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

Separation and purification process of recombinant human erythrogenin

A recombinant human erythropoietin, separation and purification technology, applied in the direction of erythropoietin, animal/human protein, growth factor/inducing factor, etc., can solve the problems of protein denaturation and toxicity, and achieve high price, short service life, The effect of increasing production costs

Inactive Publication Date: 2006-02-22
NANJING NORMAL UNIVERSITY
View PDF1 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Most of the currently reported process methods use reversed-phase chromatography and organic solvents in the process, causing protein denaturation during the chromatography process and considering the elimination of organic solvent residues in the product, resulting in toxic effects on the human body

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Recombinant human erythropoietin (rHuEPO) engineered cell line, grown in 10% calf serum DMEM medium, then converted to serum-free medium for production culture. 35L of the cell culture harvest supernatant was filtered through a 0.65 micron membrane to remove cell debris in the supernatant. Blue-Sepharose.FF chromatography column (7.5×20cra) 3.0 column volume (CV) equilibrated with 20mM PB buffer solution, at 30ml / min flow rate, flush the column to baseline with 20mM PB buffer solution, and then use Elute with 20mM PB+0.75M NaCL buffer and collect the eluted protein peak.

[0038] 2. Metal chelate affinity chromatography column (5×15cm) uses 0.1M EDTA-Na 2 +1M NaCl equilibrate 4.0C.V, rinse the column 3.0C.V with water for injection, 50% isopropanol+1% TFA equilibrate 3.0.C.V, rinse with water for injection 3.0C.V, 0.1M NaAc.HAC+0.2M CuSO 4 .5H 2 O equilibrate 3.0CV, rinse 3.0CV with water for injection, after equilibrate 3.0CV with 20mM PB+0.5M Nacl, directly load the sa...

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

No PUM Login to View More

Abstract

The separation and purification process of recombinant human erythrogenin includes the following steps: Blue sepharose F. F. chromatography; metal chelating affinity column chromatography; Sephadex G-25 column chromatography; and Source Q column chromatography, collecting destination protein and 0.22 micron membrane filtering. The process has low cost, and using no reverse chromatography and organic solvent avoids denaturation of protein during chromatography and residue of organic solvent. The combined purification process including Blue sepharose Fast Flow chromatography, metal chelating affinity column chromatography Sephadex G-25 column chromatography and Source Q column chromatography successively can prepare medicine level rHuEPO protein in large scale.

Description

Technical field [0001] The invention relates to a protein production method, in particular to a separation and purification process of recombinant human erythropoietin (rhEPO). Background technique [0002] In 1997, Miyake et al. (Miyake. et al., J. Bid, Chem, 252, P5558 (1997)) isolated and purified human erythropoietin (EPO) from human urine for the first time, thus confirming its presence in the human body . In 1984, it was discovered that EPO is the main control factor for the differentiation, proliferation and maturation of erythroid progenitor cells. In 1985, Jacobs et al. (Jacobs et al., Nature, 313, p806 (1985)) first successfully isolated the cDNA of human EPO gene from human embryonic liver cells. In the same year, Lin et al. (Lin et al., Proc Natl Acad Sci USA, 82, P7580 (1985)) cloned the human EPO gene and obtained high-efficiency expression in CHO cells. In 1986, Law et al. (Law ML, et al., Proc Natl Acad Sci USA, 83, P6920 (1986)) determined the nucleic acid sequen...

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 Patents(China)
IPC IPC(8): C07K14/505C07K1/36
Inventor 胡云龙张双全
Owner NANJING NORMAL UNIVERSITY
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