CN101519445B - 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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- CN101519445B CN101519445B CN2009100489549A CN200910048954A CN101519445B CN 101519445 B CN101519445 B CN 101519445B CN 2009100489549 A CN2009100489549 A CN 2009100489549A CN 200910048954 A CN200910048954 A CN 200910048954A CN 101519445 B CN101519445 B CN 101519445B
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Classifications
-
- 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 [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Reproductive Health (AREA)
- Endocrinology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pregnancy & Childbirth (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gynecology & Obstetrics (AREA)
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 comprised 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-occurs.
The β subunit of FSH is comprised of 111 amino acid, and molecular weight is about 18000 D, and wherein the 7th and 24 locational l-asparagines are that the glycosylated amino acid of N-occurs.And the β subunit of LH is comprised of 121 amino acid, and molecular weight is about 14800 D; The β subunit of CG then has 145 amino acid, molecular weight 22000-39000D.
FSH is mainly used in treating 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 such as the Pergonal of Serono company, it is that FSH and LH ratio are about 1 mixture.But, need not add the patient with exogenous LH for the LH that more amount is arranged in the body, the too high normal development that can affect 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 easily causes polycystic ovary syndrome (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 with pure FSH treatment to POOS patient, 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, such 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 of high specific activity, 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 so that 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 as starting raw material take low-purity urinary follicle stimulating hormone or HMG (HMG), pass through hydrophobic chromatography, or monoclone antibody immune chromatography method and rp-hplc method come purifying, but the specific activity 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 specific activity of the FSH of acquisition is 6200IU/mg.
Therefore, this area is in the urgent need to developing a kind of FSH of high specific activity, and corresponding preparation method, and obtains to contain thus the preparation of high reactivity FSH, 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 specific activity of described urine follicle-stimulating hormone with high specific activity (pFSH) 〉=7000 international unit/mg albumen.
In another preference, the specific activity of described urine follicle-stimulating hormone with high specific activity (pFSH) 〉=8000 international unit/mg albumen; More preferably, the specific activity of described urine follicle-stimulating hormone with high specific activity (pFSH) 〉=8500 international unit/mg albumen.
In another preference, the specific activity 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 dye affinity chromatography.
In another preference, described chromatogram also comprises cation-exchange chromatography.
In another preference, described method comprises step:
The solution 1 that (a) will contain the low-purity urinary follicle stimulating hormone obtains overhead product 1 through the cation-exchange chromatography purifying; With
The solution 2 that (b) 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 dye ligand 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 time.
In a third aspect of the present invention, a kind of pharmaceutical composition is provided, contain 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.
Accordingly, the invention provides a kind of FSH of high specific activity, and corresponding preparation method, and obtain to contain thus the preparation of high reactivity FSH, 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 the FSH that can obtain at present known the highest specific activity by cation exchange resin chromatography and the affine resin chromatography of dyestuff.
Particularly, the present invention urinates or HMG or low-purity urinary follicle stimulating hormone are starting raw material, by effective cation exchange resin chromatography and the affine resin chromatography purification step of dyestuff, obtains the FSH of high specific activity, purification process is simply effective, and the FSH specific activity of acquisition is high, impurity is few.
As used herein, term " follicular stimulating hormone " and " FSH " are used interchangeably, and refer to that a class is used for promoting that sperm or ovarian follicle produce, hormone or its variant of ovary development accelerating, and it can be secreted by prepituitary gland in natural situation.
As used herein, " urinary follicle stimulating hormone " and " pFSH " is used interchangeably, and refers to extract the follicular stimulating hormone that obtains from urine, and described urine comes from Mammals, preferably comes from the people, more preferably is the urine of menopausal women.
Used raw material low-purity pFSH can adopt this area any mode commonly used to obtain among the present invention, 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 the low-purity pFSH by hydrophobic chromatography or the conventional meanses such as affinity chromatography by anti-LH antibody and/or anti-CG antibody, such as the described method of CN101307103A.。The specific activity of low-purity pFSH generally is lower than 2000IU/mg albumen, preferred 200-500IU/mg albumen.
The low-purity pFSH 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 basically only contains the foreign protein of FSH and other non-LH 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 pFSH of the present invention, adopt this area ordinary method that raw material low-purity pFSH is carried out preliminary purification, to separate other impurity except 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 with hydrophobic chromatography or removal LH wherein, obtain highly active FSH thereby then be prepared by the disclosed method of this patent.
As used herein, term " luteotropic hormone " and " LH " are used interchangeably, refer in the raw material preparation that contains FSH or acquisition process, be doped in wherein have the hormone of same or similar structure and function with natural LH.
The specific activity of urine follicle-stimulating hormone with high specific activity provided by the invention 〉=7000IU/mg albumen, preferred 〉=8500IU/mg albumen.The biological value of LH is generally less than 1LHIU/50 FSH IU in the high specific activity pFSH provided by the invention, more preferably, and less than 1 LH IU/100 FSH IU.
