CN102268073B - Method for preparing somatostatin - Google Patents
Method for preparing somatostatin Download PDFInfo
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- CN102268073B CN102268073B CN 201110200804 CN201110200804A CN102268073B CN 102268073 B CN102268073 B CN 102268073B CN 201110200804 CN201110200804 CN 201110200804 CN 201110200804 A CN201110200804 A CN 201110200804A CN 102268073 B CN102268073 B CN 102268073B
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Abstract
The invention discloses a method for preparing somatostatin, and the method comprises the following steps: 1) dissolving reduced somatostatin crude peptide obtained by solid-phase synthesis in water for injection, carrying out gradient elution and purification with octadecyl-silane-bonded silica gel as a stationary phase, trifluoroacetic acid aqueous solution as a phase A and chromatographically pure acetonitrile as a phase B, and collecting a reduced refined peptide solution; 2) diluting the reduced refined peptide solution obtained in the step 1) with purified water till about 1g of peptide is in every liter of purified water, adjusting the pH value to 6-8 with dilute ammonia solution, and adding an appropriate amount of H2O2 for oxidation to obtain an oxidized crude peptide solution; 3) concentrating the oxidized crude peptide solution to a certain volume, carrying out gradient elution and purification with octadecyl-silane-bonded silica gel as a stationary phase, trifluoroacetic acid aqueous solution as a phase A and chromatographically pure acetonitrile as a phase B, and collecting an oxidized refined peptide solution; and 4) converting the oxidized refined peptide solution into an acetate. The method provided by the invention has the advantage of high yield, is simple to operate, meets industrial requirements, and is conducive to the promotion and the prepared product has high purity.
Description
Technical field
The invention belongs to the polypeptide field, be specifically related to a kind of method for preparing Somatostatin.
Background technology
Somatostatin, namely somatostatin, be that a kind of endogenous that exists in human body is regulated hormone, and different physiological roles is had restraining effect widely.Somatostatin English name: Somatostatin, molecular formula is: C
76H
104N
18O
19S
2, molecular weight 1637.89.Amino acid structure is abbreviated as:
Its molecular structural formula is as follows:
Somatostatin is synthetic by the delta cell of hypothalamic median eminence and pancreas islet, is separated from the hypothalamus of sheep by doctor Guillemin of Germany in 1973 to obtain and to illustrate its structure.Therefore doctor Guillemin obtains Nobel's medical science in 1977 and physiology prize.The Somatostatin chemical structure be in molecule with the tetradecapeptide of a pair of disulfide linkage, mainly be present in the central nervous system peripheral organs (as: stomach, intestines, pancreas, skin etc.) of unifying in human body, peak concentration sees with hypothalamus, but maximum with digestive tube content.
Internal secretion and external secretion, the inhibition stomach mucous membrane that the Somatostatin biological function can suppress body of gland growth hormone releasing, inhibition thyrotrophic hormone(TH) and adrenocortical hormone, inhibition pancreas discharges gastrin, suppresses intestinal mucosa release secretin and kidney release feritin; reducing portal venous pressure, loose biliary tract mouth expands approximately flesh, stimulates mononuclear phagocyte system and alleviate endotoxemia; the release of Platelet Activating Factor; directly or indirectly regulate the cytokine chain and produce cytoprotection, and can strengthen the antiproliferative effect of cancer therapy drug.The clinical serious acute esophageal varices bleeding that often is applied to, serious acute Stomach duodenum ulcerative hemorrhage, acute erosive gastritis or hemorrhagic gastritis, pancreas, courage and intestines assisting therapy repeatly, the assisting therapy of diabetic ketoacidosis.Some other effect and purposes are just along with its fundamental research and clinical deeply constantly being found.Therefore having a extensive future of Somatostatin, market demand increases just year by year.
Summary of the invention
The object of the present invention is to provide a kind of method for preparing Somatostatin, at first the method adopts high performance liquid chromatography to carry out purifying to the thick peptide of reduced form Somatostatin and obtains reduced form Somatostatin essence peptide, then reduced form Somatostatin essence peptide is carried out liquid-phase oxidation and obtain the oxidized form Somatostatin, and then adopt the high performance liquid chromatography purifying to obtain the high purity Somatostatin.
The object of the present invention is achieved like this, its method steps is:
1) the thick peptide of solid phase synthesis gained reduced form Somatostatin is dissolved with water for injection, take octadecylsilane chemically bonded silica as stationary phase, take trifluoroacetic acid aqueous solution as the A phase, trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying, collects reduced form essence peptide solution.
2) the first step gained reduced form essence peptide solution is diluted to approximately 1g peptide/1L purified water with purified water, regulates between PH to 6-8 with weak ammonia, add appropriate H
2O
2Oxidation obtains the thick peptide solution of oxidized form.
3) the thick peptide solution of oxidized form is concentrated into certain volume, take octadecylsilane chemically bonded silica as stationary phase, take phosphoric acid triethylamine solution as the A phase, trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying, collects oxidized form essence peptide solution.
