CN101519445A - Urine follicle-stimulating hormone with high specific activity and method for preparing same - Google Patents
Urine follicle-stimulating hormone with high specific activity and method for preparing same Download PDFInfo
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- CN101519445A CN101519445A CN200910048954A CN200910048954A CN101519445A CN 101519445 A CN101519445 A CN 101519445A CN 200910048954 A CN200910048954 A CN 200910048954A CN 200910048954 A CN200910048954 A CN 200910048954A CN 101519445 A CN101519445 A CN 101519445A
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- stimulating hormone
- specific activity
- follicle
- high specific
- resin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses a urine follicle-stimulating hormone with high specific activity. The specific activity of the hormone is not lower than 7,000 international units/mg protein. Simultaneously, the invention discloses a method for preparing the urine follicle-stimulating hormone with high specific activity.
Description
Technical field
The present invention relates to protein purification and biomedicine field.Particularly, the present invention relates to highly active follicular stimulating hormone (FSH) and preparation method thereof, and the pharmaceutical composition that contains it.
Background technology
Follicular stimulating hormone (Follicle-stimulating hormone is called for short FSH) is the hormone that is produced by hypophysis, and it is made up of α chain and two subunits of β chain.The α subunit of FSH and luteotropic hormone (leuteinizing hormone, be called for short LH) and chorionic gona dotropin (chorionicgonadotropin, abbreviation CG) α subunit is identical, have 92 amino acid, molecular weight is about 14500D, and the 52nd and 78 locational l-asparagines are that the glycosylated amino acid of N-takes place.
The β subunit of FSH is made up of 111 amino acid, and molecular weight is about 18000D, and wherein the 7th and 24 locational l-asparagines are that the glycosylated amino acid of N-takes place.And the β subunit of LH is made up of 121 amino acid, and molecular weight is about 14800D; The β subunit of CG then has 145 amino acid, molecular weight 22000-39000D.
FSH is mainly used in treatment infertility and external supplementary reproduction clinically.FSH can extract from the urine of menopausal women, also can prepare by the DNA recombinant technology.
The first-generation product that contains FSH is Menotropins (HMG), and as the Pergonal of Serono company, it is that FSH and LH ratio are about 1 mixture.But, need not add patient with exogenous LH for the LH that more amount is arranged in the body, the too high normal development that can influence ovarian follicle of LH level, inopportune inhibition meiosis inhibitory factor can cause the aging of ovum, is fertilized and the chance of implantation thereby reduce.Therefore for this part patient, be more suitable in using pure FSH preparation.On the other hand, too much LH causes polycystic ovary syndrome easily (Polycystic Ovarian Syndrome POOS), studies show that LH too much has disadvantageous effect to reproductive function, as causes hypomenorrhea, anovulation, infertile and miscarriage.Therefore more safer than HMG to POOS patient with pure FSH treatment, can reduce ovarian hyperstimulation syndrome (OvarianHyperstimulation Syndrome, OHSS) danger.The Metrodin that Serono company releases is exactly a kind of FSH preparation that contains minute quantity LH, is fit to POOS patient's treatment.
But Metrodin has removed LH, but has kept the foreign protein (foreign protein accounts for 80-90%) in a lot of urine source, and the existence of these foreign proteins may cause certain side effect, as anaphylaxis.Therefore just tending to exploitation and using highly purified FSH on the market in recent years, it is that FSH with low-purity carries out further purifying, remove most foreign proteins, obtain the FSH that height ratio is lived, it can overcome the anaphylaxis to human body that causes because of a large amount of foreign proteins in the usual production, and because these advantages make it can adopt subcutaneous injection, make things convenient for patient's use, palliate the agonizing sufferings.
The preparation method of present urine source FSH mainly is to be starting raw material with low-purity urinary follicle stimulating hormone or gonadotropin in climacteric (HMG), pass through hydrophobic chromatography, or monoclone antibody immune chromatography method and rp-hplc method come purifying, but the ratio work of the FSH that obtains also is not very good, as in patent WO98/20039, monoclone antibody immune chromatography method and the rp-hplc method described with anti-FSH come purifying FSH, and the ratio of the FSH of acquisition is lived and is 6200IU/mg.
