CN101307103A - Purification method of follicle stimulating hormone - Google Patents
Purification method of follicle stimulating hormone Download PDFInfo
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- CN101307103A CN101307103A CNA2007100457680A CN200710045768A CN101307103A CN 101307103 A CN101307103 A CN 101307103A CN A2007100457680 A CNA2007100457680 A CN A2007100457680A CN 200710045768 A CN200710045768 A CN 200710045768A CN 101307103 A CN101307103 A CN 101307103A
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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
The present invention relates to protein purification and biomedicine field.Particularly, the present invention relates to the purification process of follicular stimulating hormone (FSH), 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 (leuteinizinghormone, be called for short LH) and chorionic gona dotropin (chorionic gonadotropin, 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 hypophysis or 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.
Because mentioned above, this area presses for to develop and do not contain the active FSH preparation of LH fully, and is required with the treatment of satisfying the patient.
The Metrodin that Serono company releases is a kind of FSH preparation that contains minute quantity LH, and Metrodin-HP thereafter then is highly purified FSH preparation.
In addition, also develop a kind of highly purified Menotropins (pHMG) in recent years on the market, Menopur as Ferring company, it is with the high purity FSH behind the purifying and high purity LH or high purity hCG (human chorionic gonadotrophin, human chorionic gonadotropin) allocates the product that forms in 1: 1 ratio, be mainly used in and substitute common HMG product, and overcome the anaphylaxis that causes because of a large amount of foreign proteins in the common HMG product human body.
U.S. Patent number 3,674,865 have described the method for purifying FSH from the urine extract, and this method comprises 21 different steps altogether, and obtaining is the mixture of FSH and LH, and FSH and LH ratio are between 3: 1～6: 1.Its purifying process is comparatively complicated, and the ratio of gained FSH is also lower.
U.S. Patent number 6,162,905 have described the method with multistep method purifying FSH such as ion exchange chromatography, dyestuff chromatographies, also have only 313: 1 but the ratio of its FSH and the immunizing potency of LH is the highest, wherein still have a spot of LH to exist.
In recent years, the means by immune response purification of target thing are gradually ripe.Immune response is meant the specificity association reaction that is taken place between antigen and the corresponding antibodies, why both can carry out specificity in conjunction with being based on two kinds of intermolecular complementary structures and affinity, and this two specific character is that the primary structure by antigen and antibody molecule determines.Antibody is by a kind of sphaeroprotein that produces in the human or animal body, generally form by 4 peptide chains, can be folded into some spherical functional zone by intrachain disulfide bond, the hypervariable region wherein and the epi-position of corresponding antigens are the specificity complementary relationship on space structure, as the relation of key and lock, thereby narrow spectrum association reaction can take place in both.
In patent WO98/20039, monoclone antibody immune chromatography method and the rp-hplc method described with anti-FSH come purifying FSH, but because its immunochromatography is the absorb-elute process, eluent is the 1M ammoniacal liquor of pH 11, cause the inactivation of FSH and coming off of monoclonal antibody during wash-out easily, there is certain influence in product.
For this reason, this area press for develop a kind ofly can be used for simply, the method for purifying FSH efficiently, and obtain to contain hardly the active FSH preparation of LH thus.
Summary of the invention
The object of the present invention is to provide a kind of advantages of simplicity and high efficiency FSH purification process, and obtain to contain hardly the active FSH product of LH by this method.
In a first aspect of the present invention, a kind of method for preparing follicular stimulating hormone FSH is provided, said method comprising the steps of:
(A) raw material that contains FSH is contacted with antibody, wherein said raw material contains as the luteotropic hormone LH of impurity and/or chorionic gona dotropin CG, and described antibody is to combine with LH and/or CG specifically, thereby makes described antibody and LH and/or CG form antigen-antibody complex;
(B) antigen-antibody complex that forms is removed from described raw material, thus the FSH of acquisition purifying.
In one embodiment, described antibody is the antibody of anti-LH and/or the antibody of anti-CG.
In another embodiment, described follicular stimulating hormone is HFSH or its variant.
In another embodiment, described HFSH or its variant are that the people urinates the follicular stimulating hormone in source or the HFSH of reorganization.
