A method for preparing insulin glargine crystals

An insulin glargine and crystallization technology is applied in the field of preparing insulin glargine crystals, which can solve the problems of short centrifugation time and freeze-drying time, influence the quality of insulin products, and bring risks to the crystallized products, and achieve simple operation, uniform size and shape, easy-to-control effects

Active Publication Date: 2020-11-24
YICHANG HEC CHANGJIANG PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Chinese patent CN102219851B discloses a method for preparing insulin glargine crystals from a crystallization solution containing insulin glargine, organic solvents, zinc, phenol derivatives, salts, and organic acids under the condition of pH 7.0 to 9.0. The prepared crystal has the advantages of stable solid molecular form, easy mass production operation, small sedimentation volume, easy separation from the supernatant, short centrifugation time and freeze-drying time, and high production efficiency. However, there are a large number of organic reagents in the crystallization process. Residues of poor and highly toxic organic solvents will seriously affect the quality of insulin products
This will not only bring risks to the final crystallized product, but also bring a lot of trouble to subsequent operations

Method used

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  • A method for preparing insulin glargine crystals
  • A method for preparing insulin glargine crystals
  • A method for preparing insulin glargine crystals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The insulin glargine crystal liquid is prepared, and the contents of each component in the crystal liquid are: insulin glargine: 2.53 g / L, phenol: 0.1%, zinc chloride: 0.05%, citric acid: 0.5%.

[0036] Weigh 10.11g of insulin glargine and dissolve it in 2L of water to make insulin glargine solution, add 3.99g of phenol, 2.02g of zinc chloride, and 20.11g of citric acid to the crystallization solution, add water to make up to 4L, and make crystallization liquid. Stir the crystallization solution at room temperature at a low speed, adjust the pH value to 5.0 with sodium hydroxide, stir for 4 hours, let it stand at room temperature, and then take the supernatant for testing. The content of insulin glargine in the supernatant is 0.04mg / ml .

[0037] Take the crystal suspension for microscopic examination, and magnify 1000 times to see obvious hexahedral crystals with high transparency ( figure 1 ).

Embodiment 2

[0039] Insulin glargine crystal liquid is prepared, the content of each component in the crystal liquid is: insulin glargine: 2.07g / L, phenol: 0.01%, zinc chloride: 0.007%, citric acid: 0.05%.

[0040] Weigh 2.07g of insulin glargine and dissolve it in 500ml of water to make insulin glargine solution, add 2ml of 5% phenol, 1.75ml of 4% zinc chloride, and 0.5g of citric acid to the crystallization solution, add water to make up to 1L, Made into a crystallization solution. Stir the crystallization solution at room temperature at a low speed, adjust the pH to 6.8 with sodium hydroxide, stir for 6 hours, let it stand at room temperature, and then remove the supernatant for testing. The content of insulin glargine in the supernatant is 0.01 mg / ml.

[0041] Take the crystal suspension for microscopic examination, magnification of 400 times shows obvious crystals, and the transparency is higher ( figure 2 ).

Embodiment 3

[0043] Insulin glargine crystal liquid was prepared, and the content of each component in the crystal liquid was: insulin glargine: 3.17g / L, phenol: 0.7%, zinc chloride: 1.5%, citric acid: 1.8%.

[0044] Weigh 6.34g of insulin glargine and dissolve it in 1L of water to make an insulin glargine solution. Add 14g of phenol, 30g of zinc chloride, and 36g of citric acid to the crystallization solution, add water to make the volume to 2L, and make a crystallization solution. Stir the crystallization solution at room temperature at a low speed, adjust the pH to 7.0 with sodium hydroxide, stir for 9 hours, let it stand at room temperature, and then remove the supernatant for detection. The content of insulin glargine in the supernatant is 0.04 mg / ml.

[0045] Take the crystal suspension for microscopic examination, and magnify 400 times to see obvious crystals, and the transparency is higher ( image 3 ).

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Abstract

The invention relates to a method for preparing a recombinant glycine-arginine insulin crystal. The method specifically comprises the following steps of (1) dissolving recombinant glycine-arginine insulin, a phenol derivative, a zinc-containing substance and organic acid in water so as to obtain crystal fluid; and (2) taking a pH regulating agent for regulating the pH value of the crystal fluid to 4.5-7.0, performing stirring for 3-9 hours, and performing separation so as to obtain the recombinant glycine-arginine insulin crystal, wherein the concentration of the recombinant glycine-arginine insulin in the crystal fluid is 1-3.5g / L, the mass / volume percentage of the phenol derivative is 0.01-1.00%, the mass / volume percentage of the zinc-containing substance is 0.005-1.5%, and the mass / volume percentage of the organic acid is 0.01-2.0%. The crystal fluid prepared by the method does not contain organic solvents, the operation process is safe, the technology is simple and easy to control, poisonous and harmful substances in chromatography in the previous step can be effectively removed, the time for filtration, collection, crystal washing and freeze drying of products is shortened, the cost is reduced, and the method is suitable for industrialized production of the recombinant glycine-arginine insulin.

Description

technical field [0001] The present invention relates to the field of crystallization of insulin. Specifically relates to a method for preparing insulin glargine crystals. Background technique [0002] Insulin is a protein hormone secreted by pancreatic β cells stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, and glucagon. It was first discovered by Canadians F.G. Banting and C.H. Best in 1921. It began to be used clinically in 1922 to save the diabetic patients who died in the past. In recent years, the prevalence of diabetes in the world has been increasing rapidly, and the demand for insulin is increasing. Simple extraction from animal cells can no longer meet the needs of patients, and the production of insulin and insulin analogues is imminent. One of the newly developed long-acting insulin analogues, insulin glargine, is being increasingly accepted and used by doctors and patients. [0003] Insulin glargine (recombinant gl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/62C07K1/30
CPCC07K14/62C07K2299/00
Inventor 林小鹊章琛李平珠江武陈小锋李文佳
Owner YICHANG HEC CHANGJIANG PHARMA CO LTD
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