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Purification technology of recombination human interferon alpha 1b

A recombinant human interferon and process technology, applied in the directions of interferon, cytokine/lymphokine/interferon, organic chemistry, etc., can solve the problems of expensive chromatography medium, short medium service life, protein drug pollution, etc. Avoid contamination problems, high binding capacity, effective concentration effect

Inactive Publication Date: 2006-01-25
BEIJING TRI PRIME GENE PHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this process has the unavoidable defects of monoclonal antibody affinity chromatography, such as expensive chromatographic medium, easy shedding of monoclonal antibody to cause protein drug contamination, and short service life of the medium.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] (1) Ultrasonic cracking: Take 40g of engineering bacteria, suspend the bacteria with 20-40ml of TE solution (pH8.0) per gram according to the mass of the bacteria, set the output power of the ultrasonic wave to above 600W, and the crushing time is 10 minutes above. Centrifuge at 12,000 rpm at 4°C for 20 minutes to collect the inclusion body precipitate, wash once with TE solution or Tris-HCl solution (pH 8.0), and collect the precipitate by centrifuging at 12,000 rpm at 4°C for 20 minutes.

[0017] (2) Denaturation of inclusion bodies: According to the mass of inclusion bodies, add 10 ml of 6N guanidine hydrochloride to each gram of inclusion bodies to fully dissolve, and denature at room temperature for more than 2 hours.

[0018] (3) Refolding of inclusion bodies: the supernatant was collected by centrifugation at 4,000 rpm, the denatured protein solution was diluted 100 times with 0.15M boric acid solution (pH 9.0), and refolded overnight at 4°C.

[0019] (4) Phenyl...

Embodiment 2

[0024] The 10% ethylene glycol solution in the Phenyl Sepharose hydrophobic column chromatography is replaced with 20% ethylene glycol solution, and the pH value of each buffer of the CM Sepharose Fast Flow cation exchange column chromatography is adjusted to 4.6, and the remaining steps are the same as in the examples 1.

[0025] After 40g of bacteria were treated as above, a total of 45mg of pure interferon α1b was obtained. The purity was 98% as detected by SDS-PAGE electrophoresis, and the specific activity was 1.2×10 as determined by conventional WISH-VSV system. 7 IU / mg. The whole process takes 28 hours.

Embodiment 3

[0027] The 10% ethylene glycol solution in the Phenyl Sepharose hydrophobic column chromatography is replaced with 40% ethylene glycol solution, and the pH value of each buffer of the CM Sepharose Fast Flow cation exchange column chromatography is adjusted to 5.0, and the remaining steps are the same as in the examples 1.

[0028] After 40g of bacteria were treated as above, a total of 48mg of pure interferon α1b was obtained. SDS-PAGE electrophoresis detection showed that the purity was 96%, and the specific activity was 1.1×10 as determined by conventional WISH-VSV system. 7 IU / mg. The whole process takes 28 hours.

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PUM

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Abstract

The invention relates to a process for purifying recombinant human interferon alpha1b, which comprises hydrophobic column chromatography, cationic column chromatography and volume exclusion chromatography. The process requires no mono- anti-stripping affinity chromatography, thus can prevent the pollution caused by drops of mono-anti-stripping, and the technological process can be shortened.

Description

technical field [0001] The invention relates to a purification process of recombinant human interferon. Background technique [0002] As a broad-spectrum antiviral drug, interferon is more and more widely used clinically and has great market value. Since human α1b interferon was successfully expressed through genetic engineering, in order to ensure high purity, the purification process of the protein has always been separated and purified by using a monoclonal antibody affinity chromatography column combined with other chromatography columns. (Patent Application No. 93114639.9), this type of method has the characteristics of high selectivity, and can reach higher purity by a column. However, this process has unavoidable defects such as expensive chromatographic medium, easy shedding of monoclonal antibody to cause protein drug contamination, and short service life of the medium. Contents of the invention [0003] The purpose of the present invention is to avoid the monoc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/56C07K1/18
Inventor 杨大军梁天凤刘金毅刘永全孙俭波程永庆
Owner BEIJING TRI PRIME GENE PHARMA CO LTD
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