Therefore, this area presses for develops the FSH that a kind of height ratio is lived, and corresponding preparation method, and obtains to contain the preparation of high reactivity FSH thus, can be used in subcutaneous injection, make things convenient for the patient use, palliate the agonizing sufferings.
Summary of the invention
The present invention aims to provide a kind of urine follicle-stimulating hormone with high specific activity.
Another object of the present invention provides the preparation method of described urine follicle-stimulating hormone with high specific activity.
The 3rd purpose of the present invention provides the pharmaceutical composition that contains described urine follicle-stimulating hormone with high specific activity.
The 4th purpose of the present invention provides the purposes of described urine follicle-stimulating hormone with high specific activity.
In a first aspect of the present invention, provide a kind of urine follicle-stimulating hormone with high specific activity (pFSH), the ratio of described urine follicle-stimulating hormone with high specific activity (pFSH) 〉=7000 international unit alive/mg albumen.
In another preference, the ratio of described urine follicle-stimulating hormone with high specific activity (pFSH) 〉=8000 international unit alive/mg albumen; More preferably, the ratio of described urine follicle-stimulating hormone with high specific activity (pFSH) 〉=8500 international unit alive/mg albumen.
In another preference, the ratio work of described urine follicle-stimulating hormone with high specific activity is 7000-15000 international unit/mg albumen; More preferably, be 8000-12000 international unit/mg albumen.
In another preference, described follicular stimulating hormone is follicular stimulating hormone or its variant that the people urinates the source.
In a second aspect of the present invention, a kind of preparation method of aforesaid urine follicle-stimulating hormone with high specific activity is provided, described method comprises step:
The low-purity urinary follicle stimulating hormone through chromatogram purification, is obtained urine follicle-stimulating hormone with high specific activity;
Described chromatogram is a dye affinity chromatography.
In another preference, described chromatogram also comprises cation-exchange chromatography.
In another preference, described method comprises step:
(a) solution 1 that will contain the low-purity urinary follicle stimulating hormone obtains overhead product 1 through the cation-exchange chromatography purifying; With
(b) solution 2 that will contain overhead product 1 obtains urine follicle-stimulating hormone with high specific activity through the dye affinity chromatography purifying.
In another preference, the resin resin matrix medium of described cation-exchange chromatography comprises agarose, dextran, Mierocrystalline cellulose, the cross-linking agent of vinylbenzene and Vinylstyrene, the cross-linking agent of vinylformic acid and/or its derivative.
In another preference, the resin resin active group of described cation-exchange chromatography is selected from sulfonic acid propyl group (SO
3H), methyne sulfonic group (CH
2SO
3H), carboxyl (COOH), carboxymethyl (OCH
2COOH) or phenylol (C
6H
5OH).
In another preference, the active group of the resin of described cation-exchange chromatography is selected from sulfonic acid propyl group or methyne sulfonic group.
In another preference, the resin resin of described cation-exchange chromatography is SP Sepharose or CMSepharose.
In another preference, the dyestuff aglucon of the resin resin of described dye affinity chromatography is selected from CibacronBlue, Orange, Red, Green.
In another preference, the solid phase carrier of the resin resin of described dye affinity chromatography is selected from bentonite, glass microsphere, quartzy microballoon, hydroxyl calcium phosphate, aluminum oxide, polyacrylamide gel, starch gel, dextrane gel, Mierocrystalline cellulose or agarose.
In another preference, the resin resin of described dye affinity chromatography is Blue Sepharose 6B or BlueSepharose FF.
In another preference, step (a) or (b) carry out 1-3 times.
In a third aspect of the present invention, a kind of pharmaceutical composition is provided, contain the aforesaid urine follicle-stimulating hormone with high specific activity and the pharmaceutically acceptable carrier for the treatment of significant quantity in the described composition.
In a fourth aspect of the present invention, provide the purposes of a kind of aforesaid urine follicle-stimulating hormone with high specific activity in the medicine of the sterile syndromes of preparation treatment.
In view of the above, the invention provides the FSH that a kind of height ratio is lived, and corresponding preparation method, and obtain to contain the preparation of high reactivity FSH thus, can be used in subcutaneous injection, make things convenient for the patient use, palliate the agonizing sufferings.
Embodiment
The contriver is through extensive and deep research, is surprised to find by cation exchange resin chromatography and the affine resin chromatography of dyestuff and can obtains the FSH that at present known height ratio is lived.
