CN1415628A - Method for extracting luteinizing hormone from human urine - Google Patents
Method for extracting luteinizing hormone from human urine Download PDFInfo
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- CN1415628A CN1415628A CN 02136494 CN02136494A CN1415628A CN 1415628 A CN1415628 A CN 1415628A CN 02136494 CN02136494 CN 02136494 CN 02136494 A CN02136494 A CN 02136494A CN 1415628 A CN1415628 A CN 1415628A
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- lutropin
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- chromatography
- sepharose
- exchange chromatography
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Abstract
A process for extracting human uroxanthin auxin (HUA) from its coarse product includes such steps as anionic exchange chromatography, eluting, collecting the component containing HUA, ultrafilter for desalting, cationic exchange chromatography, eluting, collecting the component containing HUA, ultrafilter for desalting, hydrophobic chromatography, eluting, collecting the component containing HUA, ultrafilter, concentrating, gel filter, molecular sieve-gel chromatography, collecting, and freeze drying.
Description
Technical field:
The present invention relates to the method that a kind of people of extraction urinates lutropin.
Background technology:
It is that molecular weight is 29Kda, contains three sugar chains by the hypophysis excretory, by the non-covalent heterodimer glycoprotein that is combined into of two different subunits of α, β that the people urinates lutropin.The people is kept in the existence of sugar chain, and to urinate the lutropin biological value essential, and the variation of its terminal sialic acid content has formed different electric charge isomer.The α subunit that the people urinates lutropin has identical amino-acid sequence with Urofollitropin, thyrotropin, chorionic gonadotrophin, and the difference of β subunit has caused different biological activitys separately.
Prove after deliberation, developed country has 15～20% Mr. and Mrs not give birth to approximately, cause and do not give birth to multiple reason, the common cause of atocia is to cause ovulatory dysfunction because gonad-stimulating hormone lacks, the common cause of male sterility be sperm degenerate or quantity very little.It is all very important to the reproductive function of masculinity and femininity that the people urinates lutropin, for the male sex, it is spermatogeny and ripe necessary that the people urinates lutropin, for the women, it is that follicle maturity, egg cell division, ovulation, corpus luteum generate and keep the essential gonadotropin of progesterone secretion luteal phase that the people urinates lutropin.
Therefore, the people urinates lutropin and has therapeutic value, can be used for the treatment of the people and urinate a series of diseases that the lutropin shortage causes.And at present the people to urinate lutropin be to play a role by fixed proportion with Urofollitropin in the postmenopause urine promoted gonadofrophin preparation, clinical application has been caused very big restriction.
Summary of the invention:
The objective of the invention is to provide a kind of people of extraction to urinate the method for lutropin, urinate from postmenopause urine promoted gonadofrophin or people and extract highly purified people the lutropin crude product and urinate lutropin, to be used for clinical treatment.
