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Purifying method of recombinant human granulocyte colony stimulating factors

A colony-stimulating factor and granulocyte technology, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., to achieve the effects of reducing side effects, improving protein purity, and improving purity

Inactive Publication Date: 2009-12-09
HARBIN PHARMA GROUP BIOLOGICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention can solve the shortcomings of the existing recombinant human granulocyte colony-stimulating factor purification technology, is an innovation to the separation and purification means of genetic engineering proteins, and greatly improves the protein purity, specific activity and stability of recombinant human granulocyte colony-stimulating factor after separation and purification. sex

Method used

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  • Purifying method of recombinant human granulocyte colony stimulating factors
  • Purifying method of recombinant human granulocyte colony stimulating factors
  • Purifying method of recombinant human granulocyte colony stimulating factors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] A semi-preparative MKF-RP column was used.

[0019] Sample: 200ml of cationic chromatography product, protein concentration 2mg / ml.

[0020] 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).

[0021] First elute with eluent A, then use gradient elution of 15%-90% eluent B, and collect sample peaks in 10-55 minutes.

[0022] The collected samples were evaporated at low temperature to remove organic matter, then concentrated by dialysis and stored at 4°C.

Embodiment 2

[0024] XK 50 / 30 column was used, packed with SBC MCL GEL type reversed-phase preparative liquid chromatography packing material.

[0025] Sample: 500ml of anion chromatography product, protein concentration 2.2mg / ml.

[0026] 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.

[0027] 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

[0029] Use Butyl-Sepharose 4 Fast Flow, XK50H16 chromatography column.

[0030] Sample: 500ml of anion chromatography product, protein concentration 1.5mg / ml. Add ammonium sulfate to 0.8M.

[0031] Equilibrium solution: 20mmol / ml Tris-HCl, pH8.0, 0.8M ammonium sulfate

[0032] Pre-eluent: 20mmol / ml Tris-HCl, pH8.0, 0.5M ammonium sulfate

[0033] Eluent: 20mmol / ml Tris-HCl, pH8.0, 0.2M ammonium sulfate

[0034] Collect the eluted part of the eluate and dialyze to remove salt.

[0035] 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:

[0036] 0 months

[0037]

[0038] March

[0039]

[0040] June

[0041]

[0042] December

[0043]

[0044] 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 purifying method of recombinant human granulocyte colony stimulating factors and applies a fine separating step of opposite-phrase filling to a purifying technology of the recombinant human granulocyte colony stimulating factors for the first time. Compared with the prior art, the method adopts the opposite-phrase filling for the first time to finely separate the recombinant human granulocyte colony stimulating factors, the electrophoresis purity is increased to 99 percent from 90 percent, the HPLC purity is increased to 99 percent from 90 percent, and the specific activity is increased to 2.3*10U / mg from 6.1*10U / mg. The purifying technology of the recombinant human granulocyte colony stimulating factors is adopted to greatly increase the purity, the specific activity and the stability of the recombinant human granulocyte colony stimulating factors and can effectively reduce the clinical side effect of the recombinant human granulocyte colony stimulating factors.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to the purification of medicinal recombinant human granulocyte colony-stimulating factor protein, and in particular to the purification method of recombinant human granulocyte colony-stimulating factor by reverse phase packing chromatography. Background technique [0002] Granulocyte colony-stimulating factor (granulocyte colony-stimulating factor, referred to as recombinant human granulocyte colony-stimulating factor) can promote the proliferation and differentiation of granulocyte hematopoietic stem cells and enhance the function of mature cells. Severe neutropenia caused by neutropenia and neutropenia associated with aplastic anemia have obvious curative effects, but the source of its natural products is very limited, and the recombinant human granulocyte colony-stimulating factor of genetic engineering The biological activity is similar to the natural one, and it can be produce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/18C07K1/20
Inventor 李郑武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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