because it cannot be cleaned with alkali, it cannot be treated to remove pyrogens, and it is expensive. Not ideal for purifying t-PA

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The purification method of the recombinant human tissue-type plasminogen activator TNK mutant rhTNK-tPA provided in this example is as follows:

[0025] (1) Supernatant pretreatment: Harvest the serum-free culture supernatant of the cells in the reactor, and filter through a microporous membrane to remove cell debris to protect the purified filler;

[0026] (2) Blue Sepharose 6Fast Flow affinity column chromatography: equilibrate 2.5 times column volume with buffer A, the component of buffer A is 20mM phosphate buffer saline and 0.04% Tween 80, pH7.2; Load sample; Use Wash 2.5 times the column volume with buffer A; wash out the impurity protein with buffer B, the component of buffer B is buffer A, add 2M NaCl on the basis of adding 2M NaCl; use 2.5 times the column volume of buffer C to elute to get the target peak, The components of buffer C are 20mM phosphate, 1M NaCl, 2M urea and 0.04% Tween 80, pH7.20; the target peak is diluted 2 times with phosphate buffer and the...

Embodiment 2

[0031] The purification method of the recombinant human tissue-type plasminogen activator TNK mutant rhTNK-tPA provided in this example is as follows:

[0032] (1) After filtering the rhTNK-tPA cell culture medium, take the supernatant, and after Blue Sepharose 6FastFlow affinity column chromatography, elute with eluent a to obtain the eluent a1 containing the target peak;

[0033] (2) After the eluent a1 containing the target peak obtained in step (1) is subjected to Lysine HyperD affinity column chromatography, it is eluted with eluent b to obtain the eluent b1 containing the target peak;

[0034] (3) The eluent b1 containing the target peak obtained in step (2) is subjected to Sephadex G-25 gel filtration, and the purified recombinant human tissue-type plasminogen activator is obtained after eluting with eluent c TNK mutant rhTNK-tPA.

[0035] In the above steps:

[0036] During step (1) in Blue Sepharose 6Fast Flow affinity column chromatography, according to the binding...

Embodiment 3

[0045] The purification method of the recombinant human tissue-type plasminogen activator TNK mutant rhTNK-tPA provided in this example is as follows:

[0046] (1) Supernatant pretreatment: Harvest the serum-free culture supernatant of the cells in the reactor, and filter through a microporous membrane to remove cell debris to protect the purified filler;

[0047] (2) Take the rhTNK-tPA supernatant, and after Blue Sepharose 6Fast Flow affinity column chromatography, elute with eluent a to obtain the eluent a1 containing the target peak;

[0048] (3) After the eluent a1 containing the target peak obtained in step (1) is subjected to Lysine HyperD affinity column chromatography, it is eluted with eluent b to obtain the eluent b1 containing the target peak;

[0049] (4) Take the eluent b1 containing the target peak obtained in step (2) and carry out Sephadex G-25 gel filtration, and obtain purified recombinant human tissue-type plasminogen activator TNK after eluting with eluent ...

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PUM

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Abstract

The invention relates to a purification method for recombined human tissue type plasminogen exciter TNK mutant rhTNK-tPA. The method comprises the following steps of: 1, filtering the rhTNK-tPA cell culture medium and taking the supernatant, and after chromatography by a BlueSepharose6FastFlow compatible column, eluting by using eluent a to obtain eluent a1 containing a target peak; 2, after performing chromatography of the eluent a1 containing the target peak obtained in step 1 by a LysineHyperD compatible column, eluting by using eluent b to obtain eluent b1 containing the target peak; and 3, performing SephadexG-25 gel filtration of the eluent b1 containing the target peak obtained in step 2, and obtaining the purified recombined human tissue type plasminogen exciter TNK mutant rhTNK-tPA after elution by eluent c. The method has the characteristics of high capacity, high flow speed, easy regeneration, simple process and low cost, and is suitable for linear amplification and mass production.

Description

technical field [0001] The invention belongs to the technical field of separation and purification of recombinant protein of medical biotechnology, and specifically relates to a purification method of recombinant human tissue-type plasminogen activator TNK mutant rhTNK-tPA. Background technique [0002] Recombinant human tissue-type plasminogen activator TNK mutant (recombinant human tissue-type plasminogen activator for TNK mutant, rhTNK-tPA) is a new type of thrombolytic drug expressed in mammalian cells using gene recombination technology, clinically used Thrombolytic therapy for acute myocardial infarction is by far the safest and most effective thrombolytic emergency drug. In the production process of rhTNK-tPA, the purification process has an important impact on the quality of the product. [0003] The existing purification scheme of t-PA is mainly determined according to its molecular structure characteristics. Use ion exchange chromatography such as SP-Sepharose, C...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/48
Inventor 杨琴曹凤兰王志坚吴宜南董珣梁银冰
Owner 石药集团明复乐药业(广州)有限公司