A kind of preparation method of protein site-directed pegylation modification

A technology of PEGylation and polyethylene glycol is applied in the field of protein modification to achieve the effects of reducing commercial production costs, good stability and high uniformity

Active Publication Date: 2021-12-07
科兴生物制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Usually, this PEGylation modification reaction is carried out under weak acid conditions, and sodium cyanoborohydride is used in the reaction, but sodium cyanoborohydride is a highly toxic substance, and the storage conditions are strict, and hydrocyanic acid and borohydride are finally produced in the reaction. Sodium, which poses a huge risk to product safety, so in order to ensure the safety and cost of medication, research on the preparation method of replacing sodium cyanoborohydride to complete the PEGylation of proteins will have greater social value

Method used

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  • A kind of preparation method of protein site-directed pegylation modification
  • A kind of preparation method of protein site-directed pegylation modification
  • A kind of preparation method of protein site-directed pegylation modification

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Experimental program
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Effect test

Embodiment 1

[0047] rhG-CSF reaction solution (40ml, concentration 3mg / ml), containing 100mM sodium phosphate, pH5.0, also contains 120mM MVB6 (vitamin B6), after it is fully stirred and frozen (4 ℃), add 5 times the amount of protein Methoxypolyethylene glycol propionaldehyde (MPEG-PALD) (average molecular weight 20 kDa). The reaction mixture was continuously stirred at the same temperature.

[0048] During the reaction, the modification rate of protein was monitored by RP-HPLC. What RP-HPLC used was a C18 250×4.6mm 5 μm column (phenomenex), with 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile at 1.0 Gradient elution was performed at a flow rate of ml / min.

[0049] The RP-HPLC analysis results at 8, 9, 10, and 11 hours respectively are shown in Table 1:

[0050]

[0051]

Embodiment 2

[0053] rhG-CSF reaction solution (40ml, concentration 3mg / ml), containing 100mM sodium phosphate, pH5.0, also contains 40mM NADPH (nicotinamide adenine dinucleotide phosphate), fully stirred and frozen (4 ℃) , Add 5 times the protein amount of methoxypolyethylene glycol propionaldehyde (MPEG-PALD) (average molecular weight is 20kDa). The reaction mixture was continuously stirred at the same temperature.

[0054] During the reaction, the modification rate of protein was monitored by RP-HPLC. What RP-HPLC used was a C18 250×4.6mm 5 μm column (phenomenex), with 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile at 1.0 Gradient elution was performed at a flow rate of ml / min.

[0055] The RP-HPLC analysis results at 8, 9, 10, and 11 hours respectively are shown in Table 1:

[0056] time RP-HPLC modification rate (%) 8 77.5 9 84.4 10 86.0 11 85.8

Embodiment 3

[0058] rhG-CSF reaction solution (40ml, concentration 3mg / ml), containing 300mM sodium phosphate, pH 7.0, also contains 40mM NADPH (nicotinamide adenine dinucleotide phosphate), fully stirred and frozen (4 ℃) , Add 5 times the protein amount of methoxypolyethylene glycol propionaldehyde (MPEG-PALD) (average molecular weight is 20kDa). The reaction mixture was continuously stirred at the same temperature.

[0059] During the reaction, the modification rate of protein was monitored by RP-HPLC. What RP-HPLC used was a C18 250×4.6mm 5 μm column (phenomenex), with 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile at 1.0 Gradient elution was performed at a flow rate of ml / min.

[0060] The RP-HPLC analysis results at 8, 9, 10, and 11 hours respectively are shown in Table 1:

[0061] time RP-HPLC modification rate (%) 8 72.3 9 75.1 10 79.5 11 80.8

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Abstract

The present invention relates to the technical field of protein modification, in particular to a preparation method for protein site-directed pegylation modification. A preparation method for protein site-specific PEGylation modification. In the presence of vitamins and / or coenzymes, 5kD-40kD polyethylene glycol derivatives are reacted with proteins in an amount of 3 to 7 times the protein mass. The present invention adopts a new modification method to carry out PEGylation modification of rhG-CSF, the reaction conditions are mild, NADP+ is generated after the reaction, and no harmful substances are generated. At the same time, the polyethylene glycol modification yield of the preparation method is relatively high, and the yield is up to 80% % or more, high uniformity, the modification site is Met, the first amino acid methionine at the N-terminal of the protein rhG‑CSF, and has good stability. Commercial production costs.

Description

technical field [0001] The present invention relates to the technical field of protein modification, in particular to a preparation method for protein site-directed pegylation modification. Background technique [0002] With the rapid development of biotechnology, more and more recombinant protein drugs have been successfully applied to the treatment of human diseases. Recombinant human granulocyte colony stimulating factor (rhG-CSF) is a very successful genetic engineering drug. Human granulocyte colony stimulating factor (G-CSF) is produced by monocytes and fibroblasts, can stimulate the formation of granulocyte colonies, has a stimulating effect on neutrophils, and is often used in cancers receiving radiotherapy / chemotherapy Adjuvant therapy for patients and leukemia patients after bone marrow transplantation, Amgen was approved by the FDA in 1991, but due to the short half-life of rhG-CSF in vivo and the tendency to produce immunogenic reactions, its clinical use has be...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/107C07K14/535A61K38/19A61K47/60A61P35/02
CPCA61K38/193A61K47/60A61P35/02C07K1/1077C07K14/535
Inventor 马玉涛孔凡楼崔宁董成涛魏述亮胡贵新李思
Owner 科兴生物制药股份有限公司
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