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Purification method of pegylated recombinant human granulocyte colony stimulating factor

A technology of colony stimulating factor and pegylation, applied in the field of medicine, can solve the problems of difficult control of endotoxin content, difficult amplification of molecular sieves, difficult control of endotoxin, etc., and achieves less loss of target protein and improved protein yield and quality. , easy-to-control effects

Active Publication Date: 2013-01-02
QILU PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although molecular sieve chromatography can remove and separate various cross-linked isomers, because molecular sieves are difficult to scale up in industry, most domestic purification methods use ion-exchange chromatography for separation and purification based on the difference in surface charge. For example, CN1663962A discloses a one-step purification method of PEG-G-CSF, adopts cation exchange chromatography column SP Sepharose F.F., equilibrates with acetic acid-sodium acetate buffer after packing, and then elutes with acetic acid-sodium acetate buffer of different pH values
Although this purification process can be scaled up industrially, it is difficult to control the endotoxin in the endotoxin because it adopts a cation-exchange medium with a single function for adsorption-desorption separation, which makes it difficult to control the endotoxin content in the final product, affecting the quality of the product

Method used

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  • Purification method of pegylated recombinant human granulocyte colony stimulating factor
  • Purification method of pegylated recombinant human granulocyte colony stimulating factor
  • Purification method of pegylated recombinant human granulocyte colony stimulating factor

Examples

Experimental program
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Effect test

Embodiment 1

[0031] 1. Equilibrate the Sephadex G-25 desalting chromatography column with 100 mM phosphate buffer solution at pH 8.5, load the PEG-G-CSF cross-linking solution, and collect the desalting solution.

[0032] 2. First connect the anion exchange chromatography column and the multi-binding ion exchange chromatography column in series. The chromatography medium of the anion exchange chromatography column is agarose gel (sepharose Q Fast Flow), and the multi-binding ion exchange chromatography column The chromatographic medium used was high flow rate agarose gel (Captro adhere).

[0033] 3. Equilibrate the tandem chromatography column with pH 8.5 100mM phosphate buffer, load the collected desalted solution, and then wash with pH 8.5 100mM phosphate buffer to the baseline. Then separate the anion-exchange chromatography column from the multi-binding ion-exchange chromatography column, equilibrate the Captro adhere ion-exchange chromatography column with 100mM Tris-HCl buffer soluti...

Embodiment 2

[0037] 1. Equilibrate the Sephadex G-25 desalting chromatography column with 10 mM phosphate buffer solution at pH 6.0, load the PEG-G-CSF cross-linking solution, and collect the desalting solution.

[0038] 2. First connect the anion exchange chromatography column and the multi-binding ion exchange chromatography column in series. The chromatography medium of the anion exchange chromatography column is agarose gel (sepharose Q Fast Flow), and the multi-binding ion exchange chromatography column The chromatography medium used was high-flow agarose gel (Captro MMC).

[0039] 3. Equilibrate the tandem chromatography column with 10mM phosphate buffer solution of pH 6.0, load the collected desalted solution, and then wash to the baseline with 10mM phosphate buffer solution of pH 6.0. Then separate the anion exchange chromatography column from the multi-binding ion exchange chromatography column, equilibrate the Captro MMC ion exchange chromatography column with 100mM sodium acetat...

Embodiment 3

[0043] 1. Equilibrate the Sephadex G-25 desalting chromatography column with 20 mM phosphate buffer solution at pH 8.0, load the PEG-G-CSF cross-linking solution, and collect the desalting solution.

[0044] 2. First connect the anion exchange chromatography column and the multi-binding ion exchange chromatography column in series. The chromatography medium of the anion exchange chromatography column is agarose gel (sepharose DEAE Fast Flow), and the multi-binding ion exchange chromatography column The chromatographic medium used was high flow rate agarose gel (Captro adhere).

[0045] 3. Equilibrate the tandem chromatographic column with 20mM phosphate buffer of pH 8.0, load the collected desalted solution, and then wash to the baseline with 20mM phosphate buffer of pH 8.0. Then separate the anion-exchange chromatography column from the multi-binding ion-exchange chromatography column, equilibrate the Captro adhere ion-exchange chromatography column with 20mM Tris-HCl buffer ...

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Abstract

The present invention provides a purification method of pegylated recombinant human granulocyte colony stimulating factor. The inventor performed extensive researches of a variety of chromatographic methods, and found that high-purity pegylated recombinant human granulocyte colony stimulating factors can be obtained by using desalting chromatography, ion exchange tandem chromatography and desalting chromatography to perform sequential combined purification. The purification process of the present invention makes full use of the characteristics of the target protein in the surface charge and hydrophobicity, firstly uses an anion exchange chromatography column to effectively remove endotoxin and to increase controllability of the process; and makes full use of strong hydrophobicity of the target protein in the second-stage chromatography, realizes tandem chromatography, and combines removal of endotoxin and protein purification into a step. The process can not only effectively remove endotoxin, but also simplify operation steps, save production time, and improve production efficiency. The process is very suitable for large-scale industrial production.

Description

technical field [0001] The invention relates to a method for producing genetic engineering medicine by using recombinant DNA technology, in particular to a purification method of PEGylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF or PEG-G-CSF), belonging to the technical field of medicine. Background technique [0002] Granulocyte colony-stimulating factor (G-CSF) is one of the hematopoietic factors, which is composed of 175 amino acids and is a water-soluble protein with hydrophobicity. Its main function is to stimulate the proliferation and differentiation of hematopoietic cells of the neutrophil lineage, increase the number of neutrophils in peripheral blood, activate the function of neutrophils, and play an important role in the prevention and treatment of neutropenia and other diseases caused by multiple factors. Complications (such as fever, infection, etc.) have a significant effect. In 1991, Amgen's recombinant human granulocyte colony-st...

Claims

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Application Information

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IPC IPC(8): C07K14/535C07K1/18C07K1/16
Inventor 王晶翼孙丽霞王克波艾现伟董婷王希菊张乐王乐
Owner QILU PHARMA
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