4) convert oxidized form essence peptide solution to acetate.
The purifying scale comprises the chromatographic column of following specification: 2CM * 25CM (diameter * length), 5CM * 25CM, 15CM * 25CM
The invention has the advantages that the product purity that present method makes is high, yield is high, easy and simple to handle, be fit to very much suitability for industrialized production.
Embodiment
The below is described in further details the present invention with example:
Embodiment one:
1) the thick peptide of solid phase synthesis gained reduced form Somatostatin is dissolved with water for injection, take octadecylsilane chemically bonded silica as stationary phase, the chromatographic column specification is 2CM * 25CM (diameter * length); Take the 0.05%-0.2% trifluoroacetic acid aqueous solution as the A phase, trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying.Flow velocity: 15-20ml/min.Detect wavelength: 220nm.Gradient: B%:15%-35% 40-60min.Applied sample amount is 120-150mg.Collect reduced form essence peptide solution.
2) the first step gained reduced form essence peptide is diluted to approximately 1g peptide/1L purified water with purified water, regulates between PH to 6-8 with weak ammonia, add 0.01%-1.5%(v/v) H
2O
2Oxidation 1-3 hour, obtain the thick peptide solution of oxidized form standby.
3) the thick peptide solution of oxidized form is concentrated into 10-15ml at vacuum rotary steam below 35 ℃.Take octadecylsilane chemically bonded silica as stationary phase, the chromatographic column specification is 2CM * 25CM (diameter * length); Take PH2.5-3.5 phosphoric acid triethylamine solution as the A phase, trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying.Flow velocity: 15-20ml/min.Detect wavelength: 220nm.Gradient: B%:10%-30% 60-80min.Applied sample amount is 90-130mg.Collect oxidized form essence peptide solution.
4) convert oxidized form essence peptide solution to acetate, get the sand core funnel that 15-20g anionite-exchange resin is placed in suitable size, rinse to neutrality with ultrapure water and add oxidized form essence peptide solution, fully stir rear decompress filter and collect filtrate, add one to twice of purifying washing again in resin, decompress filter is collected filtrate, merges all filtrates and is concentrated into 10-15ml at vacuum rotary steam below 35 ℃.Be transferred in the 50ml cillin bottle, can obtain purity after lyophilize greater than 99.5% high purity Somatostatin.
Embodiment two:
1) the thick peptide of solid phase synthesis gained reduced form Somatostatin is dissolved with water for injection, take octadecylsilane chemically bonded silica as stationary phase, the chromatographic column specification is 5CM * 25CM (diameter * length); Take the 0.05%-0.2% trifluoroacetic acid aqueous solution as the A phase, trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying.Flow velocity: 80-120ml/min.Detect wavelength: 220nm.Gradient: B%:15%-35% 40-60min.Applied sample amount is 1200-1500mg.Collect reduced form essence peptide solution.
2) the first step gained reduced form essence peptide solution is diluted to approximately 1g peptide/1L purified water with purified water, regulates between PH to 6-8 with weak ammonia, add 0.01%-1.5%(v/v) H
2O
2Oxidation 1-3 hour, obtain the thick peptide solution of oxidized form standby.
3) the thick peptide solution of oxidized form is concentrated into 30-50ml at vacuum rotary steam below 35 ℃.Take octadecylsilane chemically bonded silica as stationary phase, the chromatographic column specification is 5CM * 25CM (diameter * length); Take PH2.5-3.5 phosphoric acid triethylamine solution as the A phase, trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying.Flow velocity: 80-120ml/min.Detect wavelength: 220nm.Gradient: B%:10%-30% 60-80min.Applied sample amount is 1-2g.Collect oxidized form essence peptide solution.
4) convert oxidized form essence peptide solution to acetate, get the sand core funnel that 40-50g anionite-exchange resin is placed in suitable size, rinse to neutrality with ultrapure water and add oxidized form essence peptide solution, fully stir rear decompress filter and collect filtrate, add one to twice of purifying washing again in resin, decompress filter is collected filtrate, merges all filtrates and is concentrated into 10-15ml at vacuum rotary steam below 35 ℃.Be transferred in the 50ml cillin bottle, can obtain purity after lyophilize greater than 99.5% high purity Somatostatin.
Embodiment three:
1) the thick peptide of solid phase synthesis gained reduced form Somatostatin is dissolved with water for injection, take octadecylsilane chemically bonded silica as stationary phase, the chromatographic column specification is 15CM * 25CM (diameter * length); Take the 0.05%-0.2% trifluoroacetic acid aqueous solution as the A phase, trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying.Flow velocity: 400-500ml/min.Detect wavelength: 220nm.Gradient: B%:15%-35% 40-60min.Applied sample amount is 20-30g.Collect reduced form essence peptide solution.
2) the first step gained reduced form essence peptide solution is diluted to approximately 1g peptide/1L purified water with purified water, regulates between PH to 6-8 with weak ammonia, add 0.01%-1.5%(v/v) H
2O
2Oxidation 1-3 hour, obtain the thick peptide solution of oxidized form standby.
