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A purification method of recombinant human granulocyte stimulating factor

A purification method and cationic column technology, applied in the biological field, can solve the problems of small immunogenicity, toxicity, and specific activity decline

Active Publication Date: 2014-10-01
HARBIN PHARMA GROUP BIOLOGICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, after years of research on its purification process, it has been difficult to solve its purity problem, and its specific activity has dropped significantly. For example, the existing technology "High-efficiency expression of granulocyte colony-stimulating factor (G-CSF) in E. coli and the establishment of a rapid purification process" China Journal of Biochemistry and Molecular Biology 1998, Vol. 14 described the following technical scheme: dilute the refolded protein, followed by a step of SP-Sepharose FF column chromatography to homogeneity. The specific activity of the purified G-CSF protein reached 3.4×10 8 U / mg, the total activity of G-CSF recovered per liter of expression bacteria solution reached 1.06×10 11 U / mgU. The N-terminal amino acid sequence analysis of the purified product showed that the removal of methionine was complete, and it may have less immunogenicity and toxicity when used in humans

Method used

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  • A purification method of recombinant human granulocyte stimulating factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Anion column chromatography (XK50 / 30 chromatography column, Q sepharose FF filler)

[0040] Use the balance solution (20mmol / L pH8.2 Tris-HCl buffer solution), adjust the pump flow rate to 30.0-49.9ml / min. Equilibrate to 8-10 times the column bed volume.

[0041] Sample pretreatment: adjust the pH value of the renaturation product to 8.2 with 2mol / L pH8.2 Tris-HCl, then concentrate it by ultrafiltration to 1 / 10 of the renaturation volume, which is the sample solution.

[0042] Sample loading: The pump flow rate is 8.0-49.9ml / min when loading the sample. Equilibrate 2-4 column bed volumes with equilibration solution after sample loading.

[0043] Elution: use pre-wash solution (20mmol / L pH7.8 NaH 2 PO 4 -Na 2 HPO 4Buffer) for pre-washing, adjust the pump flow rate to 30.0-49.9ml / min, and elute 2-4 column bed volumes. Then change the inlet tube to the eluent (20mmol / L pH7.8 NaH2PO4-Na2HPO4 buffer, 0.1MNaCL), start the elution of the target peak, and collect the OD ...

Embodiment 2

[0048] Anion column chromatography (XK50 / 30 chromatography column, Q sepharose FF filler)

[0049] Use the balance solution (20mmol / L pH8.2 Tris-HCl buffer solution), adjust the pump flow rate to 30.0-49.9ml / min. Equilibrate to 8-10 times the column bed volume.

[0050] Sample pretreatment: adjust the pH value of the renaturation product to 8.2 with 2mol / L pH8.2 Tris-HCl, and then concentrate it by ultrafiltration to 1 / 10 of the renaturation volume, which is the sample solution.

[0051] Sample loading: The pump flow rate is 8.0-49.9ml / min when loading the sample. Equilibrate 2-4 column bed volumes with equilibration solution after sample loading.

[0052] Elution: use pre-wash solution (20mmol / L pH7.8 NaH 2 PO 4 -Na 2 HPO 4 Buffer) for pre-washing, adjust the pump flow rate to 30.0-49.9ml / min, and elute 2-4 column bed volumes. Then change the inlet tube to the eluent (20mmol / L pH7.8 NaH2PO4-Na2HPO4 buffer, 0.1MNaCL), start the elution of the target peak, and collect th...

Embodiment 3

[0059] Cation column chromatography (XK50 / 30 chromatography column, CM Sepharose FF filler)

[0060] Balance: use balance solution (20mmol / L pH4.5 HAc-NaAc buffer), adjust the pump flow rate to 30.0-49.9ml / min, and balance 8-10 times the column bed volume.

[0061] Sample pretreatment: adjust the pH value of the refolded product to 4.5 with glacial acetic acid, and then concentrate it by ultrafiltration to 1 / 10 of the refolded volume, which is the sample solution.

[0062] Sample loading: adjust the pump flow rate to 30.0-49.9ml / min, and start sample loading. After loading the sample, switch to the balance solution and balance 2-4 column bed volumes until the recording pen returns to the baseline. Pre-wash with pre-extraction solution (20mmol / L pH4.5HAc-NaAc buffer, 0.15 MNaCLp), and adjust the pump flow rate to 30.0-49.9ml / min until the impurity peak is washed down. Change to the eluent (50mmol / L pH4.5HAc-NaAc buffer, 0.2 MNaCLp) to elute the target peak, and collect the OD...

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Abstract

The invention relates to a purifying method for a recombinant human granulocyte stimulating factor. The method comprises the following steps of a. performing fermentation culture by selecting engineering bacteria as bacterial strains; b. separating and washing inclusion bodies of the bacterial strains obtained from the fermentation culture to obtain refined inclusion bodies; c. performing renaturation on the refined inclusion bodies to obtain a renaturation preoduct; d. performing anionic column chromatography treatment on the renaturation product to obtain an anionic column chromatography product; e. performing cation column chromatography treatment on the anionic column chromatography product to obtain a cation column chromatography product; and f. processing the cation column chromatography product by using a reverse phase packing column chromatography, and performing desalinizing treatment to obtain the recombinant human granulocyte colony-stimulating factor.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to a purification method of recombinant human granulocyte stimulating factor. The present invention adopts the fine separation and purification step of the reverse phase packing in the purification process. Compared with the prior art, the purity, specific activity and stability of the recombinant human granulocyte stimulating factor can be greatly improved. Background technique: [0002] Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a hematopoietic growth factor that specifically acts on granulocyte progenitor cells and promotes their proliferation and differentiation into mature neutrophils. Has the following functions: [0003] 1. Promote the increase of neutrophil count after bone marrow transplantation. [0004] 2. Neutropenia caused by cancer chemotherapy. [0005] 3. Neutropenia associated with myelodysplastic syndrome. [0006] 4. Neutropenia associa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00C07K1/20C07K1/18
Inventor 王伟权陆刚陈玉军庞睿李邦东牛国玲于俊清杨凤铎陶永宝赵海丹宋成君
Owner HARBIN PHARMA GROUP BIOLOGICAL ENG
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