In another embodiment, the antibody of described anti-LH is immunoreactive antibody to take place with LH or LH β subunit or their variant, the antibody that comprises anti-people LH or its variant, the antibody of anti-people LH β subunit or its variant, the antibody of anti-animal LH or its variant, the antibody of anti-animal LH β subunit or its variant.
In another embodiment, the antibody of described anti-LH comprises the antibody of the anti-LH that uses polyclonal antibody technology, monoclonal antibody technique or the preparation of DNA recombinant technology.
In another embodiment, the antibody of described anti-LH comprises: contain the mixture of anti-LH antibody or the anti-LH antibody that purifying is crossed.
In another embodiment, the described mixture that contains anti-LH antibody comprises: the animal serum, animal ascites or the cell culture fluid that contain anti-LH antibody.
In a preference, the anti-LH antibody that described purifying is crossed comprises: animal serum, animal ascites or the cell culture fluid that will contain anti-LH antibody carries out the product behind the purifying.
In another embodiment, the antibody of described anti-CG is immunoreactive antibody to take place with CG or CG β subunit or their variant, the antibody that comprises anti-people CG or its variant, the antibody of anti-people CG β subunit or its variant, the antibody of anti-animal CG or its variant, the antibody of anti-animal CG β subunit or its variant.
In another embodiment, the antibody of described anti-CG comprises: the antibody that uses the anti-CG of polyclonal antibody technology, monoclonal antibody technique or the preparation of DNA recombinant technology.
In another embodiment, the antibody of described anti-CG comprises: contain the mixture of anti-CG antibody or the anti-CG antibody that purifying is crossed.
In a preference, the described mixture that contains anti-CG antibody comprises: the animal serum, animal ascites or the cell culture fluid that contain anti-CG antibody.
In a preference, the anti-CG antibody that described purifying is crossed comprises: animal serum, animal ascites or the cell culture fluid that will contain anti-CG antibody carries out the product behind the purifying.
In another embodiment, described purge process comprises that use immuno-precipitation and immunochromatographic method carry out purifying.
In another embodiment, in the described follicular stimulating hormone, the biological value ratio of FSH and LH was not less than 100: 1; In the preferred described follicular stimulating hormone, the ratio of the immunizing potency of FSH and LH was not less than 1000: 1; In the more preferably described follicular stimulating hormone, the ratio of the immunizing potency of FSH and LH was not less than 10000: 1.
In a second aspect of the present invention, the preparation of drug combination method of a kind of FSH of containing is provided, said method comprising the steps of:
(A) raw material that contains FSH is contacted with antibody, wherein said raw material contains as the luteotropic hormone LH of impurity and/or chorionic gona dotropin CG, and described antibody is specificity in conjunction with LH and/or CG but the antibody of cross reaction does not take place with FSH basically, thereby makes described antibody and LH and/or CG formation antigen-antibody complex;
(B) antigen-antibody complex that forms is removed from described raw material, thus the FSH of acquisition purifying;
(C) it is formulated together to treat the FSH of the purifying that makes in the step (B) of significant quantity, pharmaceutically acceptable carrier and optional other activeconstituents, thereby forms described pharmaceutical composition.
In a preference, the content of the FSH of described purifying is the 0.001-99.9 weight % of described pharmaceutical composition gross weight; Preferred 0.01-99 weight %, more preferably 0.02-95 weight %.
In another preference, described pharmaceutical composition does not contain LH.
In another preference, other activeconstituents in the step (C) is selected from: LH and or hCG.
In a preference, described pharmaceutical composition also comprises the LH of purifying, and wherein the biological value of FSH and LH is than being 0.01-100: 1, and preferred 0.1-10: 1, more preferably 1-5: 1.
In another preference, described pharmaceutical composition is Menotropins (HMG) or high purity Menotropins (pHMG).
In another preference, described pharmaceutical composition is high purity pHMG, and it comprises by the FSH of the purifying of required mixed and high purity LH.The weight ratio of the FSH of described purifying and high purity LH is 10: 1-1: 10, preferred 5: 1-1: 5, and more preferably 1: 1.
In this external method for preparing follicular stimulating hormone FSH provided by the invention, described LH and/or CG are mammiferous LH and/or CG.
