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Renaturation and purification process of recombinant human granulocyte colony-stimulating factor

A colony-stimulating factor and granulocyte technology, applied in recombinant DNA technology, growth factors/inducing factors, organic chemistry, etc., can solve problems such as complex preparation process, existence of isomers and renaturation yield

Active Publication Date: 2011-12-21
NCPC NEW DRUG RES & DEV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aiming at the problems of complex preparation process, isomers and low renaturation yield in the production of recombinant human granulocyte colony-stimulating factor, the present invention designs a compound that can significantly improve the renaturation yield, reduce isomers, and require simple equipment. , low-cost refolding liquid formula and refolding scheme

Method used

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  • Renaturation and purification process of recombinant human granulocyte colony-stimulating factor
  • Renaturation and purification process of recombinant human granulocyte colony-stimulating factor
  • Renaturation and purification process of recombinant human granulocyte colony-stimulating factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Acquisition of rhG-CSF high-purity inclusion bodies

[0019] After fermenting rhG-CSF genetically engineered bacteria (provided by North China Pharmaceutical Jintan Biotechnology Co., Ltd.), take 1 kg of wet bacteria and wash with PBS buffer (60mmol / L PB, 150mmol / L NaCl, 1mmol / LEDTA, pH 7.0) , wash away impurities such as the unutilized medium carried in the thalline, centrifuge at 8000g (Avanti J-25, Beckman company), collect the precipitate, and then put it in the above solution again, under the condition that the solid-liquid ratio is 1:10 , mixed into a uniform suspension, pumped into a high-pressure homogenate pump for high-pressure homogenate crushing (APV30-30CD, APV GAULIN Company), and then obtained 120 g of wet crude inclusion bodies by centrifugation.

[0020] The crude inclusion bodies obtained above were mixed with buffer A (urea 4mol / L, Tris-HCl 20mmol / L, EDTA10mmol / L, TritonX-1000.2%, pH8.0) and buffer B (Tris-HCl 50mmol / L, EDTA10mmol / L, pH8....

Embodiment 2

[0021] Example 2: Lysis of rhG-CSF inclusion bodies

[0022] Take 5 g of high-purity inclusion bodies, cleavage with lysate (8mol / L Urea, 100mmol / L Tris-HCl, 5mmol / L DTT, 2mmol / L EDTA) at a solid-liquid ratio of 1:100, and stir gently at room temperature for 2-3 Hours, centrifuged to obtain 510ml supernatant, and the protein concentration was determined by the Lowry method.

[0023] The lysate was subjected to SephadexG-25 separation to remove excess reducing agent DTT.

Embodiment 3

[0024] Example 3: Refolding of rhG-CSF inclusion bodies

[0025] The lysate of rhG-CSF inclusion bodies obtained in Example 2 with the reducing agent removed was continuously fed and diluted with refolding solutions A, B, C, and D for 5-6 hours, and the final concentration was controlled at 0.12 mg / ml. After the feeding is completed, stir gently at 4-8°C for 10-12 hours. Then at 10-15°C, stir gently for 6-8 hours to carry out renaturation. Four kinds of refolding liquids used in this embodiment are as follows:

[0026] Component

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Abstract

The invention relates to a preparation method for recombinant human granulocyte colony stimulating factor (recombinant human granulocyte colony stimulating factor, rhG-CSF). The refolding solution consists of Guanidine-HCl, Urea, Tris-HCl, Cysteine, Cystine and Glycerol ) and other components. Using the refolding solution to refold rhG-CSF inclusion bodies, the refolding process is simple, and the finished product has high purity and good stability.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a preparation method of recombinant human granulocyte colony stimulating factor (rhG-CSF). Background technique [0002] Human granulocyte colony stimulating factor (hG-CSF) belongs to the family of hematopoietic growth factors, and the main producing cells are monocyte-macrophages, endothelial cells, fibroblasts and certain tumor cells such as 5637, CHU- 2. ST-1, etc. hG-CSF is one of the hematopoietic growth factors that specifically acts on granulocytes to promote their proliferation and differentiation into mature neutrophils. It has been reported that hG-CSF has no species specificity, it can enhance the function of neutrophils, increase the number of mature granulocytes, promote the release of neutrophils and progenitor cells from the bone marrow into the peripheral blood, and increase the number of early stage neutrophils. Progenitor cells, thereby promoting hematopoietic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/00C07K1/06C07K14/475C12N15/09
Inventor 郝爱鱼董爱华黄晓兰梁倩张卫婷宋欣孔德清高文利赵军强暴娜周兴军王惠欣任乐民姜杨
Owner NCPC NEW DRUG RES & DEV
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