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Preparation method of insulin derivative

A technology of insulin derivatives and insulin, applied in the field of preparation of insulin derivatives, can solve the problems of loss of reaction products, precipitation of reaction products, affecting product yield, etc., and achieve the effect of increasing product yield and reducing solubility

Active Publication Date: 2016-08-03
GAN&LEE PHARMA
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  • 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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Examples

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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 (500mg, 0.088mmol) to 137.5ml of 50mM / L NaHCO at 10-15°C 3 In the acetonitrile / water mixed solution (acetonitrile:water=1:1), add 3.0ml5%Na again 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, the ...

Embodiment 2

[0049] Embodiment 2 adds 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 2ml ZnCl 2 Aqueous solution (ZnCl 2 The concentration is 4.8mg / mL, Zn 2+ The molar ratio of insulin to insulin is 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 12.0 mg of the residual insulin degludec precursor in the supernatant was measured 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% (12.0mg / 170.7mg) of the control example.

[0052] The above-men...

Embodiment 3

[0054] Embodiment 3 adds 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 2ml ZnCl 2 Aqueous solution (ZnCl 2 The concentration is 12mg / 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 6.0 mg in the supernatant was measured by an external standard method. Compared with Example 1 of the control example, the loss amount of the insulin degludec precursor remaining in the supernatant was 3.5% (6.0mg / 170.7mg) of the control example.

[0057] The above-...

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Abstract

In allusion to the problem of low yield existing by an isoelectric precipitation method during the present preparation process of an insulin derivative, the invention provides a preparation method of an insulin derivative. According to the method, divalent or trivalent metal ions are added into a reaction solution after a coupled reaction between an insulin part and a modified group part. Then, a crude product of an insulin derivative or an insulin derivative precursor can be precipitated out thoroughly from the reaction solution, product loss of residues in a supernatant can decrease to below 7% of loss of a reference substance (without addition of metal ions), and loss of some products even can decrease to below 1% or cannot be detected. According to the invention, product yield is greatly raised.

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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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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