Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Purification method of polyethylene glycol recombinant human granulocyte colony-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 95% or 90%. and other problems, to achieve the effect of short purification cycle, easy operation and easy process

Active Publication Date: 2017-09-05
JIANGSU HENGRUI MEDICINE CO LTD
View PDF7 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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%

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Purification method of polyethylene glycol recombinant human granulocyte colony-stimulating factor
  • Purification method of polyethylene glycol recombinant human granulocyte colony-stimulating factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0044] The PEG-G-CSF modified product was clarified and filtered through a 0.45 μm filter element (CVHL71TP3) to obtain a clarified reaction solution;

[0045] 20mmol / L acetic acid-sodium acetate, pH4.0 buffer balance hollow fiber column UFP-10-E-55 and hollow fiber system FlexStand;

[0046] 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;

[0047] 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;

[0048] Samples were collected from the bottom val...

Embodiment 2

[0060] Step 1, PEG-G-CSF modified product buffer replacement

[0061] Same as Example 1: Step 1

[0062] Step 2, SP Sepharose HP column chromatography purification of PEG-G-CSF crude product

[0063] Equilibrate the SP Sepharose HP chromatography column with 20mmol / L acetic acid-sodium acetate, pH4.0 buffer solution, flow rate 60cm / h, equilibrate 5 times the column volume, until the UV and conductivity are stable;

[0064] Load the PEG-G-CSF crude product at a flow rate of 40cm / h;

[0065] After loading the sample, wash the chromatography column with 20mmol / L disodium hydrogen phosphate-citric acid, pH4.5 buffer solution for 5 times the column volume, and the flow rate is 70cm / h;

[0066] Use 20mmol / L disodium hydrogen phosphate-citric acid, pH4.5 solution as solution A, take 20mmol / L disodium hydrogen phosphate-citric acid, pH8.0 solution as solution B, use from 0% to 100% B solution The sample was eluted with a linear gradient at a flow rate of 70 cm / h, the gradient volum...

Embodiment 3

[0070] Step 1, PEG-G-CSF modified product buffer replacement

[0071] Same as Example 1: Step 1

[0072] Step 2, SP Sepharose HP column chromatography purification of PEG-G-CSF crude product

[0073] Operate according to the purification conditions in CN101172161B embodiment three, 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;

[0074] Load the crude PEG-G-CSF at a flow rate of 120cm / h;

[0075] 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;

[0076] Use 20mmol / L acetic acid-sodium acetate, pH4.0 solution as solution A, and 20mmol / L acetic acid-sodium acetate...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
purityaaaaaaaaaa
Login to View More

Abstract

The invention relates to a purification method of a polyethylene glycol recombinant human granulocyte colony-stimulating factor. Particularly, the method for performing cation exchange chromatographic separation and purification of the PEG-G-CSF and preparing PEG-G-CSF stock solution by proper buffer solution substitution is optimized, and the PEG-G-CSF with HPLC (high performance liquid chromatography) and electrophoresis purity of 98% or more can be obtained by one-step column chromatographic purification and buffer solution substitution. By the purification process, process routes are simplified, production time is saved, protein recovery rate is high, the obtained protein stock solution is high in purity, proper medical auxiliary materials are added, so that a medicinal preparation can be prepared, and the purification method is quite suitable for large-scale industrial enlarged 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/535C07K1/36C07K1/34C07K1/18
CPCC07K14/535
Inventor 张晨光王宏伟陈磊汪铖孙金星孔祥林汪军
Owner JIANGSU HENGRUI MEDICINE CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com