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Preparation method of semaglutide

A precursor and inclusion body technology, applied in the field of polypeptide drug preparation, can solve problems such as less research, achieve the effect of reducing production costs and improving yield

Active Publication Date: 2022-07-26
BEIJING HUIZHIHENG BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on semaglutide mainly focuses on its structure and preparation, and there are few studies on expression vectors with high expression potential, recombinant engineering bacteria and fermentation methods.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] This embodiment provides a preparation method of semaglutide.

[0059] In order to obtain the semaglutide precursor that can be expressed stably and highly and realize the improvement of industrial application value, the inventors designed a fusion protein structure containing the semaglutide precursor, and inserted it into the expression vector for fusion. Expression in the form of protein inclusion bodies. For details on synthesizing fusion polypeptide-encoding genes, constructing recombinant expression vectors, constructing recombinant engineered bacteria, and high-density fermentation of recombinant engineered bacteria, please refer to the prior patent applications CN113502296A and CN113502310A.

[0060] In this example, semaglutide is prepared on the basis of the obtained fermentation broth, and the fusion protein sequence expressed by the construct used is shown in SEQ ID NO.5, and the specific method is as follows:

[0061] (1) Homogeneous centrifugation

[006...

Embodiment 2

[0076] Embodiment 2: Recombinant EK enzyme and its engineering bacteria preparation

[0077] In order to obtain an improved in vitro renaturation efficiency of the protein, improve the solubility of the protein itself, maintain the specificity and activity of the protease, thereby increasing the yield and effectively enhancing the industrial application value, the inventors designed a bovine enterokinase. The light chain mutant has better enzymatic activity than existing commercial enzymes, and the yield of active protein is increased by more than 4 times. For details, refer to the prior patent application CN202210697582.8, in which some examples and data are shown in this example.

[0078] Specific examples of mutations are:

[0079] (1) Mutant EK L m1 (commercialized EK enzyme, Yaxin):

[0080] In view of the poor stability and low renaturation rate of wild-type bovine enterokinase in the process of renaturation and renaturation, which is not conducive to purification, th...

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Abstract

The invention relates to a preparation method of semaglutide. The preparation method comprises the following steps: collecting an inclusion body from a fermentation thallus, treating the inclusion body to prepare a fusion protein, carrying out enzyme digestion and purification treatment on the fusion protein to obtain a semaglutide precursor, and connecting the semaglutide precursor with a side chain and dipeptide to obtain semaglutide. The semaglutide precursor fusion protein prepared by the invention is subjected to enzyme digestion treatment by using a mutant EK enzyme. According to the preparation method provided by the invention, high-expression recombinant engineering bacteria and high-activity and high-yield mutant EK enzyme are combined at the same time, so that the production efficiency of semaglutide can be remarkably improved, and the production cost of semaglutide can be remarkably reduced.

Description

Technical field [0001] The invention relates to the field of polypeptide drug preparation, and in particular to a preparation method of semaglutide. Background technique [0002] With the development of society and economy, people's living standards have gradually improved, and people's dietary structure has undergone tremendous changes, which has also led to an increase in the incidence of obesity and further led to a sharp increase in the number of diabetic patients. According to statistics, the number of diabetic patients in my country exceeds 100 million, and there are also more than 150 million invisible pre-diabetic patients. Diabetes has become the third most common chronic disease. [0003] In recent years, glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1RAs) have gradually become a research focus in the treatment of T2DM. Currently, the main GLP-1 drugs on the market for the treatment of diabetes include exenatide, albiglutide, dulaglutide, liraglutide and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K14/605C07K1/36C07K1/30C07K1/20C07K1/18C07K1/10C07K1/06C07K1/04
CPCC12P21/06C07K14/605
Inventor 曹海燕林兆生董欣张海波王含
Owner BEIJING HUIZHIHENG BIOTECHNOLOGY CO LTD
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