The present invention is achieved in that urinating the lutropin crude product with postmenopause urine promoted gonadofrophin that extracts from postmenopausal women's urine of suitably tiring or people is starting raw material, wherein postmenopause urine promoted gonadofrophin or the people biological value of urinating the lutropin crude product does not wait from every milligram 1 international unit to 300 international unit, adopt suitable ion exchange layer analysis method, comprise anionic exchange medium chromatography or cation exchange medium chromatography, both can use wherein a kind of chromatography method, can use simultaneously by two kinds of chromatography methods again, and the front and back order can be exchanged, starting raw material postmenopause urine promoted gonadofrophin or people are urinated the lutropin crude product carry out preliminary purification, the anion-exchange chromatography medium, active group is diethylamino ethyl (DEAE), diethyl quaternary amine (Q), diethyl hydroxypropyl quaternary amine (QAE) etc., carrier is to be (Sepharose), dextran (Sephadex) and Mierocrystalline cellulose (Cellulose) etc., to select Q-Sepharose for use is excellent, to cation-exchange chromatography, the active group of chromatography media is sulfonic group (S), sulfonic acid propyl group (SP), carboxymethyl (CM) etc., carrier is agarose (Sepharose), dextran (Sephadex) and Mierocrystalline cellulose (Cellulose) etc., select for use headed by the SP-Sepharose, chromatography condition, ionic strength is 0.01M～0.5MNaCl or KCl, adopt 0～1M NaCl or KCl linear gradient elution, fraction collection or stepwise elution, collection or merging contain the people and urinate the lutropin active part, anion-exchange chromatography pH is controlled at 4.0～10.0, pH is at 3.8～pH7.5 in cation-exchange chromatography control, collect liquid and can or use acetone with lyophilize, organic solvent deposit methods such as ethanol obtain to be suitable for being further purified the dry-matter of usefulness, also can be with method concentrating and desalinatings such as ultrafiltration, be directly used in subsequent technique, with the ethanol sedimentation method is excellent, the alcoholic acid final concentration is 70～90% during precipitation, with 80% is excellent, dry-matter or concentrated solution with acquisition in above-mentioned are raw material, carrying out hydrophobic chromatography is further purified, chromatography media is Phenyl Sepharose or OctylSepharose or Butyl Sepharose, it is excellent selecting Phenyl Sepharose for use, adsorption conditions is pH4-8, and solution contains 0.5-2M NaCl or (NH
4)
2SO
4With NaCl is excellent, wash-out is made gradient elution or stepwise elution with the method that reduces salt concn, fraction collection contains the part that the people urinates lutropin, with lyophilize or use acetone, organic solvent deposit methods such as ethanol obtain to be suitable for being further purified the dry-matter of usefulness, also can be with method concentrating and desalinatings such as ultrafiltration, be directly used in subsequent technique, with the ethanol sedimentation method is excellent, the alcoholic acid final concentration is 70～90% during precipitation, 80% to be excellent,, with the molecular sieve gel filtration method people to be urinated lutropin and separate with remaining foreign protein from the dry-matter or the concentrated solution of above-mentioned acquisition.This step is by the pyrogen-free operation; molecular sieve gel is G-75 or G-100; gel skeleton is Sephadex or Superdex; also can be Sephacryl S-100; with SephadexG-100 at first; chromatography condition is pH4～8; damping fluid contains 0～0.5M NaCl; ultraviolet detection flows out, and fraction collection detects purity with FPLC; merge and to contain that the people urinates the lutropin activity and FPLC is the part of single component; the ultrafiltration desalination; concentrate, lyophilize, also can add dehydrated alcohol to final concentration is 70～90% precipitating proteins; precipitation is excellent with 80%; this step is dry to be excellent with freeze-drying, the final protein that obtains, and index becomes freeze-dried preparation in accordance with known methods; preparation contains appropriate excipients; can be N.F,USP MANNITOL; lactose; glycine; glucose; sucrose or their mixture also can contain adequate protective agent, as human serum albumin.
Since the present invention adopted ion exchange chromatography, hydrophobic chromatography and molecular sieve gel chromatography method thereby, just be able to from climacteric gonadotropin or people urinate and extract highly purified people the lutropin crude product and urinate lutropin, its biological value is every milligram 5000～15000 international unit, and the people urinates lutropin and the bioactive ratio of Urofollitropin greater than 100: 1.
Embodiment:
Below will describe the details that a kind of people of extraction who proposes according to the present invention urinates the method for lutropin in detail.