In a preference, described Mammals is behaved.
In another embodiment, described antibody is selected from down group:
(a) antibody of anti-LH β subunit;
(b) antibody of anti-CG β subunit; And/or
(c) their combination.
In another preference, the antibody of described anti-LH β subunit or anti-CG β subunit is selected from: anti-hCG monoclonal antibody (for example can available from the Monoclonal anti-β hcgHYB-093-05 of AntibodyShop or the Monoclonal anti-β hcg C7659 of Sigma), anti-LH monoclonal antibody (for example can available from the MS-1448-PABX of Thermo Fisher).
In another preference, described antibody is to adopt polyclonal antibody technology, monoclonal antibody technique or the preparation of DNA recombinant technology.
In another preference, described antibody is mixture or the purified anti-LH and/or the antibody of CG β subunit that comprises the antibody of anti-LH and/or CG β subunit.
In another embodiment, described antibody comprises: monoclonal antibody and the antiserum(antisera), animal ascites or the cell culture fluid that contain antibody.
In a preference, described purifying antibody by saltout, conventional purification process such as chromatography carries out.
In another embodiment, wherein said purification process is immuno-precipitation or affinity chromatography.
In another preference, can carry out further purifying and utilization to isolating LH and/or CG in the purge process.
In another embodiment, the raw material of the described FSH of containing is selected from: without the raw material of any prepurification step, removed the raw material of partial impurities or only contained the raw material of LH and/or CG through the prepurification step basically through the prepurification step.
In another embodiment, the ratio of the FSH of described purifying is lived to being not less than 500IU/mg albumen.
In a preference, the ratio of the FSH of described purifying is lived and is 500-15000IU/mg, preferred 600-12000IU/mg albumen, and more preferably 700-10000IU/mg albumen most preferably is 750-8000IU/mg albumen.
The present invention also provides a kind of antibody in the purposes from the feedstock production FSH that contains FSH, and described raw material contains as the LH of impurity and/or CG, and described antibodies specific is in conjunction with LH and/or CG but with FSH cross reaction does not take place basically.
In a preference, described antibody is selected from down group:
(a) antibody of anti-LH β subunit;
(b) antibody of anti-CG β subunit; And/or
(c) their combination.
Purification process mild condition of the present invention, easy and simple to handle, FSH yield height, purity is high and the height of tiring.
Description of drawings
Fig. 1 is the protein structure synoptic diagram of hCG.
Fig. 2 is the protein structure synoptic diagram of LH.
Embodiment
The inventor is through extensive and deep research, being surprised to find can be by immune method of purification, utilize the antibody of the β subunit of anti-LH or CG to remove impurity LH and/or CG among the FSH, thereby under mild conditions, simply and efficiently obtain the FSH purified product that high purity, height are tired.On this basis, the contriver has finished the present invention.
Particularly, described in background technology part, available technology adopting the antibodies FSH of anti-FSH come purifying FSH.But this method is easy to cause coming off of FSH inactivation and antibody when wash-out, thereby has influenced purification effect.
In exploring the process of improving art methods, the inventor notices that FSH, LH and CG have following characteristics: their α subunit is identical, and the β subunit has structure separately.Wherein, the β subunit molecular structure of the β subunit of FSH and LH and CG differs greatly, and therefore the antibody of the β subunit of anti-LH and/or CG with FSH cross reaction does not take place basically.
And the β subunit of LH and CG has bigger similarity on molecular structure, the β subunit of CG is only Duoed 24 amino acid than the β subunit of LH, and these 24 amino acid have neither part nor lot in the combination of intrachain disulfide bond, therefore on space structure both very near (seeing Fig. 1 and Fig. 2), so some antibody have certain cross reaction to the two.Therefore, use the antibody of anti-LH, perhaps using has the anti-CG antibody of cross reactivity immune the combination to be taken place with LH to LH, thereby reaches the purpose that efficient removal LH also removes the CG that may exist simultaneously.
The immune method of purification that the present invention adopts, utilized the antibody capable and the LH in the target compound (or CG) of the β subunit of anti-LH or CG that this characteristic of specificity association reaction takes place just, both guaranteed the efficient removal of LH (or CG), and guaranteed again that purification condition is gentle and do not destroy the activity of FSH.
