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Method for purifying interferon protein

A technology for separation and purification of interferon, applied in the field of biopharmaceuticals, can solve problems such as high cost, affecting product purity, difficulty in producing affinity chromatography ligands, etc., and achieve the effect of reducing side effects and improving purity

Inactive Publication Date: 2010-03-03
HARBIN PHARMA GROUP BIOLOGICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production of affinity chromatography ligands is difficult and expensive, and ligand shedding affects product purity

Method used

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  • Method for purifying interferon protein
  • Method for purifying interferon protein
  • Method for purifying interferon protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] A semi-preparative MKF-RP column was used.

[0018] Sample: 200ml of cationic chromatography product, protein concentration 2mg / ml.

[0019] Prepare eluent A as buffer containing 50mmol NaCl and 20mmol Tris-HCl (pH 8), and eluate B as containing 50mmol NaCl solution 20mmol Tris-HCl 60% acetonitrile (pH 8).

[0020] First elute with eluent A, then use gradient elution of 15%-90% eluent B, and collect sample peaks in 10-55 minutes.

[0021] The collected samples were evaporated at low temperature to remove organic matter, then concentrated by dialysis and stored at 4°C.

Embodiment 2

[0023] XK 50 / 30 column was used, packed with SBC MCL GEL type reversed-phase preparative liquid chromatography packing material.

[0024] Sample: 500ml of anion chromatography product, protein concentration 2.2mg / ml.

[0025] The eluent A is 20% phosphate buffer, the eluent B is 30% methanol solution containing 0.02% tween80, and the flow rate is 15ml / min.

[0026] Gradient elution, eluting with 20%-75% eluent B in 10-80 minutes, and collecting sample peaks. When collecting samples, collect the middle section. The collected samples were evaporated at low temperature to remove organic matter, then concentrated by dialysis and stored at 4°C.

Embodiment 3

[0028] Use Butyl-Sepharose 4 Fast Flow, XK50H16 chromatography column.

[0029] Sample: 500ml of anion chromatography product, protein concentration 1.5mg / ml. Add ammonium sulfate to 0.8M.

[0030] Equilibrium solution: 20mmol / ml Tris-HCl, pH8.0, 0.8M ammonium sulfate

[0031] Pre-eluent: 20mmol / ml Tris-HCl, pH8.0, 0.5M ammonium sulfate

[0032] Eluent: 20mmol / ml Tris-HCl, pH8.0, 0.2M ammonium sulfate

[0033] Collect the eluted part of the eluate and dialyze to remove salt.

[0034] Under the condition of 4 DEG C, the samples of Examples 1, 2, and 3 were stably investigated, and the investigation results were as follows:

[0035] 0 months

[0036]

[0037] March

[0038]

[0039] June

[0040]

[0041] December

[0042]

[0043] The above data shows that compared with Example 3, the samples of Example 1 and 2 obtained by the reverse packing method have electrophoretic purity and HPLC purity greater than 97% within 12 months, and the specific activity reach...

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Abstract

The invention relates to a method for purifying interferon protein. The method adopts a reverse phase filler refining and separating purification step in a purification process. Compared with the prior art, the method purifies an interferon by using a reverse phase filler refining and separating method for the first time and improves the electrophoresis purity of the interferon to 98 percent from90 percent, HPLC purity to 98 percent from 90 percent and specific activity of the interferon to 3.5*10<8>U / mg to 0.9*10<7>U / mg. The purification process of the recombinant interferon can greatly improve the purity, specific activity and stability of human interferons and effectively reduce the side effects of the interferons in clinic.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to the purification of pharmaceutical interferon protein, and in particular to the application of the reverse phase packing chromatography method in the purification of interferon. Background technique [0002] Interferons are low molecular weight glycoproteins with similar structures and functions, and have common polypeptide components. Their molecular weight is about 15-40KD, and their size depends on their glycosylation degree. In 1957, Zssacs and Lindenmann discovered for the first time that virus-infected cells produce a substance that protects other cells against infection by various viral toxins, and named it Interferon (IFN). Interferon has very high biological activity, 1mg has 1 billion active units. Its antiviral effect is non-specific, and the interferon produced by cells induced by an inducer can inhibit the replication of various viruses, and it is a broad-spectrum ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/20C07K1/18
Inventor 李郑武高晶庞睿于淼陈静赵华南李会成陈玉军冷国庆梁秋波
Owner HARBIN PHARMA GROUP BIOLOGICAL ENG
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