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Preparation method, product and application of single-modified polyethylene glycol recombinant human erythropoietin

A technology of polyethylene glycol and erythropoietin, which is applied to the preparation method of peptides, medical preparations with non-active ingredients, medical preparations containing active ingredients, etc., can solve the problems of high multiple substitution rate, difficult purification, single substitution Low efficiency and other problems, to achieve the effect of simple process, conducive to purification, high recovery rate

Active Publication Date: 2016-08-03
SHANDONG EHUA BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The existing methods for chemically modifying recombinant human erythropoietin (EPO) with PEG have defects such as low single substitution rate, high multiple substitution rate, and difficult purification. Therefore, it is urgent to develop a new single-modified polysaccharide Preparation method of ethylene glycol recombinant human erythropoietin, in order to increase the single substitution rate and reduce the multi-substitution rate at the same time

Method used

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  • Preparation method, product and application of single-modified polyethylene glycol recombinant human erythropoietin
  • Preparation method, product and application of single-modified polyethylene glycol recombinant human erythropoietin
  • Preparation method, product and application of single-modified polyethylene glycol recombinant human erythropoietin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Preparation of Preliminary Example 1 Recombinant Human Erythropoietin (EPO) Stock Solution

[0037] EPO stock solution is made by using recombinant DNA technology to transfect the recombinant plasmid with human erythropoietin gene into CHO-dhfr- (dihydrofolate reductase gene-deficient cells) cell line, and make it after cell culture, separation and high purification. details as follows:

[0038] 1. Basic requirements of raw materials and auxiliary materials

[0039] Production and testing facilities, raw materials and auxiliary materials, water, utensils, animals, etc. should meet the relevant requirements of the current version of the "Chinese Pharmacopoeia" three "general rules".

[0040] 2. Preparation of EPO stock solution

[0041] 2.1 Engineered cells

[0042] 2.1.1 Name and source

[0043] The polyethylene glycol recombinant human erythropoietin engineering cell line is a CHO-dhfr- (dihydrofolate reductase gene-deficient cell) cell line transfected with a reco...

Embodiment 2

[0085] Example 2 Identification of single-modified polyethylene glycol recombinant human erythropoietin—PEG modification reaction

[0086] The PEG modification ratio of the single-modified polyethylene glycol recombinant human erythropoietin prepared in Example 1 was identified by reversed-phase-high performance liquid chromatography. Mobile phase A is trifluoroacetic acid aqueous solution (0.3% TFA); Mobile phase B is trifluoroacetic acid and acetonitrile aqueous solution (0.2% TFA, 84% acetonitrile); Flow rate is 1ml / min; Detection wavelength is 220nm; Column temperature is 60 ℃ ; The injection volume is 5-20μg. The chromatographic column adopts Agilent Poroshell300SBC8 (5μm, 300A, 2.1x75mm).

[0087] Elution gradient:

[0088]

[0089]

[0090] The identification results of PEG modification ratio are shown in image 3 And table 1, target product single modification PEG-EPO (structural formula sees Figure 4 ) was 42.91%.

[0091] Chromatographic peak results of t...

Embodiment 3

[0093] Example 3 Identification of Single Modified Polyethylene Glycol Recombinant Human Erythropoin—Purification of PEG-EPO

[0094] The monomodified polyethylene glycol recombinant human erythropoietin prepared in Example 1 was purified. Assemble an ion-exchange chromatography column, choose strong cationic medium MacroCapSP as filler, the column height is about 15cm, connect an online ultraviolet detector behind the column, and carry out chromatographic purification in an environment of 2-8°C.

[0095] Equilibrate not less than 5 column volumes with 30 mM NaAc (sodium acetate), pH 4.0 buffer. Load the modified reaction solution prepared in Example 1, and the loading amount should not exceed 2 mg protein / ml medium (optimally about 1 mg protein / ml medium). Equilibrate no less than 5 column volumes with 30mM NaAc, pH 4.0 buffer after loading.

[0096] Wash 3-4 column volumes with 100mM NaCl, 30mMNaAc, pH4.0; elute with 175mMNaCl, 30mMNaAc, pH4.0 for 3 column volumes, collect...

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Abstract

The invention discloses a preparation method, a product and an application of single-modified polyethylene glycol recombinant human erythropoietin. According to the preparation method, recombinant human erythropoietin with normal in vivo activity is chemically modified with polyethylene glycol to obtain the single-modified polyethylene glycol recombinant human erythropoietin with an erythropoiesis promotion effect and long-term in vivo activity. By optimizing the pH value of a buffer system of a modification reaction, the protein concentration of the recombinant human erythropoietin, a protein and polyethylene glycol raw material ratio of the recombinant human erythropoietin and the like, the prepared single-modified polyethylene glycol recombinant human erythropoietin is high in yield, a multi-modified proportion is low, and the purification is facilitated. Compared with protein of recombinant human erythropoietin before modification, the single-modified polyethylene glycol recombinant human erythropoietin prepared with the method is high in in-vivo biological specific activity and remarkably prolonged in half-life period, and can be applied to preparation of drugs for treating anemic diseases, especially renal anemia.

Description

technical field [0001] The present invention relates to the preparation of polyethylene glycol modified products of recombinant human erythropoin protein, in particular to the preparation method of single-modified polyethylene glycol recombinant human erythropoin and the prepared single-modified polyethylene glycol recombinant human erythropoin , the present invention also relates to the application of the monomodified polyethylene glycol recombinant human erythropoietin in the preparation of drugs for treating anemia, especially renal anemia, which belongs to the preparation and application of polyethylene glycol recombinant human erythropoietin field. Background technique [0002] Renal disease is a relatively serious chronic disease, and it is not easy to be completely cured. The annual incidence of chronic nephritis in China is about 0.25%, and a considerable number of patients will eventually turn into renal failure. Anemia is the main complication of patients with re...

Claims

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Application Information

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IPC IPC(8): C07K14/505C07K1/107A61K38/18A61K47/48A61P7/06
CPCA61K38/1816C07K14/505
Inventor 刘海峰胥国立庞甲佩周祥山解福生田守生马立平王扬张建岭史兆松
Owner SHANDONG EHUA BIOLOGICAL PHARMA
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