3) the thick peptide solution of oxidized form is concentrated into 80-120ml at vacuum rotary steam below 35 ℃.Take octadecylsilane chemically bonded silica as stationary phase, the chromatographic column specification is 15CM * 25CM (diameter * length); Take PH2.5-3.5 phosphoric acid triethylamine solution as the A phase, trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying.Flow velocity: 400-500ml/min.Detect wavelength: 220nm.Gradient: B%:10%-30% 60-80min.Applied sample amount is 16-25g.Collect oxidized form essence peptide solution.
4) convert oxidized form essence peptide solution to acetate, get the sand core funnel that 250-300g anionite-exchange resin is placed in suitable size, rinse to neutrality with ultrapure water and add oxidized form essence peptide solution, fully stir rear decompress filter and collect filtrate, add one to twice of purifying washing again in resin, decompress filter is collected filtrate, merges all filtrates and is concentrated into 150-200ml at vacuum rotary steam below 35 ℃.Be transferred in the 50ml cillin bottle, every bottle of 15-20ml can obtain purity greater than 99.5% high purity Somatostatin after lyophilize.
Claims (1)
1. method for preparing Somatostatin is characterized in that method steps is:
1) the thick peptide of solid phase synthesis gained reduced form Somatostatin is dissolved with water for injection, take octadecylsilane chemically bonded silica as stationary phase, take trifluoroacetic acid aqueous solution as the A phase, trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying, collects reduced form essence peptide solution; Described trifluoroacetic acid aqueous solution concentration is 0.05%-0.2%;
2) the first step gained reduced form essence peptide solution is diluted to 1g peptide/1L purified water with purified water, regulates between PH to 6-8 with weak ammonia, add appropriate H
2O
2Oxidation obtains the thick peptide solution of oxidized form, H
2O
2Concentration is volume percent 0.01%-1.5%, and the time of oxidation is 1-3 hour;
3) the thick peptide solution of oxidized form is concentrated into certain volume at vacuum rotary steam below 35 ℃, take octadecylsilane chemically bonded silica as stationary phase, take phosphoric acid triethylamine solution as the A phase, trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying, collects oxidized form essence peptide solution; Phosphoric acid triethylamine solution pH value is 2.5-3.5;
4) convert oxidized form essence peptide solution to acetate, get anionite-exchange resin and be placed in sand core funnel, rinse to neutrality with ultrapure water and add oxidized form essence peptide solution, fully stir rear decompress filter and collect filtrate, then add purifying washing one to twice in resin, decompress filter is collected filtrate, merge all filtrates, concentrated at vacuum rotary steam below 35 ℃, concentrated solution is transferred in cillin bottle, can obtain purity after lyophilize greater than 99.5% high purity Somatostatin.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102702344A (en) * | 2012-06-14 | 2012-10-03 | 上海吉尔多肽有限公司 | Method for purifying tesamorelin |
| CN102827258A (en) * | 2012-10-08 | 2012-12-19 | 吉尔生化（上海）有限公司 | Method for purifying Enfuvirtide |
| CN103275189B (en) * | 2013-06-06 | 2014-08-06 | 深圳翰宇药业股份有限公司 | Cracking liquid for peptide resin, and application thereof in synthesizing somatostatin by solid phase cracking |
| CN108774285B (en) * | 2018-07-03 | 2020-06-02 | 北京市新里程医药科技有限公司 | Preparation method of somatostatin and pharmaceutical composition thereof |
| CN109180803B (en) * | 2018-10-18 | 2022-02-08 | 成都天台山制药有限公司 | Somatostatin and preparation method and pharmaceutical composition thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1508152A (en) * | 2002-12-17 | 2004-06-30 | 常州市第四制药厂有限公司 | Somatostatin full-synthesis method |
| CN1749748A (en) * | 2005-07-14 | 2006-03-22 | 天津药业研究院有限公司 | Method for analysizing amino acid |
| CN101787071A (en) * | 2010-02-26 | 2010-07-28 | 深圳翰宇药业股份有限公司 | Purification method of vapreotide |
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2011
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1508152A (en) * | 2002-12-17 | 2004-06-30 | 常州市第四制药厂有限公司 | Somatostatin full-synthesis method |
| CN1749748A (en) * | 2005-07-14 | 2006-03-22 | 天津药业研究院有限公司 | Method for analysizing amino acid |
| CN101787071A (en) * | 2010-02-26 | 2010-07-28 | 深圳翰宇药业股份有限公司 | Purification method of vapreotide |
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Effective date of registration: 20151111 Address after: 330000, Jiangxi, Nanchang hi tech Zone planning five North, planning three West Patentee after: JIANGXI HAORAN BIO-PHARMA CO., LTD. Address before: Private science and Technology Park in Jiangxi province Nanchang City 330000 min Fu Road No. 209 Patentee before: Nanchang Baitai Biotechnology Co., Ltd. |