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Purification method for recombinant human interferon beta-1a

A technology of recombinant human interferon and purification method, which is applied in the field of new purification technology of recombinant human interferon beta-1a to achieve the effect of high biological activity

Active Publication Date: 2011-10-19
ZHEJIANG HISUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] An invention patent (production method of recombinant human IFNβ, application number 200510025595.7) discloses a chromatographic purification method of recombinant human IFNβ, which can be cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, affinity chromatography, Hydrophobic chromatography and its combination, but the patent fails to illustrate how it is combined and how effective it is

Method used

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  • Purification method for recombinant human interferon beta-1a
  • Purification method for recombinant human interferon beta-1a
  • Purification method for recombinant human interferon beta-1a

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Expression of recombinant human IFNβ in mammalian cells

[0042] 1) Construction of cell lines

[0043] We optimized the human IFNβ gene sequence in Genbank by adopting mammalian biased codons, and artificially synthesized the IFNβ gene sequence (Sequence No.2) while keeping the encoded amino acid sequence (Sequence No.1) unchanged. ). Insert this artificially synthesized gene fragment into a high-efficiency eukaryotic expression plasmid vector pCG-IFNβ constructed by us, transfect CHO-K1 cells, and use methionyl sulfate to screen and establish a stably transfected cell line CHO-K1- 9A3, the expression of the protein was detected by immunoblotting and ELISA, and a cell line capable of secreting and expressing the protein at a high level was obtained. Then through the acclimatization of serum-free culture, a suspension growth cell strain suitable for serum-free culture was obtained.

[0044] 2) Identification of recombinant human IFNβ-1a cell line

[0045] ...

Embodiment 2

[0050] Purification route A of embodiment 2 recombinant human IFNβ-1a protein

[0051] In this embodiment, the dye-affinity filler based on hydroxylated polymethacrylic resin is preferred to replace the affinity filler based on dextran. The former has the advantages of high hardness of the filler, low back pressure, and can withstand a larger process flow rate. Secondly, choose two ion-exchange chromatography methods, first use strong anion exchange chromatography, and then use weak cation exchange chromatography to replace reversed-phase chromatography and other chromatography methods that are complex in process and easily lead to protein denaturation. The schematic diagram of its method is as figure 1 shown.

[0052] Materials and Instruments

[0053] AF-Blue HC 650M filler (TOSOH company product), CM-sepharose FF filler (GE healthcare company product), Q-sepharose FF filler (GE healthcare company product). Pellicon 2 ultrafiltration membrane (molecular weight cut off 10...

Embodiment 3

[0067] Example 3 Purification Route B of Recombinant Human IFNβ-1a Purification

[0068] This embodiment includes two ion-exchange chromatography methods, preferably using weak cation-exchange chromatography first, followed by strong anion-exchange chromatography. The schematic diagram of its method is as figure 1 shown.

[0069] Materials and Instruments

[0070] Capto-Blue (product of GE healthcare company), CM-sepharose FF filler (product of GE healthcare company), Q-sepharose FF filler (product of GE healthcare company). Pellicon 2 ultrafiltration membrane (molecular weight cut off 10K), 0.5M 2 (Millipore company product), Pellicon ultrafiltration system (Millipore company product), Amicon ultrafiltration centrifuge tube (Millipore company product), AKTA Purifier chromatography system (GE healthcare company product).

[0071] experimental method

[0072] 1) Concentration of the supernatant

[0073] Clean the ultrafiltration system, concentrate the supernatant, and r...

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Abstract

The invention discloses a purification method for recombinant human interferon beta-1a. The method comprises three steps: affinity chromatography, strong anion exchange chromatography and weak cation exchange chromatography. Blue dyeing affinity chromatography and interferon beta antibody affinity chromatography are adopted during affinity chromatography, Q-sepharose chromatographic column is adopted during strong anion exchange chromatography, and CM-sepharose chromatographic column is adopted during weak cation exchange chromatography. With the method, reverse phase chromatography and metal-chelating chromatography steps influencing protein activity are abandoned, two-time ionic exchange chromatography is adopted, thus obtaining the recombinant human interferon beta-1a with high purity and good bioactivity.

Description

technical field [0001] The invention relates to a protein purification method. Specifically, the invention discloses a new purification process of recombinant human interferon beta-1a. The present invention abandons these steps of reversed phase chromatography and metal chelation chromatography which greatly affect protein activity, designs a new purification process, adopts a purification process comprising one affinity chromatography and two ion exchange chromatography, and obtains Recombinant human interferon β-1a with high purity and good biological activity was obtained. Background technique [0002] Interferon (IFN) is a class of important cytokines with anti-virus, anti-tumor and immunomodulatory effects. According to comprehensive factors such as interferon-producing cells, receptors and activity, it can be divided into two types: type I and Type II, interferon beta (IFNβ) is a type I interferon, and IFNβ is mainly produced by fibroblasts. The human IFNβ gene is 7...

Claims

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Application Information

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IPC IPC(8): C07K14/565C07K1/22C07K1/18
Inventor 应跃斌王海彬运雪莲潘晨晓杨承刚白骅
Owner ZHEJIANG HISUN PHARMA CO LTD
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