Particularly, the present invention is to be starting raw material with urine or HMG or low-purity urinary follicle stimulating hormone, by effective cation exchange resin chromatography and the affine resin chromatography purification step of dyestuff, obtains the FSH that height ratio is lived, purification process is simply effective, and the FSH of acquisition is higher than living, impurity lacks.
As used herein, term " follicular stimulating hormone " and " FSH " are used interchangeably, and refer to that a class is used to promote that sperm or ovarian follicle produce, promote hormone or its variant of the development of ovary, and it can be secreted by prepituitary gland under natural situation.
As used herein, " urinary follicle stimulating hormone " and " urine source property FSH " is used interchangeably, and is meant the follicular stimulating hormone that extraction obtains from urine, and described urine comes from Mammals, preferably comes from the people, more preferably is the urine of menopausal women.
Raw material low-purity urine source property FSH used among the present invention can adopt this area any way commonly used to obtain, for example can with traditional method from the menopausal women urine by kaolin absorption, wash-out, acetone precipitation, the ethanolic soln extracting, ion exchange chromatography chromatogram (comprising positively charged ion, anion chromatographic), even hydrophobic chromatography obtains HMG, again HMG is obtained low-purity urine source property FSH by hydrophobic chromatography or the conventional meanses such as affinity chromatography by anti-LH antibody and/or anti-CG antibody, as the described method of CN101307103A.。Ratio general be lower than 2000IU/mg albumen, the preferred 200-500IU/mg albumen alive of low-purity urine source property FSH.
The low-purity urine source property FSH raw material that can be used among the present invention can be: the raw material of HMG being removed LH through the prepurification step, or by traditional extraction process from the menopausal women urine by kaolin absorption, wash-out, acetone precipitation, the ethanolic soln extracting, ion exchange chromatography chromatogram (comprising positively charged ion, anion chromatographic), and then the raw material that only contains the foreign protein of FSH and other non-LH basically that obtains by hydrophobic chromatography or the ordinary methods such as affinity chromatography by anti-LH antibody and/or anti-CG antibody.
Preferably before adopting method purifying low-purity urine source property FSH of the present invention, adopt this area ordinary method that raw material low-purity urine source property FSH is carried out preliminary purification, to separate other impurity except that LH.
HMG can obtain by traditional method, as from the menopausal women urine by kaolin absorption, wash-out, acetone precipitation, ethanolic soln extracting, ion exchange chromatography chromatogram (comprising positively charged ion, anion chromatographic), even hydrophobic chromatography.Then HMG is carried out follow-up purifying, can obtain highly active FSH thereby be prepared by this patent disclosed method then with hydrophobic chromatography or removal LH wherein.
As used herein, term " luteotropic hormone " and " LH " are used interchangeably, refer in feedstock production that contains FSH or acquisition process, be doped in wherein have the hormone of same or similar 26S Proteasome Structure and Function with natural LH.
Ratio work 〉=7000IU/mg the albumen of urine follicle-stimulating hormone with high specific activity provided by the invention, preferred 〉=8500IU/mg albumen.The biological value of LH is generally less than 1LHIU/50 FSH IU among the height ratio provided by the invention urine source property FSH alive, more preferably, and less than 1LH IU/100 FSH IU.
The preparation method of height ratio provided by the invention urine source property FSH alive comprises step:
Low-purity is urinated source gamogenetic egg bubble yield stimulant or gonadotropin in climacteric (HMG) process chromatogram purification, obtain height ratio urine source property FSH alive; Described chromatogram is cation-exchange chromatography and dye affinity chromatography.
Preferably, described method comprises step:
(1) solution 1 that will contain low-purity FSH obtains overhead product 1 through the cation-exchange chromatography purifying; With
(2) solution 2 that will contain overhead product 1 obtains urine follicle-stimulating hormone with high specific activity (pFSH) through the dye affinity chromatography purifying.
The skeleton medium of the resin of the cation-exchange chromatography that uses among the preparation method of the present invention comprises the cross-linking agent of agarose, dextran, Mierocrystalline cellulose, vinylbenzene, vinylformic acid and/or derivative; The active group of the resin of described cation-exchange chromatography is selected from sulfonic acid propyl group (SO
3H), methyne sulfonic group (CH
2SO
3H), carboxyl (COOH), carboxymethyl (OCH
2COOH) or phenylol (C
6H
5OH), preferably be selected from sulfonic acid propyl group or methyne sulfonic group; The resin of described cation-exchange chromatography is SP Sepharose or CM Sepharose.
