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Urine follicle-stimulating hormone with high specific activity and method for preparing same

A follicle-stimulating hormone and high ratio technology, which is applied in the field of protein purification and biomedicine, and can solve the problems such as the unsatisfactory specific activity of FSH

Active Publication Date: 2013-03-27
SHANGHAI TECHWELL BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The current preparation method of urine-derived FSH is mainly based on low-purity urinary follicle-stimulating hormone or menopausal gonadotropin (HMG) as the starting material, through hydrophobic chromatography, or monoclonal antibody immunochromatography and reversed-phase HPLC. Purify, but the specific activity of the FSH that obtains is not very ideal yet, as in patent WO98 / 20039, have described to purify FSH with anti-FSH monoclonal antibody immunochromatography and reverse phase HPLC method, the specific activity of the FSH that obtains 6200IU / mg

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  • Urine follicle-stimulating hormone with high specific activity and method for preparing same

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preparation example Construction

[0045] The preparation method of the high specific activity urea-derived FSH provided by the invention comprises steps:

[0046] The low-purity urea-derived follicle-stimulating hormone or menopausal gonadotropin (HMG) is chromatographically purified to obtain high specific activity urea-derived FSH; the chromatogram is cation exchange chromatography and dye affinity chromatography.

[0047] Preferably, the method comprises the steps of:

[0048] (1) purifying solution 1 containing low-purity FSH through cation exchange chromatography to obtain distillate 1; and

[0049] (2) The solution 2 containing distillate 1 was purified by dye affinity chromatography to obtain high specific activity urinary follicle-stimulating hormone (pFSH).

[0050] The skeleton medium of the resin of the cation-exchange chromatography used in the preparation method of the present invention comprises the cross-linked product of agarose, dextran, cellulose, styrene, acrylic acid and / or derivative; The...

Embodiment 1

[0090] Preparation of High Specific Activity Urofollicle Stimulating Hormone I

[0091] Low-purity urinary follicle-stimulating hormone (purchased from Shanghai Tianwei Biopharmaceutical Co., Ltd.) was used as the starting material, wherein the biological potency of FSH was 315 IU / mg, and the biological potency of LH was ≤3 IU / mg.

[0092] Dissolve 10 g of the above-mentioned low-purity urinary follicle-stimulating hormone with 300 mL of equilibrium solution (0.03 M sodium dihydrogen phosphate, pH 5), and then put it on a 250 mL CM-Sepharose chromatography column (provided by Amersham), which has been previously used with the same equilibrium solution Well balanced. After sample loading, wash 10 times column volume with washing solution (0.1M sodium acetate, pH5), then carry out 0-100% linear gradient elution with washing solution and eluent (0.1M sodium acetate+1M NaCl, pH5) ( The volume ratio concentration of the eluent, within 2 hours), monitor the 280nm place with an ultrav...

Embodiment 2

[0098] Preparation of High Specific Activity Urofollicle Stimulating Hormone II

[0099] Dissolve 5 g of low-purity urinary follicle-stimulating hormone (the starting material is the same as in Example 1) with 150 mL of equilibrium solution (0.03 M sodium dihydrogen phosphate, pH 4.8), and then put it on a 130 mL SP-Sepharose chromatography column (provided by Amersham) , this column has been equilibrated with the same equilibrium solution in advance. After sample loading, wash 10 times the column volume with washing solution (0.1M sodium acetate, pH4.8), then perform 0-100% linear gradient washing with washing solution and eluent (0.1M sodium acetate+1M NaCl, pH5) Remove (the volume ratio concentration of the eluent, within 2 hours), monitor the 280nm place with an ultraviolet detector, distribute and collect each distillate peak, detect its FSH immune titer, combine about 0.2L of active ingredients, add pre-cooled non-toxic Precipitate with water and ethanol overnight, coll...

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Abstract

A follicle-stimulating hormone(FSH) with high specific activity and preparation method thereof are disclosed, wherein specific activity of the follicle-stimulating hormone is not less than 7000 international unit (IU) / mg protein. The method for preparing the follicle-stimulating hormone with high specific activity comprises using cation exchange chromatography(CEX) to purify low-purity follicle-stimulating hormone, and using dye affinity chromatography to purify FSH after the CEX step sequentially. Thereby the follicle-stimulating hormone with high specific activity is obtained.

Description

technical field [0001] The invention relates to the fields of protein purification and biomedicine. Specifically, the present invention relates to highly active follicle-stimulating hormone (FSH), a preparation method thereof, and a pharmaceutical composition containing it. Background technique [0002] Follicle-stimulating hormone (FSH) is a hormone produced by the pituitary gland, which consists of two subunits, the α chain and the β chain. The α subunit of FSH is exactly the same as the α subunit of luteinizing hormone (LH) and chorionic gonadotropin (CG), with 92 amino acids and a molecular weight of about 14500D, at positions 52 and 78 The asparagine on is an amino acid that undergoes N-glycosylation. [0003] 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 β subunit of LH consists of 121 amino acids with a molecular weight of about 14800 D...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/59C07K1/16A61K38/24A61P15/08
CPCC07K14/59A61K38/00A61P15/08
Inventor 季晓铭高霄梁季斌严惠敏洪云海
Owner SHANGHAI TECHWELL BIOPHARMACEUTICALS CO LTD