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Method for preparing stable insulin aspart crystal

A technology for the crystallization of insulin aspart, which is applied in the field of purification and preparation of artificial insulin, can solve problems such as inability to form crystals, and achieve the effects of high production efficiency, small sedimentation volume, and shortened freeze-drying time

Active Publication Date: 2013-10-09
ZHUHAI UNITED LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insulin lispro and insulin aspart are both monomeric insulin analogues, and their structure and mode of action have certain similarities. However, when the crystallization method of insulin lispro is applied to insulin aspart, crystals cannot be formed, but amorphous precipitates

Method used

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  • Method for preparing stable insulin aspart crystal
  • Method for preparing stable insulin aspart crystal
  • Method for preparing stable insulin aspart crystal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Prepare recombinant insulin aspart crystallization liquid, each component and concentration are: experiment A is 1.2M glycine system, experiment B is 1.5M glycine system, other components are the same: recombinant insulin aspart: 3.0g / L, acetonitrile: volume fraction content 12%, phenol: volume fraction content is 0.2%, zinc acetate: the molar ratio of zinc ion to insulin aspart is 7.35:1, sodium chloride: 0.34M (20g / L).

[0038] Prepare a recombinant insulin aspart solution with a concentration of 6.0 g / L, in which the volume fraction of acetonitrile is 24%, and adjust the pH value to 6.0 for use. Weigh 1.36g and 1.69g of glycine and put them in 50ml Erlenmeyer flasks, respectively add 0.3g of sodium chloride, add 7.3ml of purified water to dissolve it, then add 7.5ml of 6.0g insulin aspart samples, and then add 30ul of water-saturated phenol, adjust the pH value to 6.0, and finally add 160ul of zinc acetate solution with a concentration of 0.355M to start the crystall...

Embodiment 2

[0040] Prepare recombinant insulin aspart crystallization liquid, each component and concentration are: experiment A contains 0.5M sodium chloride, experiment B contains 0.2M sodium chloride, other ingredients are the same: glycine: 1.2M, recombinant insulin aspart: 6.0g / L, acetonitrile: volume fraction content is 12%, phenol: volume fraction content is 0.4%, zinc acetate: insulin aspart content molar ratio is 3:1.

[0041] Prepare a recombinant insulin aspart solution with a concentration of 12.0 g / L, in which the volume fraction of acetonitrile is 24%, and adjust the pH value to 6.0 for use. Weigh 0.45g and 0.18g of sodium chloride and put them in 50ml Erlenmeyer flasks, respectively add 1.36g of glycine, add 7.3ml of purified water to dissolve it, then add 7.5ml of 12.0g of insulin aspart samples, and then add 60ul of water-saturated phenol, adjust the pH value to 6.2, and finally add 130ul of zinc acetate solution with a concentration of 0.355M to start the crystallizatio...

Embodiment 3

[0042] Embodiment 3 (comparative embodiment)

[0043] Preparation of recombinant insulin aspart crystallization solution, the components and concentrations are: Experiment A According to the Chinese patent CN95106555.6 insulin lispro crystallization process, a 15ml crystallization system was prepared, containing acetic acid: 1M, recombinant insulin aspart: 2.5g / L, acetic acid Zinc: 200mg / L, phenol: 0.2% volume fraction, pH6.0; Experiment B added acetonitrile on the basis of Experiment A, and the volume fraction content was 12%; Experiment C added sodium chloride on the basis of Experiment B, The concentration is 0.34M (20g / L). The result is as image 3 Shown: 40X magnification, Experiments A and B are amorphous precipitates (see image 3 a and image 3 b), Insulin aspart monoclinic needle-like crystals formed in experiment C have relatively poor uniformity, and the needle-like crystals are fragile and difficult to follow up (see image 3 c).

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Abstract

The invention discloses a method for preparing stable insulin aspart crystal, and belongs to the field of purification and preparation of artificial insulin. The method comprises the following steps of: preparing an organic solvent, phenolic substance and crystalline liquid at 10-30 DEG C, wherein the organic solvent comprises 0.2-1.5M of glycine and 3.0-10.0g / L of recombined insulin aspart, the volume fraction content of the organic solvent is 10-30%, the volume fraction content of the phenolic substance is 0.2-0.4%, and the crystalline liquid comprises 0.2-0.5M of salt, regulating the pH value of the crystalline liquid to be 6.0-6.5, adding zinc ion, crystallizing for 3-6 hours, then cooling to 2-8 DEG C, standing for 12-18 hours, and obtaining the stable insulin aspart crystal. According to the method, the freeze drying time for products is shortened, the product stability is improved, and the method is applicable to commercial production of the recombined insulin aspart.

Description

technical field [0001] The invention belongs to the field of purification and preparation of artificial insulin, and in particular relates to a method for preparing stable insulin aspart crystals. Background technique [0002] Diabetes mellitus is a metabolic disorder characterized by hyperglycemia resulting from defective or insufficient insulin secretion or action. According to statistics in November 2012, China has become the country with the largest number of diabetic patients in the world. The number of diabetic patients in my country has increased by 400 per hour and 10,000 per day. The total number of patients has reached 94.2 million. As high as 9.7%, its harmfulness is second only to cardiovascular and cerebrovascular diseases and tumors. About 3 / 4 of diabetic patients in my country suffer from complications, and their chronic hyperglycemia state is significantly related to long-term complications, which can easily lead to damage, dysfunction and failure of kidneys,...

Claims

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Application Information

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IPC IPC(8): C07K14/62
Inventor 马瑞肖拥军蒋振东曹春来潘太健周翠杨晓森
Owner ZHUHAI UNITED LAB
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