The method that the present invention urinates lutropin for a kind of people of extraction is promptly urinated from the people and is extracted the method that highly purified people urinates lutropin the lutropin crude product, and implementation step is as follows:
One, urinates lutropin crude product (LH activity 208IU/mg) through kaolin absorption, ethanol extracting, silicic acid filter absorption back preparation people after 55 years old～70 years old healthy women urine in 5～20 years after the collection menopause
Two, carry out the anion-exchange chromatography method crude product is carried out preliminary purification, earlier through Q-Sepharose FF (Amersham-Pharmacia company product) chromatography, chromatography condition is elected 0.01M Tris damping fluid as, pH9.5,0～1M NaCl linear gradient elution, collection contains the people and urinates the lutropin component, the ultrafiltration desalination;
Three, carrying out the cation-exchange chromatography method is further purified crude product, use SP-Sepharose FF (Amersham-Pharmacia company product) chromatography then, chromatography condition is elected 0.01M NaAc as, pH5.2,0～1M NaCl linear gradient elution, collection contains the people and urinates the lutropin component, the ultrafiltration desalination;
Four, carrying out the hydrophobic chromatography method is further purified, re-use phenyl-SepharoseFF (Amersham-Pharmacia company product) and carry out purifying, chromatography condition is elected 0.01M Tris damping fluid as, 1M NaCl, pH7.2, with 1M～0M NaCl linear gradient elution, collection contains the people and urinates lutropin component, ultrafiltration and concentration;
Five, carry out the molecular sieve gel chromatography method and the people is urinated lutropin separate with remaining foreign protein, use Sephadex G-100 gel-filtration at last, moving phase is 0.02M NH
4Ac, pH7.0.Collection contains the active ingredient that the people urinates lutropin, and lyophilize obtains the high purity people and urinates lutropin.
The people of 2000 editions appendix of product employing Chinese Pharmacopoeia urinates lutropin bioassay method (young rat seminal vesicle weightening finish method) mensuration people and urinates lutropin and tire, tire and be 9300IU/mg, adopt Urofollitropin biologic assay (young rat ovary weightening finish method) to measure Urofollitropin and tire, less than 90IU/mg.The people urinates the ratio of lutropin and Urofollitropin greater than 100 to 1.
Claims (1)
1. one kind is extracted the method that the people urinates lutropin, it is characterized in that urinating and extract highly purified people the lutropin crude product and urinate lutropin from postmenopause urine promoted gonadofrophin or people, it is through kaolin absorption by healthy women urine after the menopause that postmenopause urine promoted gonadofrophin or people urinate the lutropin crude product, the ethanol extracting, make after the pure aluminium silicate absorption, the biological value that postmenopause urine promoted gonadofrophin or people urinate the lutropin crude product is from every milligram 1 international unit to 300 international unit, at first postmenopause urine promoted gonadofrophin or people are urinated the lutropin crude product and carry out preliminary purification with the method for suitable anion-exchange chromatography and cation-exchange chromatography, anion-exchange chromatography, the active group of the chromatography media of anion-exchange chromatography can be diethylamino ethyl (DEAE), diethyl quaternary amine (Q), diethyl hydroxypropyl season (QAE) and so on, carrier is agarose (Sepharose), dextran (Sephadex) and Mierocrystalline cellulose (Cellulose) and so on, with Q-Sepharose is excellent, chromatography condition is pH4.0～10.0, ionic strength is 0.01M～0.5M NaCl or KCl, adopt 0～1MNaCl or KCl linear gradient elution, fraction collection or stepwise elution, merge the people and urinate the lutropin active part, cation-exchange chromatography, the active group of chromatography media can be sulfonic group (S), sulfonic acid propyl group (SP), carboxymethyl (CM) and so on, carrier can be elected agarose (Sepharose) as, dextran (Sephadex) and Mierocrystalline cellulose (Cellulose) and so on, with SP-Sepharose is excellent, chromatography condition is pH3.8～pH7.5, ionic strength is 0.01M～0.5M NaCl or KCl, adopt 0～1MNaCl or KCl linear gradient elution, fraction collection or stepwise elution, merge the people and urinate the lutropin active part, the order of cation-exchange chromatography or anion-exchange chromatography can be exchanged, also can only select wherein a kind of chromatography method for use, secondly be further purified with the hydrophobic chromatography method, medium is phenyl-sepharose or octyl-sepharose or butyl-sepharose, with phenyl-Sepharose is excellent, chromatography condition is pH4-8, and solution contains 0.5-2M NaCl or (NH