Therefore, the present invention adopts the antibody at impurity LH and CG, by the impurity among the method removal FSH that forms impurity-antibody conjugates, has reached the purpose of purifying FSH.Simultaneously the contriver finds that also pleasantly surprisedly purification process of the present invention has purification condition gentleness, easy and simple to handle, FSH yield height, purity height and the advantages of higher of tiring.
FSH, LH and CG
As used herein, term " follicular stimulating hormone " and " FSH " are used interchangeably, 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, its can be under natural situation by the prepituitary gland secretion, can extract from the urine of menopausal women maybe can be by the recombinant technology acquisition.Described FSH has identical or similar structure and function with natural FSH.
Used raw material FSH can adopt this area any way commonly used to obtain among the present invention, for example natural generation, obtains or synthetic by recombinant technology, wherein contains LH or CG as impurity.In an embodiment of the invention, described follicular stimulating hormone is HFSH or its variant, and preferred people urinates the follicular stimulating hormone in source or the HFSH of reorganization.
Can be used for containing FSH and comprising as the LH of impurity and/or the raw material of CG among the present invention can be: without the raw material of any prepurification step, removed the raw material of partial impurities or only contained the raw material of FSH and impurity LH and/or CG through the prepurification step basically through the prepurification step.
Preferably before adopting method purifying FSH of the present invention, adopt this area ordinary method that raw material FSH is carried out preliminary purification, to separate other impurity except that LH or CG.
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.
Similarly, term " chorionic-gonadotropin hormone " and " CG " 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 CG.
The antibody of anti-LH and/or CG β subunit
As used herein, term " antibody of anti-LH and/or CG β subunit " and " antibody of anti-LH and/or CG " is meant and can immune association reaction takes place with the β subunit of LH and/or CG, but immunological cross-reaction do not take place or with FSH the antibody of immunoreactive ability far below its ability of reacting with LH and/or CG takes place with FSH basically.Term used herein " far below " be meant with the avidity or the cross reacting rate of antigen-antibody and represent that described antibody and FSH immunoreactive ability take place for itself and below 1/10 of LH and/or CG generation immune response ability, and is preferred below 1/100.
The antibody that uses in the process of the present invention is preferably Mammals antibody, the antibody of more preferably anti-people LH or its variant, or the antibody of anti-people CG or its variant.Term " variant " is meant that aminoacid sequence compared certain difference with people LH or CG, but still has with people LH or CG is identical or the protein of similar structures and/or function.
Antibody used in the present invention can obtain by routine techniques, for example adopts polyclonal antibody technology, monoclonal antibody technique or recombinant DNA technology, or by commercially available acquisition.The antibody that is used for the present invention can be those antibody of being put down in writing in the prior art document, Schwarz for example, the The antigenic surfaceof human chorionic gonadotropin as mapped by murine monoclonalantibodies of S (" the antigenicity surface of the human chorionic gonadotropin by mouse monoclonal antibody mapping ", Endocrinology, 1986, those antibody of being put down in writing in 118:189-97).The antibody that is used for the present invention also can be by commercially available anti-LH that buys or CG antibody.
In preferred implementation of the present invention, described antibody is selected from: anti-hCG monoclonal antibody (for example can available from the Monoclonal anti-β hcg HYB-093-05 of AntibodyShop or the Monoclonal anti-β hcg C7659 of Sigma), anti-LH monoclonal antibody (for example can available from the MS-1448-PABX of Thermo Fisher).Also can obtain to express the cell strain of described monoclonal antibody by monoclonal antibody technique known in the art.
Can with directly use described antibody maybe can by saltout, ordinary method such as chromatography carries out being used for the inventive method behind the purifying.
In an embodiment of the invention, can adopt the antibody of following form: the monoclonal antibody of anti-LH and/or CG β subunit, contain animal serum, animal ascites, cell culture fluid or their purified product of anti-LH and/or CG β subunit antibody.
The immune purification process of FSH
In the present invention, can adopt multiple antibody mediated immunity purification process, for example comprise: immuno-precipitation and immunochromatographic method.
