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A kind of preparation method of insulin derivative

A technology of insulin derivatives and insulin, which is applied in the field of preparation of insulin derivatives, can solve the problems of loss of reaction products, affecting product yield, and the final yield of insulin derivatives is only 12%.

Active Publication Date: 2021-06-15
GAN&LEE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this isoelectric precipitation method cannot completely precipitate the reaction product, and there will still be a considerable amount of insulin derivatives or insulin derivative precursors that are not precipitated and remain in the supernatant, resulting in a lot of loss in this step. The reaction product directly affects the final product yield
For example, in Example 4 of patent 200480021733.8, the final yield of its insulin derivatives is only 12%

Method used

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  • A kind of preparation method of insulin derivative
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Preparation of insulin degludec without adding metal ions (comparative example).

[0043] The raw material Des(B30)-human insulin was prepared according to the method described in Example 11 of Chinese Patent No. 94193852.2.

[0044] The raw material tert-butylhexadecanedioyl-Glu(OSu)-OtBu was prepared according to the method described in Example 4 of Chinese Patent 200480021733.8.

[0045] Add des(B30) human insulin (500 mg, 0.088 mmol) to 137.5 ml of 50 mM / L NaHCO at 10-15°C 3 In the mixed solution of acetonitrile / water (acetonitrile:water=1:1), add 3.0ml 5% Na 2 CO 3 (W / V) aqueous solution to adjust the solution to clear (pH=10-11). In addition, tert-butylhexadecanedioyl-Glu(OSu)-OtBu (111mg, 0.176mmol) was dissolved in 5ml of acetonitrile, and then added to the above des(B30) human insulin solution at 10-15°C for coupling reaction . React for 1 hour.

[0046] After the coupling reaction, the pH of the reaction solution was adjusted to 6-7 with HCl, t...

Embodiment 2

[0049] Example 2 Adding Zn 2+ Ion preparation insulin degludec (Zn 2+ The molar ratio to insulin is 0.8:1).

[0050] According to the method of Example 1, des(B30) human insulin and tert-butylhexadecanediyl-Glu(OSu)-OtBu were subjected to a coupling reaction.

[0051] After the coupling reaction, adjust the pH of the reaction solution to 6-7 with HCl, then add 2 ml ZnCl 2 Aqueous solution (ZnCl 2 At a concentration of 4.8 mg / mL, Zn 2+ The molar ratio of insulin to insulin was 0.8:1) and the precipitate was collected by centrifugation and freeze-dried to obtain 566 mg of crude insulin degludec precursor. The centrifuged supernatant was subjected to chromatographic analysis, and the residual insulin degludec precursor in the supernatant was determined to be 12.0 mg by the external standard method. Compared with Example 1 of the control example, the loss of insulin degludec precursor remaining in the supernatant was 7% (12.0 mg / 170.7 mg) of the control example.

[0052] The...

Embodiment 3

[0054] Example 3 Adding Zn 2+ Ion preparation insulin degludec (Zn 2+ The molar ratio to insulin is 2:1).

[0055] According to the method of Example 1, des(B30) human insulin and tert-butylhexadecanediyl-Glu(OSu)-OtBu were subjected to a coupling reaction.

[0056] After the coupling reaction, adjust the pH of the reaction solution to 6-7 with HCl, then add 2 ml ZnCl 2 Aqueous solution (ZnCl 2 At a concentration of 12 mg / mL, Zn 2+ The molar ratio of insulin to insulin is 2:1), and the precipitate was collected by centrifugation and freeze-dried to obtain 569 mg of crude insulin degludec precursor. The centrifuged supernatant was subjected to chromatographic analysis, and the residual insulin degludec precursor in the supernatant was determined to be 6.0 mg by the external standard method. Compared with Example 1 of the control example, the loss of insulin degludec precursor remaining in the supernatant was 3.5% (6.0 mg / 170.7 mg) of the control example.

[0057] The abov...

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Abstract

The present invention aims at the problem of low yield in the isoelectric precipitation method used in the preparation process of existing insulin derivatives, and provides a preparation method of insulin derivatives. After the reaction is completed, divalent or trivalent metal ions are added to the reaction solution, so that the crude product of insulin derivatives or insulin derivative precursors can be completely precipitated from the reaction solution, and the product loss remaining in the supernatant It can be reduced to less than 7% of the control example (without adding metal ions), and some can even be reduced to less than 1% or undetectable, greatly improving the product yield.

Description

technical field [0001] The invention relates to a preparation method of insulin derivatives. Background technique [0002] Diabetes is a common endocrine and metabolic disease. In recent years, the prevalence of diabetes in the world has been increasing. In China, with the change of people's lifestyle and the acceleration of the aging process, the prevalence of diabetes is increasing rapidly. At present, the number of diabetic patients in China has exceeded 1.14 billions, bringing heavy burdens to individuals, families and society. [0003] Diabetic patients generally have relative or absolute insufficient insulin secretion, so insulin therapy has been regarded as an important means to treat diabetes and make blood sugar well controlled. In the 1970s, people developed recombinant human insulin through genetic engineering technology. Later, in order to obtain drugs with different action time curves such as quick-acting or long-acting, people successively developed a series...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/62C07K1/107
Inventor 董长青张一宁孙海瑜邵东王晓霞韩杰丽张愫华蔡莲芝王冲
Owner GAN&LEE PHARMA
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