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Efficent polyethylene glycol activating process and activate use for protein modification

A polyethylene glycol and activated product technology, applied in the field of protein chemistry, can solve the problems of long half-life and low clearance rate

Inactive Publication Date: 2005-02-16
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] (2) Free PEG injected intravenously into the human body is easily excreted through the kidneys [viii]
The complexity of the PEG chain also affects the clearance of the PEG-protein conjugate by the kidneys. Therefore, the protein conjugated with branched chain PEG has a lower renal clearance rate and longer half-life than the protein conjugated with linear PEG.

Method used

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  • Efficent polyethylene glycol activating process and activate use for protein modification
  • Efficent polyethylene glycol activating process and activate use for protein modification
  • Efficent polyethylene glycol activating process and activate use for protein modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Activation of monomethoxypolyethylene glycol 5000 (mPEG5000) with 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP)

[0064] Add 250ml of toluene and 50g of mPEG5000 into a 1000ml round bottom flask, connect the water separator, reflux condenser and drying tower to the round bottom flask in turn, and then heat the toluene solution to 140°C. Keep at reflux for two hours. Then change the reflux device to a vacuum distillation device, and steam 200ml of toluene. Add 500ml of diethyl ether to the round-bottomed flask again, and cool down naturally under the condition of stirring until the solid completely precipitates out. The ether was then removed by filtration, and the precipitate was placed in a vacuum desiccator to remove residual ether. Dry anhydrous mPEG5000 was obtained. Then measure the moisture content in the dried anhydrous mPEG5000 with a Karl Fischer moisture analyzer, and the moisture content is required to be lower than 50ppm.

[0065] Weigh and dry 2...

Embodiment 2

[0068] Conjugate recombinant human granulocyte colony-stimulating factor (rhG-CSF) with FMP-mPEG5000

[0069] G-CSF was dissolved in 0.1M boric acid-borax buffer solution, pH value was 8.6, and the concentration was 2.3 mg / ml. Take 40ml of the solution and pour it into a 200ml Erlenmeyer flask and shake it in a constant temperature shaker at 25°C. Then 0.98 g of FMP-mPEG5000 was added to the Erlenmeyer flask. After the reaction lasted for 30 minutes, the pH value of the reaction system was adjusted to 4.0 to terminate the coupling reaction. Then use gel filtration chromatography to remove unreacted FMP-mPEG, and the coupled product collected is analyzed by SDS-PAGE for its molecular weight change, the results are as follows Figure 4 As shown, the molecular weight of the product modified with FMP-mPEG increased, indicating that a coupling reaction occurred between FMP-mPEG and G-CSF, and a coupled product was obtained.

Embodiment 3

[0071] Conjugate recombinant human α-interferon (rhα-Interferon) with FMP-mPEG5000

[0072] Dissolve α-interferon in 0.1M boric acid-borax buffer solution, the pH value is 7.6, and the concentration is 3.5 mg / ml. Take 40ml of the solution and pour it into a 200ml Erlenmeyer flask and place it in a constant temperature shaker at 25°C for gentle shaking. Then 3.1 g of FMP-mPEG5000 was added to the Erlenmeyer flask. After the reaction lasted for 30 minutes, the pH value of the reaction system was adjusted to 4.0 to terminate the coupling reaction. Then gel filtration chromatography was used to remove unreacted FMP-mPEG, and the collected coupled products were analyzed by SDS-PAGE for molecular weight changes. The result is as Figure 4 As shown, the molecular weight of the product modified with FMP-mPEG increases, indicating that a coupling reaction between FMP-mPEG and α-interferon occurred, and a coupled product was obtained.

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Abstract

The invention provides the effective method of the modification reaction of the biological active molecule of the activation technique of the hydroxyl at the end of the polyethylene glycol, its product, protein and other nucleophilic groups and the reaction products. Its characteristics are as follows: (1) heat the PEG and toluene to 140deg.C to get rid of the water in the PEG. (2) The anhydrous PEG and 2-fluorin-1-methyl pyridine onium toluene-4-sulfonate reacts in the anhydrous polar organic solvent. (3) Add organic base to keep the pH value between 8 and 9 during the reaction. (4) Deposit the PEG activates with aether and purify the activates through isopropyl alcohol recrystallization. (5) The PEG activates reacts with the protein and other biological active molecules with nucleophilic groups in the buffer solution to get the PEG and its coupling materials. The activation technique of the PEG in the invention has high activation rate (95%), high purity (99%), good controllability of the modification speed and good chemical stability of the modified products. The activated PEG of the invention is especially adaptable to the modifications of the polypeptides and proteins.

Description

field of invention [0001] The invention belongs to the field of protein chemistry, and in particular relates to a protein modifier and its preparation method and application. Background technique [0002] Chemical modification of proteins with polyethylene glycol (PEG) is attracting increasing attention as a way to improve the application of proteins in the fields of medicine and biotechnology. After proper chemical modification, many features of proteins can be changed while effectively maintaining their main biological functions, such as enzymatic activity or receptor recognition ability. PEG modification can mask the surface of the protein and increase its molecular volume, thereby reducing the chance of being filtered by the glomerulus; it can also prevent the approach of antibodies, antigens, enzymes, etc., and reduce the degradation by proteases. PEG can also endow proteins with their own physical and chemical properties, such as changing the...

Claims

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Application Information

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IPC IPC(8): C07K14/52C07K14/555C07K14/805C07K17/02C07K17/06C07K17/08C08F8/30C08F283/00
Inventor 苏志国马光辉贠强
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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