Purification method of recombinant human granulocyte colony-stimulating factor

A colony-stimulating factor and purification method technology, which is applied in the downstream protein purification field of biomedicine and bioengineering, can solve the problems of affecting the stability of protein samples and increasing the process operation time, etc., and achieves short purification cycle, shortened process time, and good feasibility Effect

Active Publication Date: 2017-09-22
JIANGSU HENGRUI MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the loading, equilibrating and elution flow rates of this purification method are all 10cm / h
Such a low flow rate will greatly increase the operation time of the process and may also affect the stability of the protein sample

Method used

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  • Purification method of recombinant human granulocyte colony-stimulating factor
  • Purification method of recombinant human granulocyte colony-stimulating factor
  • Purification method of recombinant human granulocyte colony-stimulating factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of rhG-CSF refolding solution

[0049] The bacterial strain used in the process is Escherichia coli DH5α strain transformed with pBV220 / G-CSF plasmid. After fermentation, the bacterial cells are collected, and the cells are crushed with a high-pressure homogenizer to obtain crude inclusion bodies, and then the crude inclusion bodies are washed to obtain preliminary purified inclusion bodies.

[0050] Take 30 g of initially purified inclusion bodies, and use denaturing solution (20mmol / LTris-HCl, 5mmol / L EDTA, 8mol / L Urea, 10mmol / L DTT, pH8.2 ) for denaturation and dissolution, and stirred at room temperature for 15-18 hours to obtain 600 ml of denatured protein solution. Afterwards, the protein solution is subjected to two-stage filtration and clarification at 10 μm and 0.45 μm, and then the buffer is replaced with a 5KD hollow fiber column to remove the reducing agent DTT. During the replacement process, the transmembrane pressure (TMP) is contro...

Embodiment 2

[0052] Example 2 Capto MMC purification of rhG-CSF

[0053] Adjust the pH of the refolded rhG-CSF solution to 4.0 with 0.5 mol / L hydrochloric acid, and let it stand for 10 minutes. Use a filter cartridge with a pore size of 0.22 μm for filtration.

[0054] Equilibrium: equilibrate the column with equilibrium buffer (20mmol / L citric acid / disodium hydrogen phosphate, pH4.0), flow rate 300cm / hour, until the pH is stable at 4.0;

[0055] Sample loading: load the filtered refolding liquid sample at a flow rate of 300cm / hour;

[0056] Re-equilibration: After loading the sample, equilibrate the chromatography column again with an equilibration buffer (20mmol / L citric acid / disodium hydrogen phosphate, pH4.0) at a flow rate of 300cm / hour;

[0057] Washing impurities: 10mmol / L citric acid / disodium hydrogen phosphate buffer (pH7.5) to elute impurities, flow rate 180cm / hour;

[0058] Elution: The target protein was eluted with 50 mmol / L citric acid / disodium hydrogen phosphate buffer (p...

Embodiment 3

[0059] Example 3 Screening of chromatography filler

[0060] The process of preparing rhG-CSF refolding liquid is as in Example 1. In order to obtain a chromatography medium suitable for the purification of rhG-CSF refolding solution, this study compared the ability of three fillers with weak cationic ligands to purify the refolding solution. The purification process was referred to in Example 2, and the results are shown in Table 1. HPLC purity of samples obtained with CM FF was lower than mixed mode Capto MMC and HCX, and the flow rate is slow and the yield is low. In comparison, Capto MMC and HCX can better meet the pharmacopoeia standard of rhG-CSF, and is suitable for large-scale industrial scale-up.

[0061] Table 1: Comparison of the purification effects of three fillers on rhG-CSF refolding solution

[0062]

[0063]

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Abstract

The invention provides a purification method of a recombinant human granulocyte colony-stimulating factor. The method specifically comprises the steps of selecting a cation chromatographic column to purify renaturation solution of the recombinant human granulocyte colony-stimulating factor (rh-G-CSF), and then performing proper buffer solution replacement to prepare high purity rh-G-CSF stock solution, wherein fillers in the cation chromatographic column is in a cationic type with a mixed mode. According to the purification technology provided by the invention, a technical path is simplified, production time is saved, protein recovery rate is high, the acquired protein stock solution is high in purity, a medicine preparation can be produced by adding proper pharmaceutical adjuvant, and the purification technology is very suitable for large-scale industrial amplification production. Meanwhile, the invention also provides a method for preparing pegylated recombinant human granulocyte colony-stimulating factor.

Description

technical field [0001] The present invention relates to the field of downstream protein purification of biomedicine and bioengineering, and specifically relates to a purification method of recombinant human granulocyte colony stimulating factor (rhG-CSF). The present invention is simple in operation and short in time. It is suitable for industrial scale-up production. Background technique [0002] Human granulocyte colony stimulating factor (hG-CSF) can specifically act on granulocyte progenitor cells, promote their proliferation and differentiation into mature neutrophils, and is the main factor regulating granulocyte hematopoietic cells in the bone marrow. one of the cytokines. It has obvious curative effect on neutropenia caused by cancer chemotherapy, acute leukemia chemotherapy, myelodysplastic syndrome and aplastic anemia and neutrophil increase during bone marrow transplantation. [0003] The source of hG-CSF is very limited, therefore, large-scale industrial produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/535C07K1/18C07K1/107
CPCC07K14/535
Inventor 陈磊王宏伟马宣宣翟紫凝朱壮志张晨光孙金星
Owner JIANGSU HENGRUI MEDICINE CO LTD
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