Immuno-precipitation is meant that antigen, the antibody in the sample to be purified association reaction takes place produces precipitation in homogeneous system, then by centrifugal, filter or other method is separated antigen-antibody complex, to reach purification effect.
Immunochromatographic method is a kind of purification process that development is recently come out, it be with antibody immobilization to chromatographic resin, then with sample on the sample to be purified to this resin, make antigen wherein be bonded on the immobilized antibody, thereby reach antigen and the isolating effect of target compound, collect the effect that antigen reaches purifying antigen thereby perhaps carry out wash-out with suitable elutriant.Antibody can be by conventional method immobilization to chromatographic resin (as agarose), for example by methods such as divinylsulfone or bromize hydrogen activating combinations.
The present invention has adopted and has made impurity (LH, CG) and antibody generation immune response, thereby with impurity isolated method from required product (FSH), has realized the efficiently purifying of FSH with the reaction conditions of gentleness.In preferred implementation of the present invention, described chromatography is to use the gentle chromatographic solution (0.1M Tris-HCl, 0.2M NaCl) of pH 7.0 to carry out, and has guaranteed the high reactivity of gained FSH.
Product behind immune purifying can pass through ethanol sedimentation, vacuum-drying or lyophilize subsequently, or carries out further purifying, obtain highly purified FSH product with and dosage form.
Pharmaceutical composition
The present invention also provides a kind of pharmaceutical composition, and described pharmaceutical composition contains the high purity FSH 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 FSH 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.
As used herein, term " unit dosage " is meant for easy administration, and preparation of compositions of the present invention is become the required formulation of single administration, includes but not limited to various solid formulation (as tablet), liquid agent, capsule, sustained release dosage, powder injection.In another preferred implementation of the present invention, described composition is unit dosage or multi-form, and wherein the content of FSH is 0.001～2000mg/ agent, preferred 0.003-500mg/ agent, more preferably 0.005-50mg/ agent.In another preference of the present invention, use 1～6 dose of composition of the present invention every day, preferably use 1～3 dose; Most preferred, the dosage of using every day is 1 dose.
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, aqueous injection) or other suitable shape.
High purity Menotropins (pHMG)
In an embodiment of the invention, described pharmaceutical composition is Menotropins (HMG) or high purity Menotropins (pHMG), more preferably pHMG.
Described pHMG makes by high purity LH and high purity FSH of the present invention are allocated in proportion.
Wherein, the weight ratio of the FSH of described purifying and high purity LH is 10: 1-1: 10, preferred 5: 1-1: 5, and more preferably 1: 1.
Method of application
Composition of the present invention can be used by conventional route, comprising (but being not limited to): oral, intramuscular injection, subcutaneous injection etc.Preferred subcutaneous injection is used.Composition forms should be complementary with method of application.The amount of application of the present composition is pressed active substance and is calculated, and is generally about 0.00001-400mg/kg body weight every day, preferably about 0.00006～10mg/kg body weight, more preferably 0.0001～1mg/kg body weight.
Composition of the present invention can directly use, and also can unite use with other therapeutical agent or assistant agent.In preferred implementation of the present invention, composition of the present invention also can be co-administered with the material of group under being selected from of significant quantity (as the 0.005-1000mg/kg body weight/day): hCG, luteinizing hormone-releasing hormone (LHRH) analogue.
When two or more medication combined administration, generally have and be better than the individually dosed respectively effects of two kinds of medicines.Preferably, co-administered medicine or other preparation therapeutic activity of not disturbing FSH effective constituent of the present invention.
Advantage of the present invention
The invention has the advantages that:
(1) provide a kind of method that can simply and efficiently remove LH in the raw material that contains FSH and/or CG impurity, this method cost is low, step is easy, mild condition, is suitable for industrialization and generates high purity FSH;
(2) the FSH yield height of the inventive method acquisition, purity height and active high are applicable to that preparation contains the pharmaceutical composition of high purity FSH.
Embodiment
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 per-cent and umber calculate by weight.
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 among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Embodiment 1
The raw material that contains FSH
In the present embodiment, as the raw material that contains FSH, wherein the biological value of FSH is 521IU/mg with HMG (available from Shanghai Tianwei Biological Pharmaceutical Corp.), and the biological value of LH is 483IU/mg.