4)
2SO
4With NaCl is excellent; wash-out is made gradient elution or stepwise elution with the method that reduces salt concn; fraction collection contains the part that the people urinates lutropin; above-mentioned from anion-exchange chromatography; the processing of the collection liquid that obtains in cation-exchange chromatography and the hydrophobic chromatography method; can or use acetone with lyophilize; organic solvent deposit methods such as ethanol obtain to be suitable for being further purified the dry-matter of usefulness; also can be with method concentrating and desalinatings such as ultrafiltration; be directly used in subsequent technique; with the ethanol sedimentation method is excellent; the alcoholic acid final concentration is 70～90% during precipitation; with 80% is excellent; with the molecular sieve gel chromatography method people being urinated lutropin at last separates with remaining foreign protein; molecular sieve gel is G-75 or G-100; gel skeleton is Sephadex or Superdex; also can be Sephacryl S-100; with Sephadex G-100 is optimum; chromatography condition is pH4～8; damping fluid contains 0～0.5M NaCl; ultraviolet detection flows out; fraction collection; merging contains the people and urinates the lutropin active part; the ultrafiltration desalination; concentrate; lyophilize; also can add dehydrated alcohol to final concentration is 70～90% precipitating proteins; precipitation is excellent with 80%; this step is dry to be excellent with freeze-drying; the people who obtains by above method urinates lutropin; can make freeze-dried preparation in accordance with known methods; preparation contains appropriate excipients; can be N.F,USP MANNITOL; lactose; glycine; glucose; sucrose or their mixture; also can contain adequate protective agent; as human serum albumin; urinate the people that extraction obtains the lutropin crude product through above step from postmenopause urine promoted gonadofrophin or people and urinate lutropin; biological value is every milligram 5000～15000 international unit, and the people urinates lutropin and the bioactive ratio of Urofollitropin greater than 100: 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02136494 CN1415628A (en) | 2002-08-14 | 2002-08-14 | Method for extracting luteinizing hormone from human urine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02136494 CN1415628A (en) | 2002-08-14 | 2002-08-14 | Method for extracting luteinizing hormone from human urine |
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| CN1415628A true CN1415628A (en) | 2003-05-07 |
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| CN 02136494 Pending CN1415628A (en) | 2002-08-14 | 2002-08-14 | Method for extracting luteinizing hormone from human urine |
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Cited By (6)
| 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 |
| CN101348518B (en) * | 2007-12-14 | 2011-09-28 | 顾铭 | Method for purifying sea-mussel mucin by carboxymethyl ion exchange chromatography |
| CN101638427B (en) * | 2008-08-01 | 2014-03-05 | 上海泽润生物科技有限公司 | Method for purifying virus antigens |
| CN106866812A (en) * | 2017-02-27 | 2017-06-20 | 日照岚山生化制品有限公司 | A kind of method that various Urine proteins are extracted in the urine from women |
| CN107698676A (en) * | 2017-11-07 | 2018-02-16 | 江西浩然生物医药有限公司 | A kind of extraction preparation method of high-purity menotropins |
-
2002
- 2002-08-14 CN CN 02136494 patent/CN1415628A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101348518B (en) * | 2007-12-14 | 2011-09-28 | 顾铭 | Method for purifying sea-mussel mucin by carboxymethyl ion exchange chromatography |
| CN101638427B (en) * | 2008-08-01 | 2014-03-05 | 上海泽润生物科技有限公司 | Method for purifying virus antigens |
| 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 |
| CN106866812A (en) * | 2017-02-27 | 2017-06-20 | 日照岚山生化制品有限公司 | A kind of method that various Urine proteins are extracted in the urine from women |
| CN107698676A (en) * | 2017-11-07 | 2018-02-16 | 江西浩然生物医药有限公司 | A kind of extraction preparation method of high-purity menotropins |
| CN107698676B (en) * | 2017-11-07 | 2020-07-14 | 江西浩然生物医药有限公司 | Method for extracting and preparing high-purity menotrophin |
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