The dyestuff aglucon of the resin of the dye affinity chromatography that uses among the preparation method of the present invention is selected from Cibacron Blue, Orange, Red, Green; The solid phase carrier of the resin of described dye affinity chromatography is selected from bentonite, glass microsphere, quartzy microballoon, hydroxyl calcium phosphate, aluminum oxide, polyacrylamide gel, starch gel, dextrane gel, Mierocrystalline cellulose or agarose; The resin of described dye affinity chromatography is Blue Sepharose.
In a preference of the present invention, described cation-exchange chromatography purification step comprises:
(i) first sample solution that contains the low-purity urinary follicle stimulating hormone with pH4-7 is gone up sample, and the concentration of low-purity urinary follicle stimulating hormone is 1.0-5.0w/v% (g/ml) in described first sample solution, preferred 2.0-4.0w/v% (g/ml);
(ii) use first washings of pH4-6 to wash; With
(iii) use first washings of pH4-6 and first elutriant of pH4-6 to carry out wash-out, obtain overhead product 1.
More preferably, step is carried out gradient elution in (iii), in 0.5 to 5 hour, and the concentration of first elutriant (v/v) from 0 to 100%.
The salt concn of described first sample solution is 0-0.5M, and described salt is selected from hydrochloride, phosphoric acid salt and/or acetate; The salt concn of described first washings or first elutriant is 0.05-3M, and described salt is selected from hydrochloride, phosphoric acid salt and/or acetate.The metal ion of described salt is selected from sodium ion or potassium ion.
In a preference of the present invention, described dye affinity chromatography purification step comprises:
(i ') goes up sample with second sample solution that pH5-7 contains overhead product 1; The concentration of overhead product 1 is 0.5-2.0w/v% (g/ml) in described second sample solution
(ii ') washs with second washings of pH8-11; With
(iii ') carries out wash-out with second elutriant of pH8-11, obtains urine follicle-stimulating hormone with high specific activity.
The salt concn of described second sample solution is 0-0.05M, and described salt is selected from hydrochloride, phosphoric acid salt and/or acetate; The salt concn of described second washings or second elutriant is 0.01-5M, and described salt is selected from hydrochloride, phosphoric acid salt and/or acetate and/or glycinate.The metal ion of described salt is selected from sodium ion or potassium ion.
The present invention also provides a kind of pharmaceutical composition, and described pharmaceutical composition contains the urine source property FSH alive of the height ratio with the inventive method preparation of treatment significant quantity, and pharmaceutically acceptable carrier.
As used herein, term " contain " or " comprising " comprised " comprising ", " basically by ... constitute " and " by ... constitute ".As used herein, term " treatment significant quantity " is meant and can produces function or amount active and that can be accepted by people and/or animal to people and/or animal.
As used herein, the composition of term " pharmaceutically acceptable " or " acceptable on the bromatology " is applicable to people and/or animal and does not have excessive bad side reaction (as toxicity, stimulation and transformation reactions), the material of rational benefit/risk ratio is promptly arranged.
As used herein, term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various vehicle and thinner.This term refers to some medicament carriers like this: they itself are not necessary activeconstituents, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art." Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences, Mack Pub.Co. can find discussing fully about pharmaceutically acceptable vehicle in N.J.1991).
Described " pharmaceutically acceptable carrier " can contain liquid, as water, salt solution, glycerine and ethanol.In addition, also may there be complementary material in these carriers, as weighting agent, disintegrating agent, lubricant, glidant, effervescent, wetting agent or emulsifying agent, correctives, pH buffer substance etc.Usually, these materials can be formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably, pH is about 6-8.
In preferred implementation of the present invention, the urine source property FSH alive of the height ratio in the described pharmaceutical composition accounts for 0.001-99.9wt% of composition total weight; Being preferably 0.01-99wt% of composition total weight, more preferably is 0.02-95wt%, more preferably 0.05-90wt%.Surplus is materials such as pharmaceutically acceptable carrier and other additive.