Purification process
Get 10mL sepharose 4B (available from GE Amersham), after activating with divinylsulfone, be suspended in the 10mL 0.5M sodium carbonate solution, add 10mL and contain the solution of anti-hCG monoclonal antibody (available from AntibodyShop, HYB-093-05,1mg/mL), make antibodies to the activatory resin, thereby obtain immune resin.
With the anti-hCG-agarose of the 10mL immunity resin chromatography column of packing into, with balance liquid (pH 7.2 for 0.05M Tris-HCl, 0.2M NaCl) balance.
(wherein the biological value of FSH is 521IU/mg with 35mg HMG, the biological value of LH is 483IU/mg) dissolve with the above-mentioned balance liquid of 10mL, go up sample then to the immunochromatography post, wash (2 times of column volumes) with balance liquid behind the end of the sample, collect the about altogether 20mL of effluent liquid (being directly used in the mensuration of immunizing potency),-10 ℃ of dehydrated alcohols that add 5 times of volumes while stirring, centrifugal collecting precipitation, with the dehydrated alcohol dehydration, vacuum-drying gets 30mg dry product (being directly used in the mensuration of biological value).
The measuring method of immunizing potency and result thereof
Adopt double antibody sandwich method (ABBOTT AXSYM, FSH and LH immunoassay kit), indicate the immunizing potency of measuring the gained sample according to manufacturers instruction.Measurement result is as shown in table 1:
Table 1. immunochromatography is collected the immunizing potency of liquid
The measuring method of biological value and result thereof
The measuring method of biological value is pressed the method check of 2005 editions appendix XII of Chinese Pharmacopoeia M, XII N.Press Lowry method mensuration than the protein content determination of living in measuring.
The biological value of table 2. immunochromatography gained dry product
| Component | FSH unit tire (IU/mg) | FSH is than live (IU/mg albumen) | LH unit tire (IU/mg) | FSH/LH |
| The raw material that contains FSH | 521 | 742 | 483 | 1.1 |
| Dry product through the immunochromatography step | 536 | 763 | ＜5 | ＞100 |
The above results shows: adopt method of the present invention to carry out purifying, can remove the LH of the overwhelming majority among the HMG, make the purity of FSH improve greatly.
The raw material that contains FSH
In the present embodiment, as the raw material that contains FSH, wherein the biological value of FSH is 2251IU/mg with HMG (available from Shanghai Tianwei Biological Pharmaceutical Corp.), and the biological value of LH is 437IU/mg, and the biological value ratio of FSH and LH is 5: 1.
Purification process
Get 10mL sepharose 4B (available from GE Amersham), with bromize hydrogen activating good after, be suspended in the 10mL0.1M sodium hydrogen carbonate solution (pH8.5), add 10mL and contain the solution of anti-LH monoclonal antibody (available from Thermo Fisher, MS-1448-PABX 1mg/mL), transfers pH about 8.5, make wherein antibodies to the activatory resin, thereby obtain immune resin.
With the anti-LH-agarose of the 10mL immunity resin chromatography column of packing into, with balance liquid (pH 7.2 for 0.05M Tris-HCl, 0.2M NaCl) balance.
25mg HMG (wherein the biological value of FSH is 2251IU/mg, and the biological value of LH is 437IU/mg) with the above-mentioned balance liquid dissolving of 10mL, is gone up sample to the immunochromatography post then.Wash (2 times of column volumes) with balance liquid behind the end of the sample, collect the about altogether 20mL of effluent liquid (being directly used in the mensuration of immunizing potency), add-10 ℃ of dehydrated alcohols of 5 times of volumes while stirring, centrifugal collecting precipitation, with the dehydrated alcohol dehydration, vacuum-drying gets the 20mg dry product.
The measuring method of immunizing potency and result thereof
Adopt embodiment 1 used measuring method to measure the immunizing potency that gained is collected liquid.Measurement result is as shown in table 3:
The immunizing potency of the effluent liquid of collecting in table 3. immunochromatography
The above results shows: adopt method of the present invention to carry out purifying, can remove the LH of the overwhelming majority among the HMG, make the purity of FSH improve greatly.
