Menopausal gonadotropin with high specific activity as well as preparation method and application thereof

A gonadotropin and menopause technology, applied in the field of protein purification and biomedicine, can solve the problems of inaccessibility, separation, and difficulty in pHMG purification.

Active Publication Date: 2010-10-06
SHANGHAI TECHWELL BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the purification of pHMG is very difficult, because it contains FSH and LH at the same time, and the two components need to be formed in a certain proportion, and the structural properties of FSH and LH are not the same, so it is easy in the later purification process. Separating the two leads to an undesired ratio, which is especially prominent in the purification of pHMG, and there is no report on the method of directly obtaining HMG with high specific activity

Method used

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  • Menopausal gonadotropin with high specific activity as well as preparation method and application thereof
  • Menopausal gonadotropin with high specific activity as well as preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0082] high specific activity menopausal gonadotropin I

[0083] In this example, low specific activity HMG (purchased from Shanghai Tianwei Biopharmaceutical Co., Ltd.) was used as the starting material of pHMG, wherein the biological potency of FSH was 273IU / mg, and the biological potency of LH was 319IU / mg .

[0084] 500 mL of Blue Sepharose FF (purchased from GE Amersham) was loaded into a chromatographic column and equilibrated with a balance solution (0.02M sodium acetate, pH 7.0).

[0085] Dissolve 10 g of low specific activity HMG with 1 L of balance solution, adjust the pH to about 7.0 with HCl or NaOH solution, and then load the sample onto the above-mentioned Blue Sepharose FF chromatography column. After loading, wash 5 times the column volume with balance solution, and then use Wash with 0.05M Glycin-NaOH pH10 for 8 times the column volume, then wash with 0.05M Tris-HCl, 0.4M NaCl, pH9 for 8 times the column volume, and finally elute with 0.05M Tris-HCl, 4M NaCl,...

Embodiment 2

[0094] Preparation of high specific activity menopausal gonadotropin II

[0095] 500 mL of Blue Sepharose 6B (purchased from GE Amersham) was loaded into a chromatographic column and equilibrated with an equilibrium solution (0.02M sodium acetate, pH 7.0).

[0096] Dissolve 6 g of low specific activity HMG from the same source as in Example 1 with 600 mL of balance solution, adjust the pH to about 7.0 with HCl or NaOH solution, and then load it onto the above-mentioned Blue Sepharose 6B chromatography column, and use the balance solution after loading Wash 5 times column volume, then wash 5 times column volume with 0.05M Glycin-NaOH pH10, then wash 5 times column volume with 0.05M Tris-HCl, 0.2M NaCl, pH9, then use 0.05M Tris-HCl, 0.5M NaCl, Wash 7 times the column volume at pH9, and finally elute with 0.05M Tris-HCl, 4M NaCl, pH9, collect the eluate in sections, combine the active ingredients, and concentrate them to 100mL by ultrafiltration with a 10000MW membrane to obtain ...

Embodiment 3

[0105] Preparation of pHMG freeze-dried injection

[0106] An example for the manufacture of 100 vials of pHMG lyophilized injection containing 75IU FSH and 75IU LH per vial is as follows:

[0107] 1.64 mg of high specific activity menopausal gonadotropin I prepared in Example 1 was dissolved in 5 mL of pyrogen-free water for injection, sterile filtered with a 0.22 μm filter, and the filter was washed with pyrogen-free water for injection.

[0108] Dissolve 1 g of lactose in 30 mL of pyrogen-free water for injection, adjust pH to 6.5 ± 0.2 with HCl or NaOH if necessary, and sterile filter with a 0.22 μm filter. Then add it to the above FSH solution, make up to 75 mL with pyrogen-free water for injection, and mix well.

[0109] The above solution was divided into ampoules, 0.75 mL per bottle, and freeze-dried.

[0110] The resulting ampoules each contained approximately 75 IU FSH, 75 IU LH and 10 mg lactose.

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Abstract

The invention discloses menopausal gonadotropin with high specific activity as well as a preparation method and application thereof. The specific activity of the menopausal gonadotropin with high specific activity is not lower than 5000FSH international unit / mg protein. Meanwhile, the invention discloses a method for preparing the menopausal gonadotropin with high specific activity by using dye affinity chromatography.

Description

technical field [0001] The invention relates to the fields of protein purification and biomedicine. Specifically, the present invention relates to high specific activity menopausal gonadotropins and corresponding purification methods. Background technique [0002] Menopausal gonadotropins (Human Menopausal Gonadotropins, referred to as HMG) contain two active ingredients, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are glycoproteins produced by the pituitary gland Hormones are composed of two subunits, an alpha chain and a beta chain. The α subunit of FSH is exactly the same as the α subunit of LH and chorionic gonadotropin (CG), with 92 amino acids and a molecular weight of about 14500D. The asparagine at the 52nd and 78th positions is the N - Glycosylated amino acids. The β subunit of FSH consists of 111 amino acids with a molecular weight of about 18,000 D, and the asparagine at the 7th and 24th positions are N-glycosylated amino acids. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/59C07K1/22A61K38/24A61P15/08
CPCC07K14/59A61K38/24A61P5/06A61P15/08
Inventor 季晓铭高霄梁郭照晔洪云海季斌严惠敏
Owner SHANGHAI TECHWELL BIOPHARMACEUTICALS CO LTD
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