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Preparation method of insulin degludec

A technology of insulin degludec and main chain, applied in the field of preparation of insulin degludec, can solve the problems of complex process, low yield, generation of impurities and the like

Active Publication Date: 2021-12-10
NINGBO KUNPENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are many reports about the preparation of insulin degludec at home and abroad. Generally, the main chain (main chain) of insulin degludec is obtained by a gene recombination technology, and then the degludec is obtained by connecting tBuO-Pal-Glu(OSu)-OtBu substances through a liquid phase synthesis method. Insulin gludec, because the main chain of insulin degludec is in an unprotected state, the side chain is easily attached to the N-terminal amino group during the connection process, resulting in impurities, resulting in difficult purification and low yield, and the process is more complicated

Method used

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  • Preparation method of insulin degludec
  • Preparation method of insulin degludec
  • Preparation method of insulin degludec

Examples

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preparation example Construction

[0206] The preparation of insulin degludec is to first obtain the main chain of insulin degludec with lysine protected by Boc at position 29 by using gene recombination technology, and then connect the side chain tBuO-Pal-Glu(OSu)-OtBu of insulin degludec to obtain insulin degludec .

[0207] Preparation of insulin degludec

[0208] The synthesis route of insulin degludec provided by the present invention is as follows, Fmoc-modified compound 2 is prepared from the main chain of Boc-insulin degludec (compound 1), and compound 3 is obtained after de-Boc protection of compound 2, compound 3 and activated insulin degludec The side chain tBuO-Pal-Glu(OSu)-OtBu reacted to obtain compound 4, and then the Fmoc reaction was removed to obtain compound 5, and the side chain was removed from the tBu protecting group to finally obtain compound 6 insulin degludec.

[0209]

[0210] Specifically, the present invention provides a method for preparing insulin degludec, the method comprisi...

Embodiment 1

[0222] Construction and expression of embodiment 1 insulin degludec expression strain

[0223] For the construction of the insulin degludec expression vector, refer to the description in the examples in the patent application number 201910210102.9. The fusion protein FP-TEV-R-Insulin (DesB 30 , Lys 29 Boc) DNA fragment, cloned into the NcoI-XhoI site downstream of the araBAD promoter of the expression vector plasmid pBAD / HisA (purchased from NTCC company, kanamycin resistance), to obtain the plasmid pBAD-FP-TEV-R-Insulin ( DesB 30 , Lys 29 Boc). Plasmid map such as figure 1 shown.

[0224] Then the DNA sequence of pylRs was cloned into the SpeI-SalI site downstream of the araBAD promoter of the expression vector plasmid pEvol-pBpF (purchased from NTCC Company, chloramphenicol resistance), and at the same time, the pylRs was inserted downstream of the proK promoter by PCR. DNA sequence of tRNA (pylTcua) of aminoacyl-tRNA synthetase. This plasmid was named pEvol-pylRs-p...

Embodiment 2B

[0228] Example 2 Denaturation and enzymatic cleavage of Boc-insulin degludec precursor inclusion body

[0229] Add 7.5-9.0 mol / L urea solution to the inclusion body obtained in Example 1 at a weight-to-volume ratio of 1:10 (m / v), stir and dissolve at room temperature, and control the total protein concentration of the inclusion body solution to 10-25mg / L mL, NaOH to adjust the pH to 11.0-11.8, add β-mercaptoethanol and stir well. Add the inclusion body solution dropwise to the refolding buffer containing 0.2-1.0mmol / L L-cystine, dilute the inclusion body solution 5-10 times for refolding, and maintain the pH value of the fusion protein refolding solution at 10.5 -11.8, the temperature is controlled at 4-8°C, the renaturation time is 10-20h, and the renaturation rate is above 60% (see electrophoresis test). image 3 ) to obtain an insulin degludec main chain fusion protein containing a disulfide bond between the insulin degludec A chain and the insulin degludec B chain.

[02...

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Abstract

The invention provides an insulin degludec derivative and a preparation method thereof. Specifically, according to the method, insulin degludec fusion protein containing a green fluorescent protein folding unit is used for preparing insulin degludec, a Boc modified insulin degludec main chain is subjected to Fmoc modification, and side chain addition of insulin degludec is carried out through orthogonal protection. The invention also provides the Fmoc and Boc modified insulin degludec main chain involved in the process of the preparation method and the fusion protein containing the insulin degludec main chain.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a preparation method of insulin degludec. Background technique [0002] Diabetes is a major disease that threatens human health worldwide. In China, with the change of people's lifestyle and the acceleration of the aging process, the prevalence of diabetes is rising rapidly. Acute and chronic complications of diabetes, especially chronic complications that accumulate in multiple organs, cause disability and high mortality, seriously affect the physical and mental health of patients, and bring heavy burdens to individuals, families and society. [0003] The recombinant insulin degludec injection "Tresiba" developed by Novo Nordisk of Denmark is a new type of long-acting insulin analogue. In January 2013, it was approved by the European Union for the treatment of type I and type II diabetes patients. The structural feature of insulin degludec is that the lysine side chain at position ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K14/62C12N15/70C12P21/06A61K38/28A61P3/10
CPCC07K14/62C12N15/70C12P21/06A61P3/10C07K2319/60C07K2319/50C07K2319/02A61K38/00
Inventor 陈卫唐亚连骆莉
Owner NINGBO KUNPENG BIOTECH CO LTD