The effective dose that should be understood that used FSH can change with the severity of object to be administered or treatment.Particular case decides according to the individual instances (for example object body weight, age, physical appearance, the required effect that reaches) of object, and this is in the scope that skilled practitioners or nutritionist can judge.
Pharmaceutical composition of the present invention can be solid-state (as granule, tablet, lyophilized powder, suppository, capsule, sublingual lozenge) or liquid (as oral liquid, injection) or other suitable shape; Preferred powder injection or aqueous injection.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can arbitrary combination.All features that this case specification sheets is disclosed can with any composition forms and usefulness, each feature that is disclosed in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore removing has special instruction, and the feature that is disclosed only is the general example of equalization or similar features.
Major advantage of the present invention is:
1, provides a kind of highly active urine source property FSH, can be applicable to subcutaneous injection;
2, the purification process of a kind of high reactivity urine source property FSH is provided, and method is simply effective, is fit to industrialization.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio, ratio, ratio or umber by weight.
Unit in the percent weight in volume among the present invention is well-known to those skilled in the art, for example is meant the weight of solute in 100 milliliters solution.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The measuring method that biological value that the present invention relates to and ratio are lived:
Biological value
The estimation of biological potency method of FSH, LH is pressed the method check of 2005 editions appendix XII of Chinese Pharmacopoeia M, XII N.
FSH is than living
Measure (albumen optical spectroscopy) mensuration as follows:
Protein content: precision takes by weighing the about 2mg of this product, and precision adds water 5ml dissolving, is need testing solution.Precision takes by weighing the bovine serum albumin reference substance in addition, be dissolved in water and make every 1ml and contain 1.0mg, 0.8mg, 0.6mg, 0.4mg, 0.2mg, the solution of 0mg is measured the absorbancy at 280nm place respectively according to ultraviolet spectrophotometry (Chinese Pharmacopoeia version appendix in 2005 IV A), is X-coordinate with the concentration of bovine serum albumin solution, optical density at 280nm is an ordinate zou drawing standard curve, and it is linear that absorbancy and concentration relationship should be.Try to achieve the protein concentration of need testing solution from typical curve.
FSH is than living:
Be calculated as follows than work:
In the formula, C is the concentration (mg/ml) of need testing solution
Wherein, the mg in the trial-product biological value unit is meant the quality of the trial-product that takes by weighing.
Embodiment 1
Preparation urine follicle-stimulating hormone with high specific activity I
As starting raw material, wherein the biological value of FSH is 315IU/mg with low-purity urinary follicle stimulating hormone (available from Shanghai Tianwei Biological Pharmaceutical Corp.), and the biological value of LH is≤3IU/mg.
(the 0.03M SODIUM PHOSPHATE, MONOBASIC pH5) is dissolved, and goes up then to 250mL CM-Sepharose chromatography column (Amersham provides), and this post has used identical balance liquid balance good in advance with the 300mL balance liquid with the above-mentioned low-purity urinary follicle stimulating hormone of 10g.Use washings (0.1M sodium-acetate behind the end of the sample, pH5) 10 times of column volumes of washing, use washings and elutriant (0.1M sodium-acetate+1M NaCl then, pH5) carry out 0-100% linear gradient elution (volume by volume concentration of elutriant, in 2 hours), with UV-detector monitoring 280nm place, distribute to collect and respectively distillate the peak, detect its FSH immunizing potency, be associated with the about 0.4L of effective constituent, the dehydrated alcohol precipitation that adds precooling is spent the night, next day centrifugal collecting precipitation, with the dehydrated alcohol dehydration, vacuum-drying obtains 1.8 gram dry products.
With the above-mentioned dry product of 1.8g 200mL balance liquid (0.01M SODIUM PHOSPHATE, MONOBASIC, pH6.5) dissolving, go up then to 300mL Blue Sepharose FF chromatography column (Amersham provides), wash 5 times of column volumes with balance liquid behind the end of the sample, use 5 times of column volumes of 0.05M glycine buffer (pH10) washing then, use the 0.05M glycine again, 0.4M 8 times of column volumes of NaCl damping fluid (pH9) washing, use elutriant (0.05M glycine at last, 2.5M NaCl, pH9) wash-out, distributing to collect respectively distillates the peak, detects its FSH immunizing potency, is associated with the about 2L of effective constituent, after concentrating with 10,000 molecular weight membrane ultrafiltration, add the dehydrated alcohol of precooling, centrifugal collecting precipitation dewaters with dehydrated alcohol, vacuum-drying gets 266mg dry product I, i.e. urine follicle-stimulating hormone with high specific activity I.
