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A purification method of pegylated recombinant human granulocyte stimulating factor

A technology of PEGylation and stimulatory factors, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve the problem of low purity of PEG-G-CSF, which can only reach 90% or 95%. and other problems, to achieve the effect of short purification cycle, easy operation and easy process

Active Publication Date: 2020-04-24
JIANGSU HENGRUI MEDICINE CO LTD
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] CN1663962A discloses the process of using SP Sepharose FF chromatographic column to carry out PEG-G-CSF purification. The average particle size of this chromatographic medium is 90 μm, which has the advantages of fast flow rate and easy amplification, but the resolution is low, and the obtained PEG- G-CSF has low purity, and SP Sepharose HP as a high-resolution chromatography medium has an average particle size of 34 μm. We have used SP Sepharose FF chromatography column and SP Sepharose HP chromatography column in the present invention for PEG-G-CSF Compared with the purification effect, the purification effect of SP Sepharose HP chromatography column is significantly better than that of SP Sepharose FF chromatography column
[0006] CN102234310A discloses a method for separating and purifying polyethylene glycol-modified proteins through a MacroCap SP cation chromatography column. The method is applied to the separation and purification of PEG-G-CSF. The column is selected at pH 4.0-5.0, and a Lower concentration of NaCl (5%-15% 0.5-1.5mol / L NaCl solution) elutes the multi-PEG modified G-CSF, and then 20%-50% 0.5-1.5mol / L NaCl solution elutes single For the modified target peak, the purity of the protein in one-step purification can only reach 90%, and even if it is purified by MacroCap SP column chromatography again, the purity can only reach 95%

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  • A purification method of pegylated recombinant human granulocyte stimulating factor
  • A purification method of pegylated recombinant human granulocyte stimulating factor

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Step 1, PEG-G-CSF modified product buffer replacement

[0044] Equilibrate Sephadex G-25 chromatography column with 20mmol / L acetic acid-sodium acetate, pH4.0 buffer solution, flow rate 150cm / h, equilibrate 2 times column volume;

[0045] Load the PEG-G-CSF modified product clarified by the cellulose acetate filter membrane, the single loading volume shall not exceed 1 / 4 of the column volume, and the flow rate shall be 150cm / h;

[0046] Elute with 20 mmol / L acetic acid-sodium acetate, pH 4.0 buffer, collect the sample peaks, and obtain the crude PEG-G-CSF.

[0047] Step 2, SP Sepharose HP column chromatography purification of PEG-G-CSF crude product

[0048] Equilibrate the SP Sepharose HP chromatography column with 20mmol / L acetic acid-sodium acetate, pH4.0 buffer solution, flow rate 80cm / h, equilibrate 5 times the column volume, until the UV and conductivity are stable;

[0049] Load the PEG-G-CSF crude product at a flow rate of 50cm / h;

[0050] After loading the s...

Embodiment 2

[0058] Step 1, PEG-G-CSF modified product buffer replacement

[0059] The PEG-G-CSF modified product was clarified and filtered through a 0.45 μm filter element (CVHL71TP3) to obtain a clarified reaction solution;

[0060] 20mmol / L acetic acid-sodium acetate, pH4.0 buffer balance hollow fiber column UFP-10-E-55 and hollow fiber system FlexStand;

[0061] Pump the clarified reaction solution into the storage tank of the FlexStand hollow fiber system, start the system, adjust the return valve, and control the transmembrane pressure (TMP) at 20-30PSI;

[0062] Use 20mmol / L acetic acid-sodium acetate, pH4.0 buffer as the replacement solution, and replace with a constant volume, so that the rate of the replacement solution added is the same as that of the permeate, and the volume of the sample is kept constant in the hollow fiber system storage tank. The replacement ends when the dosage is about 5 times the volume of the sample;

[0063] Samples were collected from the bottom val...

Embodiment 3

[0075] Step 1, PEG-G-CSF modified product buffer replacement

[0076] Same as embodiment two: step 1

[0077] Step 2, SP Sepharose HP column chromatography purification of PEG-G-CSF crude product

[0078] Operate according to the purification conditions in CN101172161A Example 3, first wash 5 times the volume with 0.5M NaOH solution, then wash with purified water to neutrality, then balance the SP Sepharose HP layer with 20mmol / L acetic acid-sodium acetate, pH4.0 buffer solution Analysis column, flow rate 120cm / h, equilibrate 5 times the column volume, until the UV and conductivity are stable;

[0079] Load the crude PEG-G-CSF at a flow rate of 120cm / h;

[0080] After the sample loading is completed, wash the chromatography column with 20mmol / L acetic acid-sodium acetate, pH4.0 buffer solution for 10 times the column volume, and the flow rate is 120cm / h;

[0081] Use 20mmol / L acetic acid-sodium acetate, pH4.0 solution as solution A, and 20mmol / L acetic acid-sodium acetate, ...

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Abstract

The invention relates to a pegylation recombinant human granulocyte colony stimulating factor purification method. Particularly, the purification method optimizes separation and purification of PEG-G-CSFs (pegylation granulocyte colony stimulating factors) in a cation-exchange chromatography manner, PEG-G-CSF stock solution is prepared by proper buffer solution in a replacement manner, HPLC (high performance liquid chromatography) and the PEG-G-CSFs can be acquired by the aid of one-step column chromatography purification and replacement of the buffer solution, and the electrophoresis purity of the PEG-G-CSFs is 98% or more. According to a purification process, a process route is simplified, production time is saved, protein recovery rate is high, the purity of obtained stock solution is high, medicinal preparations can be prepared by adding proper medical auxiliary materials, and the purification process is quite suitable for large-scale industrial amplifying production.

Description

technical field [0001] The invention relates to the field of purification of high-purity medicinal protein solutions, in particular to the purification of PEGylated recombinant human granulocyte colony stimulating factor (PEG-G-CSF) modified products, and belongs to the field of medical technology. Background technique [0002] In recent years, with the rapid development of biotechnology, more and more protein and polypeptide drugs have been discovered and successfully applied to the treatment of human diseases. Among them, recombinant human granulocyte colony stimulating factor (recombinant human granulocyte colony stimulating factor, rhG-CSF) is a very successful genetic engineering drug. G-CSF is produced by monocytes and fibroblasts, can stimulate the formation of granulocyte colonies, and has a stimulating effect on neutrophils. Therefore, it is often used as adjuvant therapy for cancer patients receiving radiation therapy or chemotherapy and leukemia patients after bon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/535C07K1/36C07K1/34C07K1/18
CPCC07K14/535
Inventor 张晨光王宏伟孙金星汪军王亚里孔祥林封海莹
Owner JIANGSU HENGRUI MEDICINE CO LTD
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