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Recombinant erythropoietin with intracorporeal physical activity and modified by macrogol

A technology of erythropoietin and polyethylene glycol, which is applied in the field of physiologically active conjugates in vivo, can solve the problems of increasing the half-life of rhEPO plasma, and achieve the effect of reducing the cost of treatment medication, production cost and scale cost

Inactive Publication Date: 2008-01-16
CHENGDU INST OF BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although the above improvements have achieved the effect of significantly increasing the plasma half-life of rhEPO, it still adopts the eukaryotic cell expression system used in the current rhEPO drug production process, and the production cost should be comparable to the current product

Method used

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  • Recombinant erythropoietin with intracorporeal physical activity and modified by macrogol
  • Recombinant erythropoietin with intracorporeal physical activity and modified by macrogol
  • Recombinant erythropoietin with intracorporeal physical activity and modified by macrogol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] [Example 1] Preparation of recombinant EPOP expressed by prokaryotic expression system

[0036] 1. Strains and plasmids

[0037] Plasmid PBV 220 Full length 3.66Kb, tandem phage P L P R Promoter, downstream of ribosomal gene termination signal, ampicillin resistance, see the preparation method containing P L P R The establishment and application of the prokaryotic high-efficiency expression vector of the promoter, Acta Virus Sinica, 1990, 62): 11, the plasmid was donated by the author. Escherichia coli strain DH 5α Purchased from GIBCO company DH 5α standard strain.

[0038] 2. Tool enzymes and reagents

[0039] Restriction enzymes BamH I, EcoRI, T4 ligase and Taq DNA polymerase were purchased from Roche Company, plasmid purification kit (Wizard Plus Minipreps DNA Purification System) was purchased from Promega Company, protein molecular weight markers were purchased from Huamei Company, other reagents All domestic analytical reagents were used.

[0040] 3. Sy...

Embodiment 2

[0064] [Example 2] Preparation of PEG-EPOP conjugate

[0065] The present invention relates to modifying EPOP with PEG2NHS-20K, and the chemical modification reaction formula is as follows:

[0066]

[0067] Among them, X is NH or O, which represents the covalent connection method of PEG to EPOP chemical modification. It is generally the side chain amino group (NH) of lysine in the EPO peptide chain or the N-terminal amino group of EPO, or it can be the EPO peptide chain. Such as the side chain hydroxyl (OH) of serine.

[0068] Wherein i and j are integers representing the length of the PEG carbon chain;

[0069] According to the degree of modification of EPOP by PEG, n is an integer of 1 to 5, preferably 1;

[0070] The molecular structure of mPEG is CH 3 CH 2 CH 2 O k

[0071] Where k is an integer of 100-1000, and is the sum of i and j, so that the molecular weight of the mPEG part of the conjugate is 5000-40000 Daltons, preferably 10000-20000 Daltons.

[0072]...

Embodiment 3

[0082] [Example 3] One of the assays of erythropoiesis-stimulating activity of PEG-EPOP conjugates in animals

[0083] 1. Experimental method: Refer to "Establishment of the Reticulocyte Method for Detecting the Biological Activity of EPO in Vivo". Adopt the method of injecting only one dose and drawing blood on the fourth day.

[0084] 1.1. Recombinant EPOP sample: the purified EPO protein sample expressed and prepared according to the method in Example 1 contains N-terminal methionine and C-terminal arginine, a peptide chain with a total length of 167 amino acids, and the cell activity detected by UT-7 is 44000IU / ml;

[0085] 1.2. PEG-EPOP conjugate sample: PEG-modified PEG-EPOP conjugate sample prepared with reference to the method in Example 2. The specific method in this experiment is: add 50 mg of PEG2NHS-20K to 5ML of the aforementioned recombinant EPOP sample, After the modification reaction is complete, it is stored at 4°C without separation and purification for an...

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Abstract

The invention relates to a polyethylene glycol conjugate (PEG-EPOP) of the hematopoietin protein, which generates a recombinant erythropoietin (rhEPOP) with intracorporeal physical activity and modified by macrogol. The invention discloses the method of obtaining EPOP with low cost by the prokaryotic expression system so as to obtain polyethylene glycol conjugate (PEG-EPOP) with low cost for producing the medicine for treating anemic diseases and increasing erythrocyte.

Description

technical field [0001] The invention relates to a recombinant erythropoietin protein (EPOP) which has no physiological activity in vivo and is obtained by modifying recombinant erythropoietin protein (EPOP) with erythropoietic activity (PEG-EPOP). Background of the invention [0002] Human Erythropoietin (hEPO) is a glycoprotein hormone mainly synthesized and secreted by the kidneys. EPO is produced in the liver during the embryonic period and is then produced by the kidneys at 4 months after birth. Its physiological function is to stimulate the differentiation and proliferation of bone marrow erythrocyte precursor cells, which plays an important role in the process of erythrocyte development and maturation, and is an endogenous regulator of erythrocyte growth. [0003] In the early years, more hEPO could be obtained from the urine of aplastic anemia patients and the culture medium of fetal kidney cells, but it could not meet the clinical needs. In 1985, recombinant human E...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K17/08C07K14/505A61K47/48A61K38/18A61P7/06A61K47/60
Inventor 葛永红陈智勇曾献武刘兰军
Owner CHENGDU INST OF BIOLOGICAL PROD
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