Recombinant human granulocyte colony stimulating factor and one-step purifying process for chemical modifications thereof

A technology of colony-stimulating factor and granulocytes, applied in the direction of cytokines/lymphokines/interferons, chemical instruments and methods, animal/human proteins, etc. Activity effects and other issues

Inactive Publication Date: 2005-09-07
CHONGQING FAGEN BIOMEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the affinity chromatography method used for the purification of recombinant proteins in the early stage is no longer suitable for the needs of large-scale production; some recombi

Method used

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  • Recombinant human granulocyte colony stimulating factor and one-step purifying process for chemical modifications thereof
  • Recombinant human granulocyte colony stimulating factor and one-step purifying process for chemical modifications thereof
  • Recombinant human granulocyte colony stimulating factor and one-step purifying process for chemical modifications thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0015] Example 1 rhG-CSF construction, expression and renaturation

[0016] The amino acid sequence of human G-CSF is as follows:

[0017] NH 2 -Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu LysCys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu CysAla Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly IlePro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu SerGln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly leu Leu Gln Ala Leu Glu Gly Ile SerPro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Vla Ala Asp Phe Ala Thr ThrIle Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly AlaMet Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser HisLeu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro-COOH

[0018] Whole-gene synthesis of human G-CSF cDNA gene fragments, the use of recursive PCR method to obtain the 3' end containing NdeI and 5' end ...

example 4

[0043] The one-step purification of example four alkylation method PEG-G-CSF

[0044] For sample treatment, take the modified sample, dilute it to twice the volume with distilled water, adjust the pH to 4.0 with acetic acid, and centrifuge to remove the precipitate. Take the cation chromatography medium SP Sepharose F.F., take the equilibration buffer I (10mmol / L acetic acid-sodium acetate, pH4.0) after loading the column to equilibrate to the baseline, then load the sample, and then use the equilibration buffer II (10mmol / L acetic acid- sodium acetate, pH 5.0) equilibrated to baseline. Take elution buffer I (10mmol / L acetic acid-sodium acetate, pH5.6) to elute the PEG-G-CSF target protein peak. Take elution buffer II (10mmol / L acetic acid-sodium acetate, pH7.0) to elute the unmodified rhG-CSF protein peak.

example 5

[0045] One-step purification of example five acylation method PEG-G-CSF

[0046] For sample treatment, take the modified sample, dilute it to twice the volume with distilled water, adjust the pH to 4.0 with acetic acid, and centrifuge to remove the precipitate. Take the cation chromatography medium SP Sepharose F.F., take the equilibration buffer I (10mmol / L acetic acid-sodium acetate, pH4.0) after loading the column to equilibrate to the baseline, then load the sample, and then use the equilibration buffer II (10mmol / L acetic acid- sodium acetate, pH 5.0) equilibrated to baseline. Take elution buffer I (10mmol / L acetic acid-sodium acetate, pH6.2) to elute the PEG-G-CSF target protein peak. Take elution buffer II (10mmol / L acetic acid-sodium acetate, pH7.0) to elute the unmodified rhG-CSF protein peak.

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Abstract

The invention relates to a recombinant human granulocyte colony stimulating factor and one-step purifying process for chemical modifications, which employs the method of cation exchange chromatography, wherein one-step chromatography is utilized for obtaining high activity, high purity and high recovery ratio rhG-CSF and polyethylene glycol chemically modified rhG-CSF. The process is especially suitable for industrial production.

Description

1. Technical field [0001] The present invention relates to the field of purification of proteins and their modifications, in particular to the field of simple and efficient preparation of high-purity, high-activity medicinal protein solutions. The present invention relates to a method for purifying high-purity rhG-CSF or its polyethylene glycol (PEG) modification solution in one step with a relatively simple method, which is particularly suitable for the application of the protein and its polyethylene glycol modification in medicine Large-scale production processes in industry. 2. Background technology [0002] With the development of genetic engineering technology, genetic engineering recombinant protein technology has been applied more and more in the pharmaceutical industry. Many proteins that play a significant role in the prevention and treatment of human diseases have been prepared by means of genetic engineering and recombination. Effectively applied to the preventio...

Claims

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Application Information

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IPC IPC(8): C07K14/52C07K17/08C08G65/00
Inventor 范开王建张宪陈海蓉胡伟
Owner CHONGQING FAGEN BIOMEDICAL
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