The preparation method of high specific activity pFSH provided by the invention comprises step:
Low-purity is urinated source gamogenetic egg bubble yield stimulant or HMG (HMG) process chromatogram purification, obtain high specific activity pFSH; Described chromatogram is cation-exchange chromatography and dye affinity chromatography.
Preferably, described method comprises step:
The solution 1 that (1) will contain low-purity FSH obtains overhead product 1 through the cation-exchange chromatography purifying; With
The solution 2 that (2) 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 dye ligand 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) the first sample solution that contains the low-purity urinary follicle stimulating hormone with pH4-7 carries out loading, and the concentration of low-purity urinary follicle stimulating hormone is 1.0-5.0w/v% (g/ml) in described the first sample solution, preferred 2.0-4.0w/v% (g/ml);
(ii) the first washings with pH4-6 washs; With
(iii) carry out wash-out with the first washings of pH4-6 and the first elutriant of pH4-6, obtain overhead product 1.
More preferably, carry out gradient elution in the step (iii), in 0.5 to 5 hour, the concentration of the first elutriant (v/v) from 0 to 100%.
The salt concn of described the 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 the first washings or the 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 ') carries out loading with the 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 the second sample solution
(ii ') washs with the second washings of pH8-11; With
(iii ') carries out wash-out with the second elutriant of pH8-11, obtains urine follicle-stimulating hormone with high specific activity.
The salt concn of described the 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 the second washings or the 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 high specific activity pFSH with the inventive method preparation for the treatment of significant quantity, and pharmaceutically acceptable carrier.
As used herein, term " contain " or " comprising " comprised " comprising ", " basically by ... consist of " and " by ... consist of ".As used herein, term " treatment significant quantity " refers to and can produce 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 without excessive bad side reaction (such as toxicity, stimulation and transformation reactions), the material of rational benefit/risk ratio is arranged namely.
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 like this some medicament carriers: they itself are not necessary activeconstituents, and do not have undue toxicity after using.Suitable carrier is well known to those of ordinary skill in the art.In " Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences, Mack Pub.Co., N.J.1991), can find discussing fully about pharmaceutically acceptable vehicle.
Described " pharmaceutically acceptable carrier " can contain liquid, such as water, salt solution, glycerine and ethanol.In addition, also may there be complementary material in these carriers, such 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, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 usually, preferably, pH is about 6-8.
In preferred implementation of the present invention, the high specific activity pFSH in the described pharmaceutical composition accounts for the 0.001-99.9wt% of composition total weight; Being preferably the 0.01-99wt% of composition total weight, more preferably is 0.02-95wt%, more preferably 0.05-90wt%.Surplus is the 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 (such as granule, tablet, lyophilized powder, suppository, capsule, sublingual lozenge) or liquid (such 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 discloses can with any composition forms and usefulness, each feature that discloses in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore except special instruction is arranged, the feature that discloses only is the general example of equalization or similar features.
Major advantage of the present invention is:
1, provides a kind of highly active pFSH, can be applicable to subcutaneous injection;
2, a kind of purification process of high reactivity pFSH 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 for explanation the present invention 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 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 refers to 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 better implementation method described in the literary composition and material only present a demonstration.
The biological value that the present invention relates to and the measuring method of specific activity:
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.
The FSH specific activity
Measure as follows (albumen optical spectroscopy) mensuration:
Protein content: precision takes by weighing approximately 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 respectively the absorbancy at 280nm place according to ultraviolet spectrophotometry (Chinese Pharmacopoeia version appendix in 2005 IV A), take the concentration of bovine serum albumin solution as X-coordinate, optical density at 280nm is 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.
The FSH specific activity:
Be calculated as follows specific activity:
In the formula, C is the concentration (mg/ml) of need testing solution
Wherein, the mg in the trial-product biological value unit refers to 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 then goes up 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, then use washings and elutriant (0.1M sodium-acetate+1M NaCl, pH5) carry out the 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 approximately 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, then go up to 300mL Blue Sepharose FF chromatography column (Amersham provides), wash 5 times of column volumes with balance liquid behind the end of the sample, then use 5 times of column volumes of 0.05M glycine buffer (pH10) washing, use again the 0.05M glycine, 0.4M 8 times of column volumes of NaCl damping fluid (pH9) washing, use at last elutriant (0.05M glycine, 2.5M NaCl, pH9) wash-out, distributing to collect respectively distillates the peak, detects its FSH immunizing potency, is associated with approximately 2L of effective constituent, after concentrated 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 specific activity measurement result
| FSH biological value (IU/mg) | FSH specific activity (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 the FSH of very high specific activity.
