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Polyethylene glycol modified protein separating and purifying method

A polyethylene glycol, separation and purification technology, applied in the preparation methods of peptides, chemical instruments and methods, organic chemistry, etc., can solve the problems of troublesome resin cleaning and regeneration, pollution, affecting the service life of resins, etc.

Active Publication Date: 2011-11-09
HANGZHOU JIUYUAN GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The impact of the above-mentioned PEGylation process on the protein (larger molecular weight and low charge distribution) has brought difficulties to the subsequent separation and purification process. The purification efficiency and capacity of the resin also bring troubles to the cleaning and regeneration of the resin. If the cleaning is not sufficient, it may cause pollution and even affect the service life of the resin

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  • Polyethylene glycol modified protein separating and purifying method
  • Polyethylene glycol modified protein separating and purifying method
  • Polyethylene glycol modified protein separating and purifying method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Separation and purification of PEGylated recombinant human granulocyte-stimulating factor

[0043] 1.1. Pegylation modification of recombinant human granulocyte stimulating factor

[0044] The modification process refers to Chinese patent 95191454.5. Take a tube containing 140mg of mPEG-ALD (7.09μmol) with a molecular weight of 20KDa, add 20mg of rhG-CSF solution (1.06 μmol). After the mPEG-ALD was dissolved, 0.082ml of 1M sodium cyanoborocyanide was added, and the reaction was stirred at 4°C for 16h. After the reaction was completed, the reaction solution was first diluted to 50 ml with 1 mM HCl, the protein concentration was 0.5 mg / ml, and then the pH of the reaction solution was adjusted to 4.0 with 1 M HCl.

[0045] 1.2. MacroCap SP ion exchange column chromatography

[0046]Purification was carried out by MacroCap SP cation exchange column chromatography. The isoelectric point of polyethylene glycol recombinant human granulocyte-stimulating factor is...

Embodiment 2

[0048] Example 2 Separation and purification of PEGylated recombinant human granulocyte-stimulating factor

[0049] The elution condition is to first elute multiple PEG-modified rhG-CSF with 15% 0.5mol / L NaCl solution, and then elute the single-modified target peak with 30% 0.5mol / L NaCl solution. Other conditions are basically the same as those in practice. Similar to Example 1, the electrophoresis results indicated that the purified target peaks obtained more than 90%.

[0050] Further separation and purification of the purified sample of embodiment 3 embodiment 1

[0051] The specific experimental process is similar to the initial purification process. The elution condition is to first elute the impurity protein with 10% 0.5mol / L NaCl solution, and then elute the single-modified target peak with 35% 0.5mol / L NaCl solution. ( image 3 ).

Embodiment 4

[0052] Example 4 MacroCap SP Ion Exchange Chromatography Separation and Purification of PEG-exendin-4

[0053] 4.1. Preparation of PEG-Exendin-4 conjugates

[0054] Add 1M sodium acetate (pH5.0~pH6.0) buffer solution and water for injection to the Exendin-4 solution stored in the refrigerator at 2~8℃ to make the concentration of Exendin-4 2.5~5mg / ml, and the final concentration of sodium acetate is 0.1 M, add 2.5-5 times molar ratio of mPEG-ALD, after the mPEG-ALD dissolves, add a final concentration of 10-20mM NaCNBH3, and react for 16-24h. The modification rate of the modified sample should not be less than 70% by SEC-HPLC analysis.

[0055] 4.2. Separation and purification of PEG-exendin-4 by MacroCap SP ion exchange chromatography

[0056] MacroCap SP ion-exchange chromatography medium was selected, and the chromatography column was equilibrated with equilibration buffer (20mmol / L NaAc, pH4.0). Dilute 100ml of the modified reaction sample 3 times with purified water, an...

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Abstract

The invention relates to a polyethylene glycol modified protein separating and purifying method. The invention discloses a novel cation chromatography media MacroCap Sp which can induct protein surface charge distribution relation sensitively. The more positive charge on a protein surface, the stronger combination capability of the protein with the MacroCap Sp, and the higher ion gradient is needed for elution. By utilizing the above property, polyethylene glycol modified protein molecule can be effectively separated. The method comprises the following steps: (1) after modification of object protein by polyethylene glycol with active reaction group, adjusting pH value of a reaction solution, wherein the pH value should be lower than isoelectric point of the polyethylene glycol modified protein; (2) loading the reaction solution to balanced MacroCap Sp cation exchange columns; (3) adding a sodium salt solution to carry out ion gradient elution based on a balanced buffer solution, and collecting object peak. The method of the present invention is suitable for effective separation of recombinant human granulocyte stimulating factor modified by the polyethylene glycol and exenatide.

Description

technical field [0001] The invention relates to a method for separating and purifying proteins, more specifically, to a method for separating and purifying proteins modified with polyethylene glycol. Background of the invention [0002] Most protein drugs can only be administered by subcutaneous injection or intravenous injection. Due to the action of protease and glomerular filtration, the half-life of such protein drugs in the human body is relatively short, and the clearance rate in the body is high, often only a few Minutes to several hours of half-life, resulting in frequent administration, causing many difficulties and inconvenience to patients. In addition, its stability, etc. are also factors that affect its efficacy in vivo. Therefore, how to eliminate or reduce the antigenicity of such protein drugs, prolong their in vivo half-life and increase their stability is a hot topic in the field of biopharmaceuticals. [0003] Polyethylene glycol (PEG) chemical modificat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/18C07K14/535C07K14/47
Inventor 金荣汪军远周胜军王同映孙汉栋
Owner HANGZHOU JIUYUAN GENE ENG
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