The biological value of table 1 gained dry product and than measurement result alive
| FSH biological value (IU/mg) | FSH is than live (IU/mg albumen) | LH biological value (IU/mg) | |
| Dry product I behind the purifying | 9209 | 9352 | <1LH?IU/100FSH?IU |
The above results shows: adopt method of the present invention to carry out purifying, can obtain very height ratio FSH alive.
Embodiment 2
Preparation urine follicle-stimulating hormone with high specific activity II
With 5g low-purity urinary follicle stimulating hormone (starting raw material is with embodiment 1) 150mL balance liquid (0.03M SODIUM PHOSPHATE, MONOBASIC, pH4.8) dissolving, go up then to 130mL SP-Sepharose chromatography column (Amersham provides), this post has used identical balance liquid balance good in advance.Use washings (0.1M sodium-acetate behind the end of the sample, pH4.8) 10 times of column volumes of washing, use washings and elutriant (0.1M sodium-acetate+1M NaCl then, pH5) carry out 0-100% linear gradient elution (volume by volume concentration of elutriant, in 2 hours), with UV-detector monitoring 280nm place, distribute to collect and respectively distillate the peak, detect its FSH immunizing potency, be associated with the about 0.2L of effective constituent, the dehydrated alcohol precipitation that adds precooling is spent the night, next day centrifugal collecting precipitation, with the dehydrated alcohol dehydration, vacuum-drying obtains 0.93 gram dry product.
With the above-mentioned dry product of 0.93g 100mL balance liquid (0.01M SODIUM PHOSPHATE, MONOBASIC, pH6.5) dissolving, go up then to 200mL Blue Sepharose 6B chromatography column (Amersham provides), wash 5 times of column volumes with balance liquid behind the end of the sample, use 5 times of column volumes of 0.05M glycine buffer (pH10) washing then, use the 0.05M glycine again, 0.4M 10 times of column volumes of NaCl damping fluid (pH9) washing, use elutriant (0.05M glycine at last, 2.5M NaCl, pH9) wash-out, distributing to collect respectively distillates the peak, detects its FSH immunizing potency, is associated with the about 1.5L of effective constituent, after concentrating with 10,000 molecular weight membrane ultrafiltration, add the dehydrated alcohol of precooling, centrifugal collecting precipitation dewaters with dehydrated alcohol, vacuum-drying gets 120mg dry product II, i.e. urine follicle-stimulating hormone with high specific activity II.
The biological value of table 2 gained dry product and than measurement result alive
| Component | FSH biological value (IU/mg) | FSH is than live (IU/mg albumen) | LH biological value (IU/mg) |
| Dry product II behind the purifying | 8783 | 8907 | <1LH?IU/100FSH?IU |
The above results shows: adopt method of the present invention to carry out purifying, can obtain very height ratio FSH alive.
Comparative Examples
With the above-mentioned low-purity urinary follicle stimulating hormone of 5g (starting raw material is with embodiment 1) 200mL balance liquid (0.05M sodium phosphate, 1M ammonium sulfate, pH5) dissolving is gone up to 500mL Phenyl Sepharose chromatography column (Amersham provides), and this post has used identical balance liquid balance good in advance.Behind the end of the sample, with 2 times of column volumes of balance liquid washing, use the 0.05M sodium phosphate again, 0.5M ammonium sulfate, the buffer solution elution of pH5, collection distillates component, concentrate with 10,000 molecular weight membrane ultrafiltration behind the dialysis desalting, the dehydrated alcohol precipitation that adds precooling is then spent the night, next day centrifugal collecting precipitation, with the dehydrated alcohol dehydration, vacuum-drying obtains 52 milligrams of dry product III.
