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Purification method of rhTNK-tPA (recombinant human TNK tissue-type plasminogen activator for injection) cell harvesting fluid

A purification method and cell technology, applied in the field of protein purification, can solve the problems of easy agglomeration of the filler, low recovery rate, shortened service life of the filler, etc., and achieve the effects of reducing production cost, simple operation and good repeatability

Active Publication Date: 2018-08-21
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

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Problems solved by technology

However, the applicant found in practical application that the recovery rate of this method is low and the repeatability is not ideal enough
[0004] Secondly, in practical application, it is found that the packing in the column during the purification process is easy to agglomerate after loading the sample, resulting in frequent cleaning and repacking of the packing, which shortens the service life of the packing

Method used

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  • Purification method of rhTNK-tPA (recombinant human TNK tissue-type plasminogen activator for injection) cell harvesting fluid

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1, rhTNK-tPA purification

[0028] A) Filtration of the supernatant: harvest the cell supernatant in the cell reactor, and filter it with a 0.2 μm sterile filter after deep filtration;

[0029] B) Blue Sepharose 6FastFlow affinity column chromatography: Before the chromatography, the column is equilibrated with 2.5 times the column volume buffer ①, and its components are 20mM phosphate buffer and 0.04% Tween 80, pH7.2; for sample loading, use 2.5 times the column volume of buffer ② to wash out impurity proteins, the components are: 20mM phosphate buffer, 2M sodium chloride and 0.04% Tween 80, pH7.2; use 3 times The target protein was obtained by eluting with the column volume of eluent I. The components of eluent I were 20mM phosphate buffer, 1M sodium chloride, 2M urea and 0.04% Tween 80, and the pH value of the eluent was 7.0;

[0030] C) Lysine HyperD affinity column chromatography: The equilibration and sample loading process is the same as step B). Afte...

Embodiment 2

[0033] Embodiment 2, rhTNK-tPA purification

[0034] The difference between Example 2 and Example 1 is that the Capto Q anion exchange resin column chromatography step is added after step C), and the specific operation is: equilibrate and load according to step C), and use 2.0 times the column volume of the eluent IV was eluted to obtain the target protein. The composition of eluent IV was: 20mM phosphate buffer, 0.1M 6-amino-n-caproic acid, 0.2M arginine, 0.04% Tween 80, and the pH value of the eluent was 7.0. The dosage is 2.0 times the volume, and the remaining parameters and operations are as shown in Example 1.

[0035] After testing, the obtained rhTNK-tPA solution had a purity of 99.8%, a recovery rate of 88.3%, and a specific activity of 526,000 IU / mg.

Embodiment 3

[0036] Embodiment 3, rhTNK-tPA purification

[0037] The difference between embodiment 3 and embodiment 1 is that the order of step C) and step D) is reversed, and other parameters and operations are as shown in embodiment 1.

[0038] After testing, the obtained rhTNK-tPA solution had a purity of 99.3%, a recovery rate of 96.5%, and a specific activity of 625,000 IU / mg.

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Abstract

The invention belongs to the technical field of protein separation and purification and particularly relates to a purification method of an rhTNK-tPA (recombinant human TNK tissue-type plasminogen activator for injection) cell harvesting fluid. The key point is that the rhTNK-tPA cell harvesting fluid is subjected to deep filtration and sterilizing filtration before column chromatography, so thatthe problem that the conventional filler for column chromatography after filtration is prone to caking can be solved effectively, clarity of a filtrate is high (smaller than or equal to 0.3 NTU), filter loss is small (smaller than or equal to 5%), and significant progress is made as compared with the prior art.

Description

technical field [0001] The invention belongs to the technical field of protein purification, and in particular relates to a method for purifying rhTNK-tPA cell harvest liquid. Background technique [0002] Recombinant Human TNK Tissue-type Plasminogen Activator for Injection (Recombinant Human TNK Tissue-type Plasminogen Activator for Injection, rhTNK-tPA, trade name: Mingfule) is independently developed and produced by Guangzhou Mingkang Bioengineering Co., Ltd. using DNA recombination technology It is a kind of biological product, which is clinically used for thrombolytic treatment of acute myocardial infarction, and is the safest third-generation thrombolytic emergency drug so far. The production of rhTNK-tPA not only requires high-quality high-yield cell lines as the basic premise, but also requires corresponding large-scale high-density cell culture technology and large-scale and efficient purification methods. [0003] In the existing purification process, because the...

Claims

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Application Information

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IPC IPC(8): C12N9/48
CPCC12N9/6459C12Y304/21068
Inventor 吴宜南董珣钟勤陈军区叶才文
Owner 石药集团明复乐药业(广州)有限公司
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