Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation method for protein fixed-point pegylation modification

A technology of PEGylation and polyethylene glycol, applied in the field of protein modification, to achieve high modification yield, simple termination process, and good stability

Active Publication Date: 2020-12-22
科兴生物制药股份有限公司
View PDF7 Cites 1 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method for protein fixed-point pegylation modification
  • Preparation method for protein fixed-point pegylation modification
  • Preparation method for protein fixed-point pegylation modification

Examples

Experimental program
Comparison scheme
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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to the technical field of protein modification, and particularly relates to a preparation method for protein fixed-point pegylation modification. The preparation method for protein fixed-point pegylation modification comprises the step that in the presence of vitamins and / or coenzymes, 5 kD-40kD polyethylene glycol derivatives with the mass 3-7 times that of protein react with the protein. According to the preparation method, a new modification method is adopted for conducting pegylation modification of rhG-CSF, the reaction condition is mild, NADP <+> is generated afterthe reaction, and no harmful substance is generated; meanwhile, the preparation method is high in polyethylene glycol modification yield, the yield reaches 80% or above, the uniformity is high, the modification site is the first amino acid methionine Met at the N terminal of the protein rhG-CSF, and the stability is good; in addition, the modification reaction can be terminated by directly adjusting the acid, the termination process is simple, and the commercialized production cost is greatly reduced.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products