The biological value of table 3 gained dry product and than measurement result alive
| Component | FSH biological value (IU/mg) | FSH is than live (IU/mg albumen) | LH biological value (IU/mg) |
| Comparative Examples dry product III | 5960 | 5781 | <1LH?IU/100FSH?IU |
Embodiment 3
The urine follicle-stimulating hormone with high specific activity lyophilized injection
Be used to make 1000 bottles of FSH lyophilized injections, and the exemplary of every bottle of production that contains 75IU FSH is as follows:
Calculate the aequum (is unit with the biological value) of FSH, take by weighing FSH dry product I among the embodiment 1 by this amount, be dissolved in the 20mL injection apirogen water, if necessary, regulate pH6.5 ± 0.2 with HCl or NaOH, carry out sterile filtration with 0.22 μ m strainer then.
The 10g lactose is dissolved in the 200mL injection apirogen water, if necessary, regulates pH6.5 ± 0.2, carry out sterile filtration with 0.22 μ m strainer with HCl or NaOH.Join then in the above-mentioned FSH solution, be settled to 750mL with the injection apirogen water, mixing.
Above-mentioned solution branch is packed in the ampoule, and every bottle of 0.75mL carries out lyophilize.
In the resulting ampoule, every bottle contains 75IU FSH and 10mg lactose.
The above only is preferred embodiment of the present invention, be not in order to limit essence technology contents scope of the present invention, essence technology contents of the present invention is broadly to be defined in the claim scope of application, any technology entity or method that other people finish, if it is defined identical with the claim scope of application, also or a kind of change of equivalence, all will be regarded as being covered by among this claim scope.
Claims (16)
1. a urine follicle-stimulating hormone with high specific activity (pFSH) is characterized in that, it is than work 〉=7000 international unit/mg albumen.
2. urine follicle-stimulating hormone with high specific activity as claimed in claim 1 is characterized in that, it is than work 〉=8000 international unit/mg albumen; More preferably, it is than work 〉=8500 international unit/mg albumen.
3. urine follicle-stimulating hormone with high specific activity as claimed in claim 1 is characterized in that, follicular stimulating hormone is follicular stimulating hormone or its variant that the people urinates the source.
4. the preparation method as the arbitrary described urine follicle-stimulating hormone with high specific activity of claim 1-3 is characterized in that, described method comprises step:
The low-purity urinary follicle stimulating hormone through chromatogram purification, is obtained urine follicle-stimulating hormone with high specific activity;
Described chromatogram is a dye affinity chromatography.
5. preparation method as claimed in claim 4 is characterized in that described chromatogram also comprises cation-exchange chromatography.
6. as claim 4 or 5 described preparation methods, it is characterized in that described method comprises step:
(a) solution 1 that will contain the low-purity urinary follicle stimulating hormone obtains overhead product 1 through the cation-exchange chromatography purifying; With
(b) solution 2 that will contain overhead product 1 obtains urine follicle-stimulating hormone with high specific activity through the dye affinity chromatography purifying.
7. as the arbitrary described method of claim 4-6, it is characterized in that the resin resin matrix medium of described cation-exchange chromatography comprises agarose, dextran, Mierocrystalline cellulose, the cross-linking agent of vinylbenzene and Vinylstyrene, the cross-linking agent of vinylformic acid and/or its derivative.
8. as the arbitrary described method of claim 4-6, it is characterized in that the resin resin active group of described cation-exchange chromatography is selected from sulfonic acid propyl group (SO
3H), methyne sulfonic group (CH
2SO
3H), carboxyl (COOH), carboxymethyl (OCH
2COOH) or phenylol (C
6H
5OH).
9. preparation method as claimed in claim 10 is characterized in that, the active group of the resin of described cation-exchange chromatography is selected from sulfonic acid propyl group or methyne sulfonic group.
10. as the arbitrary described method of claim 4-6, it is characterized in that the resin resin of described cation-exchange chromatography is SP Sepharose or CM Sepharose.
11., it is characterized in that the dyestuff aglucon of the resin resin of described dye affinity chromatography is selected from Cibacron Blue, Orange, Red, Green as the arbitrary described method of claim 4-6.
12. as the arbitrary described method of claim 4-6, it is characterized in that the solid phase carrier of the resin resin of described dye affinity chromatography is selected from bentonite, glass microsphere, quartzy microballoon, hydroxyl calcium phosphate, aluminum oxide, polyacrylamide gel, starch gel, dextrane gel, Mierocrystalline cellulose or agarose.
