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Purifying method of interferon

A purification method and interferon technology, which is applied in the field of interferon purification, can solve the problems of specific activity decline and achieve the effect of improving purity

Active Publication Date: 2013-04-03
HARBIN PHARMA GROUP BIOLOGICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its purification process has been difficult to solve its purity problem after many years of research, and its specific activity has dropped significantly. For example, the prior art "Extraction and Purification of Recombinant Human Interferon_2b" Microbiology Immunology Progress, Volume 32, No. 1, 2004 uses ion Exchange chromatography column DEAE sepharose (FF) A column, column-shaped XK30 / 50, and sepphacryl100 / 50XK chromatography column, S-100 filler, obtained through two chromatography, after determination, its specific activity is 1.2×10 8 IU / mg, the protein purity is 95%

Method used

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  • Purifying method of interferon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Anion column chromatography (XK50 / 30 chromatography column, Q sepharose FF filler)

[0035] Use the balance solution (20mmol / L PH8.2 Tris-HCl buffer), adjust the pump flow rate to 30.0-49.9ml / min. Equilibrate to 8-10 times the column bed volume.

[0036] Sample pretreatment: adjust the pH value of the renatured product to 8.2 with 2mol / L pH8.2 Tris-HCl, and then dilute its volume four times with water for injection, which is the loading solution.

[0037] 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.

[0038] 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 the OD 280 Greater than 0.3...

Embodiment 2

[0045] Cation column chromatography (XK50 / 30 chromatography column, CM Sepharose FF filler)

[0046] Balance: use balance solution (20mmol / L PH4.5 HAc-NaAc buffer), adjust the pump flow rate to 30.0-49.9ml / min, balance 8-10 times the column bed volume.

[0047] Sample pretreatment: adjust the PH value of the renatured product to 4.5 with glacial acetic acid, and then dilute its volume four times with water for injection, which is the sample solution.

[0048] 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 MNaCL buffer), 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 MNaCL buffer) to elute the target peak, and collect the elut...

Embodiment 3

[0055] Anion column chromatography (XK50 / 30 chromatography column, Q sepharose FF filler)

[0056] Use the balance solution (20mmol / L PH8.2 Tris-HCl buffer), adjust the pump flow rate to 30.0-49.9ml / min. Equilibrate to 8-10 times the column bed volume.

[0057] Sample pretreatment: adjust the pH value of the renatured product to 8.2 with 2mol / L pH8.2 Tris-HCl, and then dilute its volume four times with water for injection, which is the loading solution.

[0058] 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.

[0059] 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 the OD 280 Greater than 0.3...

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Abstract

The invention relates to a purifying method of interferon. 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; d. 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 interferon.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to a method for purifying interferon. 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 interferon can be greatly improved. Background technique: [0002] Interferon (IFN) is a broad-spectrum antiviral agent, mainly through the action of cell surface receptors to make cells produce antiviral proteins, thereby inhibiting the replication of hepatitis B virus; at the same time, it can also enhance natural killer cells (NK cells), macrophages Cell and T lymphocyte activity, thereby play an immune regulation role, and enhance antiviral ability. Interferon is a group of active proteins (mainly glycoproteins) with multiple functions, and is a cytokine produced by monocytes and lymphocytes. They have broad-spectrum anti-virus, affect ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C07K1/20C07K1/18
Inventor 段志强杨栋陈玉军庞睿郑立运王晴吴贵海赵钰白宇张崇远杨超
Owner HARBIN PHARMA GROUP BIOLOGICAL ENG
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