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Purification method of recombinant human tissue type profibrinolysin activator modified body

A technology of plasminogen and purification method, which is applied in the preparation methods of peptides, chemical instruments and methods, organic chemistry, etc., can solve the problems of difficult clinical research and treatment of thrombosis patients, high price of rht-PA, etc., and achieves the output of finished products. High, easy to operate, high-purity effect

Active Publication Date: 2010-06-02
BEIJING SHIMAODONGRUI PHARMA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are many research reports on t-PA and its mutants in our country, but they are still in the laboratory research stage and have not entered the clinic. The application of rht-PA depends on imports, and rht-PA is expensive. Bringing great difficulties, the market urgently needs locally produced rht-PA thrombolytic drugs

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  • Purification method of recombinant human tissue type profibrinolysin activator modified body
  • Purification method of recombinant human tissue type profibrinolysin activator modified body

Examples

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Embodiment 1

[0045] This example further illustrates the preparation method of recombinant human tissue-type plasminogen activator (rhM-tPA);

[0046] A. Engineering cell construction:

[0047] In the present invention, the cell line name is CHO-D8 strain, which was constructed by Beijing Shimao Dongrui Pharmaceutical Technology Co., Ltd.; the construction method is as follows: 103 in the amino acid sequence of human tissue plasminogen activator (numbered in NCBI as P00750) Amino acid N at position 1 is replaced by T, Q at position 117 is replaced by N, KHRR at position 296-299 is replaced by AAAA, and the complete gene sequence of human tissue-type plasminogen activator containing mutation sites is obtained artificially; the sequence enzyme cut and transfected into dihydrofolate reductase-deficient Chinese hamster ovary cells (CHO-dhfr - ), cloned and pressurized to select a cell line that can highly express rhM-tPA as an engineered cell; this similar construction method is recorded in t...

Embodiment 2

[0105] This embodiment further illustrates the composition of the mobile phase in the described three-step purification process:

[0106] a. The mobile phase of the first step chromatographic purification treatment is liquid A, liquid B, liquid C:

[0107] The liquid A includes: phosphate buffer saline (PBS) 20mM, aprotinin (Aprotinin) 0.2-20mg / L, Tween 20 (Tween20) 0.043%;

[0108] The B solution includes: phosphate buffer (PB) 20mM, sodium chloride (NaCl) 0.5-5.0M, aprotinin (Aprotinin) 0.2-20mg / L, Tween 20 (Tween20) 0.043%;

[0109] The C solution includes: phosphate buffer (PB) 20mM, sodium chloride (NaCl) 0.2-3.0M, urea (Urea) 0.5-5M, aprotinin (Aprotinin) 0.2-20mg / L, Tween 20 (Tween20)0.043%;

[0110] b. The mobile phase of the second step of chromatographic purification treatment is liquid D, liquid E, liquid F:

[0111] The D solution includes: phosphate buffer (PB) 20mM, sodium chloride (NaCl) 0.1-20M, aprotinin (Aprotinin) 0.2-20mg / L, Tween 20 (Tween20) 0.043%;

...

Embodiment 3

[0122] This embodiment is a preferred scheme based on Embodiment 2. Among the flow items, the liquid A, liquid B, liquid C, liquid D, liquid E, liquid F, liquid G, liquid H, and liquid I The composition of is further optimized;

[0123] a. The mobile phase of the first step chromatographic purification treatment is liquid A, liquid B, liquid C:

[0124] Said liquid A includes: phosphate buffer saline (PBS) 20mM, aprotinin (aprotinin) 0.5mg / L, Tween 20 (Tween20) 0.043%;

[0125] The B solution includes: phosphate buffer (PB) 20mM, sodium chloride (NaCl) 0.8M, aprotinin (aprotinin) 0.5mg / L, Tween 20 (Tween20) 0.043%;

[0126] The C solution includes: phosphate buffer (PB) 20mM, sodium chloride (NaCl) 0.5M, urea (Urea) 0.8M, aprotinin (aprotinin) 0.5mg / L, Tween 20 (Tween20) 0.043 %;

[0127] b. The mobile phase of the second step of chromatographic purification treatment is liquid D, liquid E, liquid F:

[0128] The D solution includes: phosphate buffer (PB) 20mM, sodium chlo...

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Abstract

The invention provides a purification method of a recombinant human tissue type profibrinolysin activator modified body (rhM-tPA). The method sequentially comprises the following steps of: supernatant fluid filtration processing, first chromatography purification processing, second chromatography purification processing, third chromatography purification processing and fourth ultrafiltration concentration processing. The method obtains a finished rhM-tPA product by carrying out three chromatography purification processings on the recombinant human tissue typ profibrinolysin activator modified body (rhM-tPA) in a bioreactor, wherein the first chromatography purification processing adopts Blue SepharoseTM 6 Fast Flow affinity column ladder shape eluting; the second chromatography purification processing adopts Zn 2+-POROS 20 MC chelating column chromatography; and the third chromatography purification processing adopts Biosepra Lysin Hyperd affinity column chromatography. The three chromatography purification processings all adopt the affinity chromatography, and a needed finished rhM-tPA product with high purity and activity can be obtained only by simply processing the supernatant fluid obtained by filtration processing. The method has simple and convenient operation, low cost and greater economic benefits.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a method for purifying recombinant human tissue-type plasminogen activator (rhM-tPA). Chromatographic purification treatment, the second step of chromatographic purification treatment, the third step of chromatographic purification treatment, and the fourth step of ultrafiltration and concentration treatment. technical background [0002] Thrombotic diseases, especially acute myocardial infarction, pulmonary embolism, and ischemic stroke are diseases with high mortality and severe sequelae, which have attracted more and more attention. Thrombolysis is currently the most effective treatment. Human tissue plasminogen activator (rht-PA) is a new generation of thrombolytic drugs developed in the past ten years. It can activate plasminogen into plasmin , to start the thrombolytic process. Natural t-PA is a glycoprotein synthesized and secreted by vascular endothelial ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/34C07K1/22
Inventor 吴君堂王维定
Owner BEIJING SHIMAODONGRUI PHARMA TECH
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