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Method for purification and virus removal of recombinant human prourokinase

A pro-urokinase, virus technology, applied in biochemical equipment and methods, enzymes, peptidases, etc., can solve the problems of low protein purity, viral exogenous pollution, low safety, and inability to be used in the human body.

Inactive Publication Date: 2017-06-20
TASLY BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, this method still has the problems of low protein purity, exogenous contamination such as host cell DNA, host cell residual protein, and viruses.
The safety of the method of CN1680550A is not high, the main reason is that after completing the D step, it is the product stock solution, and the stock solution will be prepared as the raw material of recombinant human urokinase for injection, but the Tris-HCl buffer solution adopted in the D step will enter the Finished product, directly into the human body through intravenous injection
Tris-HCl buffer is irritating to the human body, and it is generally only used for research and not for human use. Tris-HCl buffer directly entering the human body will cause certain harm to the human body, so it is not suitable as a buffer system for the original solution

Method used

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  • Method for purification and virus removal of recombinant human prourokinase
  • Method for purification and virus removal of recombinant human prourokinase
  • Method for purification and virus removal of recombinant human prourokinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0197] Purification step 1: protein capture

[0198] Exchange chromatography with Streamline SP cation packing as the medium. The chromatography column is balanced with 0.01mol / L phosphate buffer (pH 6.0 contains 0.1mol / L NaCl), and the supernatant is taken from the fermentation broth to adjust the pH to 6.0 and then loaded, using 0.01mol / L phosphate buffer (pH6.0 contains 0.5mol / L NaCl) elution, collect the eluted protein peak according to the UV 280 ultraviolet absorption curve, which is the purified intermediate 1.

[0199] Purification step 2: Gel chromatography

[0200] Gel chromatography was performed with Sephacryl S-200 HR gel molecular sieve filler as the medium. The chromatography column was balanced with 0.01mol / L phosphate buffer (pH6.0 containing 0.09mol / L NaCl), the purified intermediate 1 was directly loaded, and 0.01mol / L phosphate buffer (pH6.0 containing 0.09 mol / L NaCl) and collect the target protein peak according to the UV 280 ultraviolet absorption curve, whi...

Embodiment 2

[0214] Purification step 1: protein capture

[0215] Exchange chromatography with Streamline SP cation packing as the medium. The chromatographic column is balanced with 0.005mol / L phosphate buffer (pH5.5 containing 0.05mol / L NaCl), and the supernatant is taken from the fermentation broth to adjust the pH to 5.5 and then loaded, using 0.005mol / L phosphate buffer (pH5.5 contains 0.3mol / L NaCl elution, collect the eluted protein peak according to the UV 280 ultraviolet absorption curve, which is the purified intermediate 1.

[0216] Purification step 2: Gel chromatography

[0217] Sephacryl S-200HR gel molecular sieve packing was used for gel chromatography. The column is balanced with 0.005mol / L phosphate buffer (pH5.5 containing 0.05mol / L NaCl), the purified intermediate 1 is directly loaded, and 0.005mol / L phosphate buffer (pH5.5 containing 0.05 mol / LNaCl) and collect the target protein peak according to the UV 280 ultraviolet absorption curve, which is the purified intermediate ...

Embodiment 3

[0231] Purification step 1: protein capture

[0232] Exchange chromatography with Streamline SP cation packing as the medium. The chromatographic column is balanced with 0.015mol / L phosphate buffer (pH6.5 contains 0.15mol / L NaCl), and the supernatant is taken from the fermentation broth to adjust the pH to 6.5 and then loaded, using 0.015mol / L phosphate buffer (pH6.5 contains 0.7mol / L NaCl elution, collect the eluted protein peak according to the UV 280 ultraviolet absorption curve, which is the purified intermediate 1.

[0233] Purification step 2: Gel chromatography

[0234] Sephacryl S-200HR gel molecular sieve packing was used for gel chromatography. The chromatography column was balanced with 0.015mol / L phosphate buffer (pH6.5 containing 0.12mol / L NaCl), and the purified intermediate 1 was directly loaded with 0.015mol / L phosphate buffer (pH6.5 containing 0.12mol / L NaCl). mol / L) and collect the target protein peak according to the UV 280 ultraviolet absorption curve, which is...

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Abstract

The invention provides a method for protein purification and virus removal of novel CHO cell-expressed recombinant human prourokinase. The method comprises the steps of protein capture, gel chromatography, cation exchange chromatography, low pH incubation, affinity chromatography, anion exchange chromatography, cation exchange chromatography and nanofiltration. By the adoption of the method, the purity of obtained protein is not lower than 98%, residues of Tris-HCl buffer solution are replaced, and clinical using risks are effectively reduced through virus removal / inactivation.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for purifying prourokinase and removing viruses. Background technique [0002] Prourokinase (pro-UK) is a type of serine protease that can activate the plasminogen in the body to be converted into active plasmin, thereby dissolving the fibrin in the thrombus. Therefore, it has the treatment of thrombolysis. Wide range of applications. The peptide bond between Lys158-Ile159 in pro-UK is easily hydrolyzed by plasmin and other enzymes to generate urokinase (UK). UK is composed of two peptide chains, A chain and B chain, connected by disulfide bonds between chains. Compared with UK, pro-UK has a higher affinity with fibrin, and the site of activation of the fibrinolytic system is often at the site of thrombosis, so the side effect of causing systemic bleeding tendency is smaller than that of urokinase, which is a superior fibrinolysis Enzyme activator. There is often ...

Claims

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Application Information

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IPC IPC(8): C12N9/72
CPCC12N9/6456C12Y304/21031
Inventor 赵侠赵国焓李雪峰莫英韩进
Owner TASLY BIOPHARMACEUTICALS CO LTD
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