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Method for purification and preparation of mono-substituted PEG-EPO (polyethylene glycol-erythropoietin)

A PEG-EPO, single substitution technology, applied in the purification process and preparation field of single substitution PEG-EPO, can solve the problems of high cost, difficulty in ion exchange to achieve purity, low yield, etc., to reduce production cost and energy consumption, The effect of long effective drug concentration time and improving the purity standard

Active Publication Date: 2013-12-25
SHENZHEN SCIPROGEN BIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the effects are not ideal, the ion exchange is difficult to reach the required purity, and the combination of ion exchange and gel filtration chromatography faces problems such as difficult amplification, high cost, and low yield (Conan J et al., Chemical Engineering Science 61, 924-939.2006)

Method used

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  • Method for purification and preparation of mono-substituted PEG-EPO (polyethylene glycol-erythropoietin)
  • Method for purification and preparation of mono-substituted PEG-EPO (polyethylene glycol-erythropoietin)
  • Method for purification and preparation of mono-substituted PEG-EPO (polyethylene glycol-erythropoietin)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Reaction and detection of EPO and GLUC-PEG

[0061] Use a 15ml plastic centrifuge tube, add 2ml of EPO stock solution (EPO content 2.5mg / ml, from Shenzhen Saibaoer Biological Pharmaceutical Co., Ltd.), add 3ml of 20mM phosphate buffer solution with pH 7.2, and the final concentration of EPO is 1mg / ml. Accurately weigh 109.4mg GLUC-PEG (35,000±3,500D, from Beijing Jiankai, see patent number: 02818455.6, structural formula as formula II) with an analytical balance, add 1094μl 2mM and pH 3.0 phosphate buffer to dissolve. After dissolving, all the GLUC-PEG-NHS solution was added into the EPO, and the pH of the EPO solution was measured to be 7.2 by test paper. The reaction mixture was placed on a shaker and reacted with shaking at room temperature for 2.5 hours.

[0062] After the reaction is completed, the reactant contains unreacted EPO, PEG, and the monosubstituted PEG-EPO (m PEG-EPO), disubstituted PEG-EPO (d PEG-EPO) and multi-substituted PEG-EPO (oliPEG-EPO) generate...

experiment example 1

[0101] Purity detection of purified product mPEG-EPO (SDS-PAGE and SEC-HPLC)

[0102] SDS-PAGE (non-reduced) purity (according to the 2010 Pharmacopoeia method) detection of the purified product obtained in the above examples, 10 μg of conventional EPO and mPEG-EPO were loaded, and Coomassie brilliant blue staining, the results are as follows Figure 5 shown.

[0103] From Figure 5 It can be seen from the results that the mPEG-EPO purified by the above chromatography (hydrophobic + desalting + ion exchange + ultrafiltration) is the only band on the SDS-PAGE gel, and the purity is greater than 98%.

[0104] SEC-HPLC analysis is carried out to purified product, and condition is the same as embodiment 1, and result is as follows Figure 6 shown.

[0105]

[0106] It can be seen from the above results that the mPEG-EPO purified by the above chromatography (hydrophobic + desalting + ion exchange + ultrafiltration) was subjected to SEC-HPLC purity analysis, and the purity rea...

experiment example 2

[0109] In vivo activity detection of the purified product mPEG-EPO

[0110] Measuring principle: The proliferation and development process of the erythrocyte system in the bone marrow: pluripotent stem cells → unipotent stem cells → primitive erythrocytes → promyeloid erythrocytes → mesenchymal erythrocytes → late immature erythrocytes → reticulocytes → mature erythrocytes. From the proliferation of primitive erythrocytes to the stage of late erythrocytes, a total of 3-4 divisions are required, which takes about 72 hours. The number of erythrocytes changes from one to 8-16, the nucleus changes from large to small and condensed, and the hemoglobin in the cytoplasm gradually increases. After metablastic cells no longer divide, the nuclei are expelled during development and become reticulocytes. Reticulocytes contain a small amount of ribonucleic acid (RNA) and become reticular when stained with brilliant tar blue, hence the name reticulocytes. Reticulocytes mature further, RNA ...

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Abstract

The invention discloses a purification technology of mono-substituted polyethylene glycol-erythropoietin (m PEG-EPO) and a preparation method thereof. The method comprises: subjecting a mixture of EPO, PEG, m PEG-EPO, di PEG-EPO and oli PEG-EPO to hydrophobic chromatography treatment so as to obtain a mixture of m PEG-EPO and PEG, then purifying the mixture of m PEG-EPO and PEG so as to obtain m PEG-EPO, and finally conducting concentration and buffer solution replacing by an ultrafiltration method. The product prepared by the method of the invention has purity up to over 95% and a yield not lower than 60%, and also has obvious and lasting erythropoiesis promotion function in animals as well as a better long-acting effect than conventional EPO. The m PEG-EPO prepared by the method of the invention has substantially improved purity index, as well as effectively reduced production cost and energy consumption.

Description

technical field [0001] The invention relates to a purification process and preparation method of cytokine proteins, in particular to a purification process and preparation method of single-substituted PEG-EPO. Background technique [0002] Conventional EPO (erythropoietin) is a hematopoietic factor that exists in the human body. In 1985, it was successfully cloned in vitro. In 1989, it was approved for marketing as an injectable drug for the treatment of anemia. Conventional EPO is the protein drug with the most successful application and the largest market sales in the current pharmaceutical protein. In 2006, the global sales amounted to 13.9 billion US dollars. However, conventional EPO is easily cleared by the renal corpuscle in the body, and the half-life is short. Patients need frequent injections to maintain the effective drug concentration, and EPO is easy to aggregate and cause antigen-antibody reactions. Chinese patent "polymer / recombinant human erythropoietin conj...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/505C07K1/36C07K1/34C07K1/20C07K1/18
Inventor 黎雄辉张向荣张涤平盛光阳刘建军黄俊龙黄海燕吴园园
Owner SHENZHEN SCIPROGEN BIO PHARMA
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