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, then go up 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, then use washings and elutriant (0.1M sodium-acetate+1M NaCl, pH5) carry out the 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 approximately 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, then go up to 200mL Blue Sepharose 6B chromatography column (Amersham provides), wash 5 times of column volumes with balance liquid behind the end of the sample, then use 5 times of column volumes of 0.05M glycine buffer (pH10) washing, use again the 0.05M glycine, 0.4M 10 times of column volumes of NaCl damping fluid (pH9) washing, use at last elutriant (0.05M glycine, 2.5M NaCl, pH9) wash-out, distributing to collect respectively distillates the peak, detects its FSH immunizing potency, is associated with approximately 1.5L of effective constituent, after concentrated 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 specific activity measurement result
| Component | FSH biological value (IU/mg) | FSH specific activity (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 the FSH of very high specific activity.
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, upper to 500mL Phenyl Sepharose chromatography column (Amersham provides), 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 again the 0.05M sodium phosphate, 0.5M ammonium sulfate, the buffer solution elution of pH5, collection distillates component, concentrated with 10,000 molecular weight membrane ultrafiltration behind the dialysis desalting, the dehydrated alcohol precipitation that then adds precooling is 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 specific activity measurement result
| Component | FSH biological value (IU/mg) | FSH specific activity (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
For the manufacture of 1000 bottles of FSH lyophilized injections, and the exemplary of every bottle of production that contains 75IU FSH is as follows:
Calculate the aequum (take biological value as unit) 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 pH 6.5 ± 0.2 with HCl or NaOH, then carry out sterile filtration with 0.22 μ m strainer.
The 10g lactose is dissolved in the 200mL injection apirogen water, if necessary, regulates pH 6.5 ± 0.2 with HCl or NaOH, carry out sterile filtration with 0.22 μ m strainer.Then join in the above-mentioned FSH solution, be settled to 750mL with the injection apirogen water, mixing.
Mentioned solution is distributed 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 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 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 (8)
1. the preparation method of a urine follicle-stimulating hormone with high specific activity in turn includes the following steps:
The solution 1 that (a) will contain the low-purity urinary follicle stimulating hormone obtains overhead product 1 through the cation-exchange chromatography purifying; With
The solution 2 that (b) will contain overhead product 1 obtains urine follicle-stimulating hormone with high specific activity through the dye affinity chromatography purifying;
The specific activity of the urine follicle-stimulating hormone with high specific activity that obtains in the step (b) 〉=8 500 international unit/mg albumen;
The 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;
The resin active group of described cation-exchange chromatography is selected from sulfonic acid propyl group, methyne sulfonic group, carboxyl, carboxymethyl or phenylol;
The dye ligand of the resin of described dye affinity chromatography is selected from Cibacron Blue, Orange, Red, Green.
2. preparation method as claimed in claim 1, wherein the cation-exchange chromatography purifying in the step (a) may further comprise the steps:
(i) the first sample solution that contains the low-purity urinary follicle stimulating hormone with pH4-7 carries out loading, and the concentration of low-purity urinary follicle stimulating hormone is 1.0-5.0w/v% in described the first sample solution, and wherein the unit of w/v is g/ml;
(ii) the first washings with pH4-6 washs; With
(iii) carry out wash-out with the first washings of pH4-6 and the first elutriant of pH4-6, obtain overhead product 1;
Dye affinity chromatography purifying in the step (b) may further comprise the steps:
(i ') carries out loading with the second sample solution that pH5-7 contains overhead product 1; The concentration of overhead product 1 is 0.5-2.0w/v% in described the second sample solution, and wherein the unit of w/v is g/ml;
(ii ') washs with the second washings of pH8-11; With
(iii ') carries out wash-out with the second elutriant of pH8-11, obtains urine follicle-stimulating hormone with high specific activity;
Wherein, salt concn is 0.01-5M in the second elutriant in the step (iii '), and described salt is selected from hydrochloride, phosphoric acid salt, acetate and/or glycinate.
3. preparation method as claimed in claim 1, the resin active group of described cation-exchange chromatography is selected from sulfonic acid propyl group or methyne sulfonic group.
4. preparation method as claimed in claim 1, the resin of described dye affinity chromatography is Blue Sepharose 6B or Blue Sepharose FF.
5. such as each described preparation method of claim 1-3, the resin of described cation-exchange chromatography is SP Sepharose or CM Sepharose.
6. such as each described preparation method of claim 1-3, 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.
7. such as each described preparation method of claim 1-3, it is characterized in that step (a) or (b) carry out 1-3 time.
8. such as each described preparation method of claim 1-3, wherein follicular stimulating hormone is the follicular stimulating hormone that the people urinates the source.
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| 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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| CN2009100489549A CN101519445B (en) | 2009-04-08 | 2009-04-08 | Urine follicle-stimulating hormone with high specific activity and method for preparing same |
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| CN101555279B (en) * | 2009-05-19 | 2013-03-27 | 上海天伟生物制药有限公司 | Urinary follicle stimulating hormone with high purity and preparation method 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 |
| 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 |
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| 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 |
| EP1809663B1 (en) * | 2004-11-09 | 2008-09-17 | Ares Trading S.A. | Method for purifying fsh |
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