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Preparation method for glycine-lysine-proline insulin crystals

A technology for insulin lispro and insulin, which is applied in the field of preparation of human insulin analog crystals, and can solve the problems of inability to form crystals of insulin lispro, lower safety, lower stability, etc.

Active Publication Date: 2019-07-02
GAN&LEE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, insulin glargine also has some problems in clinical application: 1. The onset of action is slow (the onset time is about 1.5 hours), and patients with hyperglycemia during meals, or patients who occasionally eat more meals, cannot effectively reduce their mealtimes. 2. It cannot be mixed with other insulins, because the isoelectric points of various insulins are different. Insulin glargine is stable under acidic conditions, while natural insulin and existing fast-acting insulin analogs are stable under neutral conditions. If insulin glargine is directly mixed with other insulins, insulin glargine will precipitate before injection and cannot be administered, and other insulins will also be affected by the pH change of the mixture after mixing, resulting in reduced stability and reduced safety.
However, because the isoelectric point of insulin lispro is about 5.3, it is quite different from other insulins. Using the existing insulin crystallization method, insulin lispro cannot be crystallized, only amorphous precipitates can be formed.

Method used

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  • Preparation method for glycine-lysine-proline insulin crystals
  • Preparation method for glycine-lysine-proline insulin crystals
  • Preparation method for glycine-lysine-proline insulin crystals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The preparation of recombinant insulin lispro crystals is carried out in the following four main steps:

[0065] 1) Prepare recombinant insulin lispro crystallization liquid at 25°C, the components and concentrations contained in the crystallization liquid are: recombinant insulin lispro: 15g / L; ethanol: 15%; acetic acid: 1.0M; sodium chloride : 0.3M; Phenol: 0.1% (mass volume ratio).

[0066] The preparation process is as follows: Weigh 1.5g of recombinant insulin lispro dry powder, measure 15ml of ethanol, 5.7ml of acetic acid, 1ml of 10% phenol, add 1.75g ​​of sodium chloride, add water and mix to finally dissolve to 100ml to make a crystallization solution.

[0067] 2) Add 15.69 mg of zinc chloride (0.5% Zn ion) to the crystallization solution in step 1), and adjust the pH to 8.3 with ammonium hydroxide to make the crystallization solution completely clear.

[0068] 3) Then adjust the pH to 7.4 with acetic acid, stir at a speed of 1500 rpm, and adjust the temperatu...

Embodiment 2

[0072] Steps 1)-4) of Example 1 were used to prepare recombinant insulin lispro crystals, except that the amount of Zn ions added in step 2) was 0.1%. After the crystallization was over, the supernatant was taken for HPLC detection, and the content of recombinant insulin lispro in the supernatant was 0.27 mg / ml; the crystal suspension was stirred up and examined under a microscope, and 160 times magnification showed obvious hexahedral crystals with relatively high transparency. high (see figure 2 ); the zinc content of the finally obtained crystalline product is 0.53%; the crystallization yield is 98.2%.

Embodiment 3

[0074] Steps 1)-4) of Example 1 were used to prepare recombinant insulin lispro crystals, except that the amount of Zn ions added in step 2) was 0.8%. After the crystallization was over, the supernatant was taken for HPLC detection, and the content of recombinant insulin lispro in the supernatant was 0.26 mg / ml; the crystal suspension was stirred up and examined under a microscope, and 160 times magnification showed obvious hexahedral crystals with relatively high transparency. high (see image 3 ); the zinc content of the finally obtained crystalline product is 0.81%; the crystallization yield is 98.27%.

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Abstract

The invention discloses a preparation method for glycine-lysine-proline insulin crystals. The method includes the step of adding Zn ions to a crystalline solution containing glycine-lysine-proline insulin in a method of two or more steps for two or more steps of crystallization, wherein according to the mass ratio of Zn to glycine-lysine-proline insulin, the amount of the Zn ions added in the first step of crystallization is 0.1%-0.8%, the amount of the Zn ions added in other steps of crystallization is 0.1% or more, and the total amount of the Zn ions added in all the steps is 0.4% or more. The preparation method can prepare high-quality and high-yield glycine-lysine-proline insulin crystals, and is prone to industrialized production of glycine-lysine-proline insulin.

Description

technical field [0001] The invention relates to a preparation method of human insulin analog crystals, in particular to a preparation method of insulin lispro (GlyA21-LysB28-ProB29-human insulin) 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, and the prevalence rate is as high as 9.7%, its harmfulness is second only to cardiovascular and cerebrovascular diseases and tumors. In addition, about 3 / 4 of diabetic patients in my country suffer from complications. The chronic hyperglycemia state of patients is significantly related to long-term complications, which can eas...

Claims

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Application Information

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IPC IPC(8): C07K14/62C07K1/30
CPCC07K14/62
Inventor 吴文君王大梅
Owner GAN&LEE PHARMA
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