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Method for preparing recombinant human insulin crystal

A technology of recombinant human insulin and insulin, which is applied in the field of protein crystallization to achieve the effect of short freeze-drying time, large particles and fast sedimentation speed

Inactive Publication Date: 2016-07-13
TONGHUA DONGBAO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The market for insulin and insulin analogs is huge, and there is no report on the successful crystallization of insulin or insulin analogs without the addition of phenolic substances and organic solvents during the crystallization process

Method used

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  • Method for preparing recombinant human insulin crystal
  • Method for preparing recombinant human insulin crystal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1) Add zinc chloride solution to the reverse high-efficiency chromatography solution containing recombinant human insulin, so that every OD 280 nm of recombinant human insulin makes each OD 280 nm recombinant human insulin contains zinc ions at a concentration of 14 mg / ml, add low-temperature purified water, and when the concentration of the insulin solution is diluted to 4 mg / ml, turn on the stirrer switch and stir;

[0044]2) Add 3-5M ammonia water to the insulin solution in step 1) to adjust the precipitation to promote precipitation until the pH is between 7.0, stop stirring; transfer the primary crystallization solution to a crystallization tank and let it settle. , Determination of supernatant OD 280 When nm is less than or equal to 0.1, remove the supernatant and obtain a crystal;

[0045] 3) Transfer the first knot crystal to the second knot settling tank, add 1-3M acetic acid to the second knot settling tank to dissolve, turn on the stirrer, and stop adding a...

Embodiment 2

[0054] 1) Add zinc chloride solution to the reverse high-efficiency chromatography solution containing recombinant human insulin, so that every OD 280 nm of recombinant human insulin makes each OD 280 nm recombinant human insulin contains zinc ions at a concentration of 46 mg / ml, add low-temperature purified water, and when the concentration of the insulin solution is diluted to 0.5 mg / ml, turn on the stirrer switch and stir;

[0055] 2) Add 3-5M ammonia water to the insulin solution in step 1) to adjust the precipitation and promote precipitation until the pH is between 5.5, stop stirring; transfer the primary crystallization solution to a crystallization tank and let it settle. , Determination of supernatant OD 280 When nm is less than or equal to 0.1, remove the supernatant and obtain a crystal;

[0056] 3) Transfer the first knot crystal to the second knot settling tank, add 1-3M acetic acid to the second knot settling tank to dissolve, turn on the stirrer, and stop stir...

Embodiment 3

[0064] 1) Add zinc chloride solution to the reverse high-efficiency chromatography solution containing recombinant human insulin, so that every OD 280 nm of recombinant human insulin makes each OD 280 nm recombinant human insulin contains zinc ions at a concentration of 55 mg / ml, add low-temperature purified water, and when the concentration of the insulin solution is diluted to 6 mg / ml, turn on the stirrer switch and stir;

[0065] 2) Add 3-5M ammonia water to the insulin solution in step 1) to adjust the precipitation to promote precipitation until the pH is between 7.5, stop stirring; transfer the primary crystallization solution to a crystallization tank and let it settle. , Determination of supernatant OD 280 When nm is less than or equal to 0.1, remove the supernatant and obtain a crystal;

[0066] 3) Transfer the first knot crystal to the second knot settling tank, add 1-3M acetic acid to the second knot settling tank to dissolve, turn on the stirrer, and stop stirrin...

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Abstract

The invention provides a method for preparing recombinant human insulin crystal, and belongs to the technical field of protein crystallization. According to the method, zinc ions are subjected to three times of crystallization, the obtained insulin crystal is large in particle size and good in homogeneity, phenols or organic solvents used in normal methods are not used in the method, so that residues of harmful substances in the product are avoided and the quality of the insulin crystal is improved; moreover, the method is stable in preparation process and beneficial to industrial production.

Description

technical field [0001] The invention belongs to a method for preparing recombinant human insulin crystals, in particular to a method for preparing recombinant human insulin tertiary crystals, and belongs to the technical field of protein crystallization. Background technique [0002] Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia caused by various etiologies. It is a syndrome caused by the absolute or relative lack of insulin in the body or the insensitivity of target tissues to insulin. In recent years, with the improvement of people's living standards, the incidence of diabetes has been increasing year by year, becoming the third leading cause of death after cardiovascular and tumors. The occurrence of diabetes and its complications not only brings great suffering to patients, but also brings a heavy economic burden to individuals, families and society. The use of insulin is an important means for diabetic patients to achieve blood sugar ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C07K1/30
CPCC07K14/62C07K2299/00
Inventor 冷春生李树鹏孟祥成贾冰杨旭常晓慧
Owner TONGHUA DONGBAO PHARMA
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