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Method for carrying out fixed-point polyethylene glycol long-acting modification on interferon IFN alpha-2b

A technology of polyethylene glycol and interferon, applied in the direction of interferon, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of difficult separation, decreased activity, difficult quality control, etc., and achieve easy separation and recovery, production Effects of cost reduction and high storage stability

Pending Publication Date: 2020-05-05
JIANGSU SUNAN PHARMA IND CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the current PEGylation reactions are carried out on the free terminal amino groups on the protein peptide chain. Since there are many such sites in the protein molecule, the modification of PEG is non-selective, resulting in the diversification of modified products and difficulty in their separation. The problem of difficult quality control
Therefore, it is necessary to use a method for site-directed modification of IFN α-2b to avoid the decrease in activity and the disadvantages of secondary separation and purification caused by random modification of protein drugs

Method used

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  • Method for carrying out fixed-point polyethylene glycol long-acting modification on interferon IFN alpha-2b
  • Method for carrying out fixed-point polyethylene glycol long-acting modification on interferon IFN alpha-2b

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Use the porous film with carboxyl groups to immobilize transglutaminase, the steps are as follows: the carboxylated porous film is placed in 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC ) and N-hydroxysulfosuccinimide (Sulfo-NHS) in 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution (pH 4.5, 0.1 M), in which the concentration of EDC and Sulfo-NHS Both were 10 mg / mL to activate the carboxyl groups on the porous membrane at 4 °C. After activating for 1.5 h, the film was washed with double distilled water, and then the activated porous film was placed in a phosphate buffer solution of transglutaminase to react for 12 h, and then fully washed with double distilled water, and the immobilized film was obtained. Porous film with transglutaminase.

[0025] Interferon IFN α-2b was dissolved in phosphate buffer (pH 7.4, 10 mM) to form a protein solution with a concentration of 1.5 mg / mL, and 1 times the amount of interferon IFN α-2b was added to the so...

Embodiment 2

[0028] Use porous microspheres with epoxy groups to immobilize transglutaminase, the steps are as follows: equilibrate the porous microspheres with epoxy groups in phosphate buffer (pH 8.0, 50 mM) for 2 h, centrifuge Afterwards, it was transferred to the phosphate buffer solution in which transglutaminase was dissolved for enzyme immobilization, and the whole reaction system was stirred and reacted at room temperature for 18 h to complete the enzyme immobilization. The porous microspheres were subjected to multiple centrifugation washes to remove physically adsorbed transglutaminase and by-products.

[0029] Interferon IFN α-2b was dissolved in phosphate buffer (pH 7.4, 10 mM) to form a protein solution with a concentration of 1.5 mg / mL, and 1.01 times the amount of interferon IFN α-2b was added to the solution with Polyethylene glycol (molecular weight 5kDa, branched) with primary amino groups was stirred to dissolve completely, and then immobilized transglutaminase was added...

Embodiment 3

[0032] Use porous fibers with aldehyde groups to immobilize transglutaminase, and the steps are as follows: the porous fibers with aldehyde groups are placed in phosphate buffer (pH 8.0, 50 mM) for 2 h, centrifuged to remove the supernatant and transferred The enzyme was immobilized in the phosphate buffer solution dissolved with transglutaminase, and the whole reaction system was stirred at room temperature for 18 h to complete the enzyme immobilization. Multiple centrifuge washes were performed to remove physically adsorbed transglutaminase and by-products.

[0033] Interferon IFN α-2b was dissolved in phosphate buffer (pH 7.4, 10 mM) to form a protein solution with a concentration of 1.5 mg / mL, and 1.03 times the amount of interferon IFN α-2b was added to the solution with Polyethylene glycol with primary amino groups (molecular weight 10kDa, multi-arm type) was stirred to dissolve completely, and then immobilized transglutaminase was added. The whole reaction system was s...

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Abstract

The invention relates to the field of pharmaceutical synthesis, and discloses a method for carrying out fixed-point polyethylene glycol long-acting modification on interferon IFN alpha-2b. According to the method, fixed-point polyethylene glycol modification is carried out on the interferon IFN alpha-2b by utilizing immobilized glutamine transaminase. The glutamine transaminase is transferase forcatalyzing acyl transfer reaction, and is proved to have a high fixed-point catalytic action, so that fixed-point polyethylene glycol modification of the interferon IFN alpha-2b can be realized underthe mediation action of the glutamine transaminase. On the other hand, because the immobilized glutamine transaminase is used, the chemical and pH stability is relatively high; and the operation is facilitated in the fixed-point modification process, and the repeated utilization rate is high, so that the production cost of the product can be greatly reduced.

Description

technical field [0001] The invention belongs to the field of drug synthesis, and in particular relates to a method for modifying interferon IFN α-2b with long-acting polyethylene glycol at a fixed point. Background technique [0002] Interferon (Interferon, IFN) is a cytokine with high activity and multiple biological functions secreted by monocytes under the action of a specific inducer. It has the functions of inhibiting virus replication, inhibiting cell division and immune regulation. Among the many types of interferon IFN, IFN α-2b is one of the earliest subtypes discovered, it is an antiviral and antitumor drug, and has immunomodulatory activity, and its broad-spectrum antiviral, antitumor and immune function regulation It has the strongest ability and plays an irreplaceable role in the treatment of many diseases, for example, it can be used to treat viral hepatitis B, hepatitis D, viral meningitis, viral eye infection, recurrent keratitis, kidney cancer, capillary Le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C12N11/14C12N11/12C12N11/10C12N11/082C12N11/096C12N11/089C12N9/10
CPCC07K14/56C12N11/14C12N11/12C12N11/10C12N11/08C12N9/1044C12Y203/02013
Inventor 辛渊蓉俞洋郝德顺何燕刘宏飞
Owner JIANGSU SUNAN PHARMA IND CO LTD
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