The preparation method of embodiment 3. high purity Menotropinses (pHMG)
Preparation 100mL 0.01M sodium dihydrogen phosphate (adjusting pH to about 6.5 with NaOH) adds the 4g lactose, after the stirring and dissolving, filters with 0.22 μ m strainer.Get the 15mL filtered solution, add the FSH dry product 10mg that obtains among the embodiment 2, add 5mg high purity LH (the LH 4592IU/mg that tires again, available from Shanghai Tianwei Biological Pharmaceutical Corp.), after the dissolving, carry out vacuum lyophilization fully, get 570mg high purity HMG (containing lactose) as excipient.Record its FSH and be 2358IU/mg albumen than living, LH is 2114IU/mg albumen than living.
The preparation of embodiment 4.FSH lyophilized injection
Be used to make 10000 bottles of FSH lyophilized injections, and the exemplary of every bottle of production that contains 75IU FSH is as follows:
The FSH elaboration of a certain amount of (is unit with the biological value) that calculates is dissolved in the 50mL injection apirogen water, if necessary, regulates pH 6.5 ± 0.2 with HCl or NaOH, carries out sterile filtration with 0.22 μ m strainer then.
The 100g lactose is dissolved in the 2L injection apirogen water, if necessary, regulates pH 6.5 ± 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 7.5L 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.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (17)
1. a method for preparing follicular stimulating hormone FSH is characterized in that, said method comprising the steps of:
(A) raw material that contains FSH is contacted with antibody, wherein said raw material contains as the luteotropic hormone LH of impurity and/or chorionic gona dotropin CG, and described antibodies specific ground combines with LH and/or CG, thereby makes described antibody and LH and/or CG form antigen-antibody complex;
(B) antigen-antibody complex that forms is removed from described raw material, thus the FSH of acquisition purifying.
2. the method for claim 1 is characterized in that, described antibody is anti-LH antibody and/or anti-CG antibody.
3. the method for claim 1 is characterized in that, described follicular stimulating hormone is HFSH or its variant.
4. method as claimed in claim 3 is characterized in that, described HFSH or its variant are that the people urinates the follicular stimulating hormone in source or the HFSH of reorganization.
5. method as claimed in claim 2, it is characterized in that, described anti-LH antibody is immunoreactive antibody to take place with LH or LH β subunit or their variant, the antibody that comprises anti-people LH or its variant, the antibody of anti-people LH β subunit or its variant, the antibody of anti-animal LH or its variant, the antibody of anti-animal LH β subunit or its variant.
6. method as claimed in claim 2 is characterized in that, described anti-LH antibody comprises the anti-LH antibody that uses polyclonal antibody technology, monoclonal antibody technique or the preparation of DNA recombinant technology.
7. method as claimed in claim 2 is characterized in that, described anti-LH antibody comprises: contain the mixture of anti-LH antibody or the anti-LH antibody that purifying is crossed.
8. method as claimed in claim 7 is characterized in that, the described mixture that contains anti-LH antibody comprises: the animal serum, animal ascites or the cell culture fluid that contain anti-LH antibody.
9. method as claimed in claim 7 is characterized in that, the anti-LH antibody that described purifying is crossed comprises: animal serum, animal ascites or the cell culture fluid that will contain anti-LH antibody carries out the product behind the purifying.
10. method as claimed in claim 2, it is characterized in that, described anti-CG antibody is immunoreactive antibody to take place with CG or CG β subunit or their variant, the antibody that comprises anti-people CG or its variant, the antibody of anti-people CG β subunit or its variant, the antibody of anti-animal CG or its variant, the antibody of anti-animal CG β subunit or its variant.
11. method as claimed in claim 2 is characterized in that, described anti-CG antibody comprises: the anti-CG antibody that uses polyclonal antibody technology, monoclonal antibody technique or the preparation of DNA recombinant technology.
12. method as claimed in claim 2 is characterized in that, described anti-CG antibody comprises: contain the mixture of anti-CG antibody or the anti-CG antibody that purifying is crossed.
