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Purification method of follicle stimulating hormone

A technology of follicle stimulating hormone and luteinizing hormone, which is applied in the field of protein purification and biomedicine, can solve the problems of product impact, FSH inactivation, monoclonal antibody shedding, etc., and achieve the effect of simple operation, mild conditions and high potency

Active Publication Date: 2008-11-19
SHANGHAI TECHWELL BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In the patent WO98 / 20039, it is described to use anti-FSH monoclonal antibody immunochromatography and reverse phase HPLC method to purify FSH, but because its immunochromatography is an adsorption and elution process, the eluent is 1M ammonia water with pH 11 , it is easy to cause the inactivation of FSH and the shedding of monoclonal antibodies during elution, which will have a certain impact on the product

Method used

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  • Purification method of follicle stimulating hormone
  • Purification method of follicle stimulating hormone
  • Purification method of follicle stimulating hormone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Raw materials containing FSH

[0120] In this example, HMG (purchased from Shanghai Tianwei Biopharmaceutical Co., Ltd.) was used as the raw material containing FSH, wherein the biological potency of FSH was 521 IU / mg, and the biological potency of LH was 483 IU / mg.

[0121] Purification method

[0122] Take 10 mL of agarose 4B (purchased from GE Amersham), activate it with divinyl sulfone, suspend it in 10 mL of 0.5 M sodium carbonate solution, add 10 mL of a solution containing anti-hCG monoclonal antibody (purchased from AntibodyShop, HYB-093- 05, 1 mg / mL), so that the antibody is bound to the activated resin, thereby obtaining the immunoresin.

[0123] 10 mL of immunoresin anti-hCG-agarose was loaded into a chromatographic column, and equilibrated with equilibrium solution (0.05M Tris-HCl, 0.2M NaCl, pH 7.2).

[0124] Dissolve 35mg of HMG (of which the biological potency of FSH is 521IU / mg, and the biological potency of LH is 483IU / mg) with 10mL of the above balan...

Embodiment 2

[0135] Raw materials containing FSH

[0136] In this example, HMG (purchased from Shanghai Tianwei Biopharmaceutical Co., Ltd.) was used as the raw material containing FSH, wherein the biological potency of FSH was 2251IU / mg, and the biological potency of LH was 437IU / mg. FSH and LH The ratio of biological potency is 5:1.

[0137] Purification method

[0138]Take 10 mL of agarose 4B (purchased from GE Amersham), activate it with hydrogen bromide, suspend it in 10 mL of 0.1 M sodium bicarbonate solution (pH 8.5), add 10 mL of a solution containing anti-LH monoclonal antibody (purchased from Thermo Fisher, MS-1448-PABX, 1 mg / mL), adjust the pH to about 8.5, so that the antibody therein binds to the activated resin, thereby obtaining the immune resin.

[0139] 10 mL of immunoresin anti-LH-agarose was loaded into a chromatographic column, and equilibrated with equilibrium solution (0.05M Tris-HCl, 0.2M NaCl, pH 7.2).

[0140] 25mg of HMG (wherein the biological potency of FSH i...

Embodiment 3

[0146] Embodiment 3. The preparation method of high-purity urinary gonadotropin (pHMG)

[0147] Prepare 100mL of 0.01M sodium dihydrogen phosphate solution (adjust the pH to about 6.5 with NaOH), add 4g of lactose, stir to dissolve, and filter with a 0.22μm filter. Take 15 mL of the filtrate, add 10 mg of the dried FSH product obtained in Example 2, and then add 5 mg of high-purity LH (LH titer 4592 IU / mg, purchased from Shanghai Tianwei Bio-Pharmaceutical Co., Ltd.), after completely dissolving, carry out vacuum freezing After drying, 570 mg of high-purity HMG (containing lactose as an excipient) was obtained. The specific activity of FSH was measured to be 2358IU / mg protein, and the specific activity of LH was 2114IU / mg protein.

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Abstract

The invention provides a method for preparing follicle-stimulating hormone FSH. The method comprises the steps as follows: (A) a raw material containing the FSH is contacted with an antibody, wherein, the raw material contains luteinizing hormone LH and / or chorionic gonadotropin CG as impurities, and the antibody is an antibody which specifically combines the LH and / or the CG but not crossly react with the FSH basically, thereby making the antibody form an antigen-antibody compound with the LH and / or the CG; (B) the formed antigen-antibody compound is removed from the raw material, thereby obtaining purified FSH. The invention also provides a method for preparing a drug composition containing the FSH.

Description

technical field [0001] The invention relates to the fields of protein purification and biomedicine. Specifically, the present invention relates to a method for purifying follicle stimulating hormone (FSH), 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 14800D; the β subunit of CG ...

Claims

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Application Information

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IPC IPC(8): C07K14/59A61K38/24A61P15/08
Inventor 季晓铭季斌金灿煌严惠敏
Owner SHANGHAI TECHWELL BIOPHARMACEUTICALS CO LTD
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