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Method for purifying recombinant interleukin 12

A technology of interleukin and cell culture, applied in the field of protein purification, can solve the problems of inability to meet the requirements of modern society, low protein purity and protein recovery rate, and achieve the goals of saving sample loading time, improving purity and reducing production costs Effect

Inactive Publication Date: 2017-01-25
GUANGDONG COOWAY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the purity and recovery rate of the target protein obtained by the current purification process of interleukin-12 is low, which cannot meet the requirements of modern society

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the purification method of recombinant interleukin 12

[0036] 1.1 Sample pretreatment

[0037] The cell culture solution of recombinant human interleukin 12 was centrifuged at 4000 rpm for 12 minutes, and the supernatant was collected. The recombinant human interleukin-12 cells were purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd.

[0038] 1.2 Ultrafiltration concentrated liquid replacement

[0039] Equipment: Millipore ultrafiltration membrane bag / tube with a molecular weight cut-off of 30KD

[0040] Method: Concentrate the culture solution after centrifugation by ultrafiltration. The pore size of the filter membrane is 0.45 μm. The ultrafiltration membrane is a 30KD filter membrane. ) double dilution of the concentrated solution, continue to concentrate by ultrafiltration, and perform multiple dilution again after the volume is reduced, dilute a total of 4 times according to this method, and collect the ultrafiltration concentrated solut...

Embodiment 2

[0072] Embodiment 2, the purification method of recombinant interleukin 12

[0073] 1.1 Sample pretreatment

[0074] The cell culture solution of recombinant human interleukin 12 was centrifuged at 4000rpm for 10min, and the supernatant was collected. The recombinant human interleukin-12 cells were purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd.

[0075] 1.2 Ultrafiltration concentrated liquid replacement

[0076] Equipment: Millipore ultrafiltration membrane bag / tube with a molecular weight cut-off of 30KD

[0077] Method: Concentrate the culture solution after centrifugation by ultrafiltration. The pore size of the filter membrane is 0.45 μm. The ultrafiltration membrane is a 30KD filter membrane. =6.5) Doubling dilution is carried out to concentrated solution, continue to concentrate by ultrafiltration, carry out doubling dilution again after the volume reduces, dilute altogether 4 times by this method, collect ultrafiltration concentrated solution.

[007...

Embodiment 3

[0109] Embodiment 3, the purification method of recombinant interleukin 12

[0110] 1.1 Sample pretreatment

[0111] The cell culture solution of recombinant human interleukin 12 was centrifuged at 4000rpm for 10min, and the supernatant was collected. The recombinant human interleukin-12 cells were purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd.

[0112] 1.2 Ultrafiltration concentrated liquid replacement

[0113] Equipment: Millipore ultrafiltration membrane bag / tube with a molecular weight cut-off of 30KD

[0114] Method: Concentrate the culture solution after centrifugation by ultrafiltration. The pore size of the filter membrane is 0.45 μm. The ultrafiltration membrane is a 30KD filter membrane. =6.5) Doubling dilution is carried out to concentrated solution, continue to concentrate by ultrafiltration, carry out doubling dilution again after the volume reduces, dilute altogether 4 times by this method, collect ultrafiltration concentrated solution.

[011...

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Abstract

The invention relates to the technical field of protein purification, in particular to a method for purifying recombinant interleukin 12. The method comprises the following steps: pre-treating the cell culture medium of recombinant human interleukin 12, and then performing cation exchange column chromatography, ammonium sulfate fractionation, hydrophobic chromatography, anion exchange column chromatography and gel filtration chromatography, so as to obtain the recombinant interleukin 12. According to the method, the degradation of target protein by protease can be effectively reduced through the pretreatment on the cell culture medium. During the cation exchange column chromatography, impurities which are combined with the target protein by hydrophobic force and difficult to remove and noncovalently conjugated degraded fragments of the target protein can be removed through the washing of a buffer solution containing 20 percent absolute ethyl alcohol, so that the purpose of purification is achieved. The method is high in protein recovery rate, simple and rapid to operate and low in cost.

Description

technical field [0001] The invention relates to the technical field of protein purification, in particular to a method for purifying recombinant interleukin-12. Background technique [0002] Interleukin (interleukin, IL) is a kind of cytokine that is produced by and acts on a variety of cells. Interleukin 12 (hereinafter referred to as IL-12), produced by antigen-presenting cells and B cells, is a pro-inflammatory cytokine in the form of a heterodimer, and is secreted extracellularly in this form. IL-12 can induce the production of IFN-γ, which is mainly produced by B cells and macrophages in the immune response in vivo, especially in bacterial or parasitic infections; its molecule is a heterodimer, 40kd (p40 ) and the two subunits of 35kd (p35) are linked by a disulfide bond. [0003] Interleukin originally refers to cytokines that are produced by leukocytes and play a regulatory role among leukocytes. Now interleukin refers to a class of cytokines whose molecular struct...

Claims

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Application Information

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IPC IPC(8): C07K14/54C07K1/36C07K1/34C07K1/30C07K1/18C07K1/20C07K1/16
CPCC07K14/5434
Inventor 陈松彬倪彦艳李小翠
Owner GUANGDONG COOWAY BIOTECH CO LTD
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