13. method as claimed in claim 12 is characterized in that, the described mixture that contains anti-CG antibody comprises: the animal serum, animal ascites or the cell culture fluid that contain anti-CG antibody.
14. method as claimed in claim 12 is characterized in that, the anti-CG antibody that described purifying is crossed comprises: animal serum, animal ascites or the cell culture fluid that will contain anti-CG antibody carries out the product behind the purifying.
15. the method for claim 1 is characterized in that, described purge process comprises uses immuno-precipitation and immunochromatographic method to carry out purifying.
16., it is characterized in that in the described follicular stimulating hormone, the biological value ratio of FSH and LH was not less than 100: 1 as each described method in the claim 1～15; In the preferred described follicular stimulating hormone, the ratio of the immunizing potency of FSH and LH was not less than 1000: 1; In the more preferably described follicular stimulating hormone, the ratio of the immunizing potency of FSH and LH was not less than 10000: 1.
17. a preparation of drug combination method that contains FSH said method comprising the steps of:
(A) raw material that contains FSH is contacted with antibody, wherein said raw material contains as the luteotropic hormone LH of impurity and/or chorionic gona dotropin CG, and described antibody is specificity in conjunction with LH and/or CG but the antibody of cross reaction does not take place with FSH basically, thereby makes described antibody and LH and/or CG formation antigen-antibody complex;
(B) antigen-antibody complex that forms is removed from described raw material, thus the FSH of acquisition purifying;
(C) it is formulated together to treat the FSH of the purifying that makes in the step (B) of significant quantity, pharmaceutically acceptable carrier and optional other activeconstituents, thereby forms described pharmaceutical composition.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010034198A1 (en) * | 2008-09-24 | 2010-04-01 | 上海天伟生物制药有限公司 | A method for removing/inactivating virus in glycoprotein |
| WO2010145125A1 (en) * | 2009-06-18 | 2010-12-23 | 上海天伟生物制药有限公司 | Highly purified human menopausal gonadotropins, method for preparing and uses 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 |
| CN102743742A (en) * | 2011-04-22 | 2012-10-24 | 上海天伟生物制药有限公司 | Glycoprotein composition with low solvent residues, its preparation and its application |
| 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 |
| CN103030691A (en) * | 2011-09-29 | 2013-04-10 | 长春金赛药业有限责任公司 | Method for separating and purifying gonadotropin glycoprotein subunits |
| CN102743742B (en) * | 2011-04-22 | 2016-12-14 | 上海天伟生物制药有限公司 | A kind of glycoprotein compositions of low solvent residue and its production and use |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1717042A1 (en) * | 1963-03-11 | 1972-11-09 | Serono Ist Farm | Method for obtaining gonadotropic pituitary hormones from urine |
| US5990288A (en) * | 1997-10-21 | 1999-11-23 | Vitro Diagnostics, Inc. | Method for purifying FSH |
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- 2007-09-11 CN CN 200710045768 patent/CN101307103B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010034198A1 (en) * | 2008-09-24 | 2010-04-01 | 上海天伟生物制药有限公司 | A method for removing/inactivating virus in glycoprotein |
| 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 |
| WO2010145125A1 (en) * | 2009-06-18 | 2010-12-23 | 上海天伟生物制药有限公司 | Highly purified human menopausal gonadotropins, method for preparing and uses 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 |
| CN102743742A (en) * | 2011-04-22 | 2012-10-24 | 上海天伟生物制药有限公司 | Glycoprotein composition with low solvent residues, its preparation and its application |
| WO2012142961A1 (en) * | 2011-04-22 | 2012-10-26 | 上海天伟生物制药有限公司 | Glycoprotein composition with low residual solvent level, and preparation method and use thereof |
| CN102743742B (en) * | 2011-04-22 | 2016-12-14 | 上海天伟生物制药有限公司 | A kind of glycoprotein compositions of low solvent residue and its production and use |
| CN103030691A (en) * | 2011-09-29 | 2013-04-10 | 长春金赛药业有限责任公司 | Method for separating and purifying gonadotropin glycoprotein subunits |
| CN103030691B (en) * | 2011-09-29 | 2014-10-08 | 长春金赛药业有限责任公司 | Method for separating and purifying gonadotropin glycoprotein subunits |
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