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Preparation method of insulin detemir or insulin detemir analogue

A technology for insulin detemir and its analogues, which is applied in the field of preparation of insulin analogues, can solve the problems of many by-products, high cost and low acylation efficiency, and achieves the advantages of less by-products, short reaction time and increased overall yield. Effect

Active Publication Date: 2016-03-30
SHANDONG EHUA BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing methods for preparing insulin detemir or its analogs from insulin precursors through acylation reactions have disadvantages such as low acylation efficiency, many by-products, and high costs.

Method used

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  • Preparation method of insulin detemir or insulin detemir analogue
  • Preparation method of insulin detemir or insulin detemir analogue

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 Preparation of recombinant human double-chain insulin precursor (desB30)

[0033] For the preparation of recombinant human double-chain insulin precursor (desB30), refer to relevant literature (Liu Haifeng, Research on the process of converting recombinant insulin precursor into human insulin and insulin detemir, East China University of Science and Technology, Ph.D. dissertation. 2013). details as follows:

[0034] 1.1 Test materials and instruments

[0035] Sodium chloride (NaCl), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased from Sinopharm Chemical Reagent Co., Ltd.; acetonitrile (CH 3 CN, HPLC grade), trifluoroacetic acid (TFA, HPLC grade) were purchased from J&K Chemical Company; the analytical HPLC instrument was Agilent 1260, and the analytical chromatographic column was Kromasil-C18-5μm- (4.6mm * 250mm); The chromatographic purification system is the AKTAexplorer100 chromatographic workstation of Sweden PharmaciaBiotech compa...

Embodiment 2

[0047] The preparation of embodiment 2 insulin detemir

[0048] Dissolve the lyophilized powder of recombinant human double-chain insulin precursor desB30 in disodium hydrogen phosphate at 120mmol / LpH2 and boric acid solution at 120mmol / LpH2 respectively, and stir the solution until the lyophilized powder is completely dissolved (observed with the naked eye), so that the final concentration of desB30 Both are 6mg / ml. Dissolve the solid activated ester (succinimide myristate) in pure acetonitrile, and sonicate until it is completely dissolved, so that the concentration of the activated ester is 10 mg / ml.

[0049] Use 1mol / L NaOH to uniformly adjust the pH of the desB30 precursor solution to 9.5, add pure acetonitrile so that the volume occupied by acetonitrile is 50% of the total reaction solution volume, stir slowly with a magnetic stirrer until completely mixed, and slowly add activated ester to make The molar ratio of activated ester to desB30 is about 1:2, stirred for 60 m...

Embodiment 3

[0052] The preparation of embodiment 3 insulin detemir analogs

[0053] The recombinant human double-chain insulin precursor desB30 lyophilized powder was dissolved in 100mmol / LpH3 disodium hydrogen phosphate solution, and the solution was stirred until the lyophilized powder was completely dissolved (observed with the naked eye), so that the final concentration of desB30 was about 6mg / ml. Dissolve the solid activated ester (N-succinimidyl palmitate) in ethanol, and sonicate until completely dissolved, so that the concentration of the activated ester is 10 mg / ml.

[0054] Use 1mol / L NaOH to adjust the pH of the desB30 precursor solution to 9.5, add pure ethanol so that the volume of ethanol is 40% of the total reaction solution volume, stir slowly with a magnetic stirrer until completely mixed, and slowly add activated ester to make the activated ester The molar ratio to desB30 is about 1:2, and the reaction is stirred for 60 minutes, and the reaction temperature is 25°C.

[...

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Abstract

The invention discloses a preparation method of insulin detemir or an insulin detemir analogue and belongs to the field of preparation of insulin analogues. According to the preparation method, threonine-free 30th-site human double-chain insulin precursor desB30 protein of a B chain is taken as a raw material, epsilon-amino of B29th-site lysine is selectively acylated by active ester through one step in the alkaline environment with pH ranging from 8.0 to 11 under the condition that N-terminal alpha-amino of double chains of the desB30 protein is not protected, and the insulin detemir or the insulin detemir analogue is generated efficiently. According to the preparation method of the insulin detemir or the insulin detemir analogue, parameters such as the pH value, the raw material ratio, the acylation time and the like of the acylation reaction of the insulin precursor are optimized, the yield of the insulin detemir or the insulin detemir analogue is remarkably increased, the preparation method has the advantages of high acylation efficiency, a few by-products, short acylation reaction time, a few acylation steps, simple process, low production cost and the like, and one simple and efficient method is provided for large-scale industrial preparation of the insulin detemir or the insulin detemir analogue through one-step acylation.

Description

technical field [0001] The invention relates to a method for preparing insulin analogs, in particular to a method for selectively acylation of ε-amino group of lysine at position B29 of human double-chain insulin precursor desB30 to generate insulin detemir or its analogs, which belongs to the category of insulin analogs field of preparation. Background technique [0002] Diabetes mellitus is a clinical syndrome caused by the interaction of genetic and environmental factors, and its basic pathological feature is metabolic disorder caused by absolute or relative insulin secretion deficiency. Diabetes has become the third chronic non-communicable disease that threatens human health after cardiovascular and cerebrovascular diseases and malignant tumors, especially its complications such as retinopathy, peripheral neuropathy, and diabetic nephropathy have seriously affected people's health. Quality of Life. [0003] Insulin and its analogues are the most direct and effective d...

Claims

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Application Information

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IPC IPC(8): C07K14/62
Inventor 刘海峰周祥山黄菁李志方喆应欢聂洪霞史兆松
Owner SHANDONG EHUA BIOLOGICAL PHARMA
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