13., it is characterized in that the resin resin of described dye affinity chromatography is Blue Sepharose 6B or B1ue Sepharose FF as the arbitrary described method of claim 4-6.
14. preparation method as claimed in claim 6 is characterized in that, step (a) or (b) carry out 1-3 times.
15. a pharmaceutical composition is characterized in that, contain in the described composition treatment significant quantity as arbitrary described urine follicle-stimulating hormone with high specific activity of claim 1-3 and pharmaceutically acceptable carrier.
16. one kind as the purposes of the arbitrary described urine follicle-stimulating hormone with high specific activity of claim 1-3 in the medicine of the sterile syndromes of preparation treatment.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100489549A CN101519445B (en) | 2009-04-08 | 2009-04-08 | Urine follicle-stimulating hormone with high specific activity and method for preparing same |
| PCT/CN2009/071981 WO2010115318A1 (en) | 2009-04-08 | 2009-05-26 | Follicle-stimulating hormone with high specific activity and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100489549A CN101519445B (en) | 2009-04-08 | 2009-04-08 | Urine follicle-stimulating hormone with high specific activity and method for preparing same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101519445A true CN101519445A (en) | 2009-09-02 |
| CN101519445B CN101519445B (en) | 2013-03-27 |
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|---|---|---|---|
| CN2009100489549A Active CN101519445B (en) | 2009-04-08 | 2009-04-08 | Urine follicle-stimulating hormone with high specific activity and method for preparing same |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN101519445B (en) |
| WO (1) | WO2010115318A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869827A (en) * | 2010-04-30 | 2010-10-27 | 北京九州泰康生物科技有限责任公司 | Method for preparing novel affinity medium and application thereof |
| WO2010133071A1 (en) * | 2009-05-19 | 2010-11-25 | 上海天伟生物制药有限公司 | Highly purified follicle stimulating hormone from urea and method for preparing thereof |
| CN102464713A (en) * | 2010-12-21 | 2012-05-23 | 上海丽珠制药有限公司 | Preparation method of follicle-stimulating hormone |
| CN102924588A (en) * | 2012-10-26 | 2013-02-13 | 日照新康生物科技有限公司 | Preparation method of high-specific-activity urofollitropin |
| CN103570820A (en) * | 2012-08-06 | 2014-02-12 | 齐鲁制药有限公司 | Method for purifying recombinant human follicle stimulating hormone |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5990288A (en) * | 1997-10-21 | 1999-11-23 | Vitro Diagnostics, Inc. | Method for purifying FSH |
| US20030166525A1 (en) * | 1998-07-23 | 2003-09-04 | Hoffmann James Arthur | FSH Formulation |
| CN100417663C (en) * | 2004-07-23 | 2008-09-10 | 南昌市万华生化制品有限公司 | Purifying and producing process for high purity follicle stimulating hormone in urine |
| ES2312037T3 (en) * | 2004-11-09 | 2009-02-16 | Ares Trading S.A. | METHOD FOR PURUIFYING FSH. |
-
2009
- 2009-04-08 CN CN2009100489549A patent/CN101519445B/en active Active
- 2009-05-26 WO PCT/CN2009/071981 patent/WO2010115318A1/en active Application Filing
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010133071A1 (en) * | 2009-05-19 | 2010-11-25 | 上海天伟生物制药有限公司 | Highly purified follicle stimulating hormone from urea and method for preparing thereof |
| CN101869827A (en) * | 2010-04-30 | 2010-10-27 | 北京九州泰康生物科技有限责任公司 | Method for preparing novel affinity medium and application thereof |
| CN102464713A (en) * | 2010-12-21 | 2012-05-23 | 上海丽珠制药有限公司 | Preparation method of follicle-stimulating hormone |
| CN103570820A (en) * | 2012-08-06 | 2014-02-12 | 齐鲁制药有限公司 | Method for purifying recombinant human follicle stimulating hormone |
| CN103570820B (en) * | 2012-08-06 | 2015-11-18 | 齐鲁制药有限公司 | The purification process of a kind of Gonal-F |
| CN102924588A (en) * | 2012-10-26 | 2013-02-13 | 日照新康生物科技有限公司 | Preparation method of high-specific-activity urofollitropin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101519445B (en) | 2013-03-27 |
| WO2010115318A1 (en) | 2010-10-14 |
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