Preparation method of single-modified polyethylene glycol recombinant human erythropoietin, its products and applications

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 problem of low single substitution rate, high multiple substitution rate, Purification is difficult and other problems, to achieve the effect of high recovery, simple process, and beneficial to purification

Active Publication Date: 2019-05-17
华润昂德生物药业有限公司
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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 of single-modified polyethylene glycol recombinant human erythropoietin, its products and applications
  • Preparation method of single-modified polyethylene glycol recombinant human erythropoietin, its products and applications
  • Preparation method of single-modified polyethylene glycol recombinant human erythropoietin, its products and applications

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[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 recombinant plasmid carrying a human erythropoietin gene.

[0044] 2.1.2 Establishment, subculture and preservation of cell bank

[0045] Cells from the original cell bank are passaged, amplified and frozen in liquid nitrogen. As a master cell bank, the master cell bank should not exceed 2 cell passages at most; cells from the master cell bank are passaged, expanded and then frozen in liquid nitrogen. In nitrogen, as a working cell bank, the working cell bank must be limited to 1 cell passage. The cells are frozen in liquid nitrogen and can be used for production after passing the test.

[0046] 2.1.3 Verification of master cell bank and working cell bank cells

[0047] It should comply with th...

Embodiment 1

[0082] Example 1 Preparation of Monomodified Polyethylene Glycol Recombinant Human Erythropoin (PEG-EPO)—PEG Modification Reaction

[0083] Get the qualified EPO stock solution, adjust the buffer system to 150mM K 2 HPO 4 , pH7.5, EPO concentration is 2.84mg / ml.

[0084] According to the ratio of EPO:PEG molar ratio of 1:3 (mass ratio of 1:5), weigh mPEG-SBA (methoxypolyethylene glycol succinimide butyrate, straight chain, 30KDa, molecular structure see figure 2 ), added to the adjusted EPO stock solution, stirred slowly to dissolve, and stirred at a uniform speed (80-130rpm) with an electric stirrer at room temperature (20-25° C.), and reacted for 2 hours. After the reaction, dilute 5 times and adjust the pH to 4.0 with glacial acetic acid, and store in a refrigerator at 2-8°C for no longer than 3 days; if it exceeds 3 days, it needs to be stored in a refrigerator at -20°C.

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 Poroshell 300SB C8 (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] Table 1 Chromatographic peak re...

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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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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/505C07K1/107A61K38/18A61K47/60A61P7/06
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 华润昂德生物药业有限公司
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