CN102702344A - Method for purifying tesamorelin - Google Patents
Method for purifying tesamorelin Download PDFInfo
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- CN102702344A CN102702344A CN2012101946454A CN201210194645A CN102702344A CN 102702344 A CN102702344 A CN 102702344A CN 2012101946454 A CN2012101946454 A CN 2012101946454A CN 201210194645 A CN201210194645 A CN 201210194645A CN 102702344 A CN102702344 A CN 102702344A
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Abstract
The invention discloses a method for purifying tesamorelin by using a reverse efficient liquid chromatography. The method comprises the following steps of: (1) dissolving a tesamorelin rough peptide obtained through solid phase synthesis with deionized water, performing gradient elution purification by using a reverse phase silica gel column of which fixed phase is tetraalkyl silane bonded silica gel, octadecyl silane bonded silica gel or octadecyl bonded silica gel and the moving phases consist of a triethylamine phosphate buffering solution serving as a phase A and chromatograph pure acetonitrile serving as a phase B, and collecting a peptide solution of a target peak value; (2) rotationally evaporating and concentrating the obtained peptide solution under reduced pressure, and leaving a concentrated solution for later use; (3) transforming into acetate by exchanging an anion exchange resin; and (4) performing rotary evaporation, concentration and freeze drying on a final high-purity peptide acetate solution under reduced pressure once again to obtain a powdery finished product. The invention provides a method which is suitable for industrially purifying tesamorelin. According to the method, fine peptide of which the purity is more than 98 percent can be obtained, batch production can be realized, and the requirements of high purity, high yield, low cost and high efficiency can be met.
Description
Technical field
The invention belongs to the reversed-phase HPLC technical field, especially relate to a kind of mass industrialization purifying and replace the not method of Rayleigh (Tesamorelin).
Background technology
Replacing not, the Rayleigh is a kind of somatotropin releasing factor (GRF) analogue; Be to adopt the synthetic method to produce, it comprises 44 the aminoacid sequence trans-3-hexenoyl-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH among the human body GRF
2At present to replacing not discovering of Rayleigh; Replacing not, the Rayleigh has the superfatted effect of human immunodeficiency virus (H IV) infected patient midriff that can stop lipodystrophy; This discovery, make for Rayleigh not when HIV patient's pharmacological agent this too much spinoff of the stomach fat that produces have milestone inthe.
When carrying out antiviral therapy, medicine also can disturb some normal function of human body sometimes for HIV patient.The ultimate aim of medicament research and development always hopes to invent medicine safer, more effective, that human body more is prone to tolerance.As you know, " being three fens poison of medicine ", most of AIDS patients, clinician think: present anti-AIDS drug is also imperfect, thus key is how we monitor the result of treatment that obtains maximum to his spinoff.The phenomenon that visceral adipose tissue is accumulated occurs during a lot of HIV the infected's ARTs, this process is relevant with the cardiovascular risk increase, goes down for a long time, and HIV patient can jeopardize life because of the spinoff of pharmacological agent equally.The investigator replaces the not effect of Rayleigh minimizing visceral adiposity to study to using GRFA, and being resolved with expectation has the spinoff that medicine is produced now.
Replace not nafarelin acetate salt (tesamorelin; Commodity are called Egrifta); By the development of Canadian Theratechnologies company, November 10 in 2010, the approval of Nikkei U.S. FDA was exclusively gone on the market by EMD Serono company in the U.S., was used for the treatment of HIV dependency lipodystrophy; Be the first medicine that is used to treat lipodystrophy of FDA approval, and Egrifta effective constituent is exactly to replace not Rayleigh (tesamorelin) acetate.Present domestic Dichlorodiphenyl Acetate salt for the record of Rayleigh does not seldom almost have, and more lacks the not methods of Rayleigh of replacing in a large number of producing.To the effective curative effect that is had side effects when treat HIV the Rayleigh, will be indispensable material medicine product on the market in the future for Rayleigh not, and now synthetic replace not Rayleigh bulk drug, also bring into schedule.
To replacing the not demand of Rayleigh for acetate in the market; The present invention utilize reversed-phased high performace liquid chromatographic to solid phase synthesis for Rayleigh not carrying out separation and purification; Production that not only can mass; And can obtain highly purifiedly for Rayleigh not, and not only solved in short supply for Rayleigh not of acetate in the market, also for the drug research that reduces the visceral adiposity effect for Rayleigh not more competent raw materials requirement is provided simultaneously for research institution.
Summary of the invention
The object of the present invention is to provide one, to be suitable for industrialization pure for the method for Rayleigh not, mainly solves prior art and can not separate the high purity that obtains industrialization for the technical problem of Rayleigh not.
For realizing above-mentioned purpose, technical scheme of the present invention is following: a kind of purifying replaces the not method of Rayleigh, comprises the steps:
1) the solid phase synthesis gained use deionized water dissolving for the thick peptide in Rayleigh not, stationary phase is the reverse phase silica gel post, and moving phase is two phases, and wherein phosphoric acid triethylamine buffered soln is the A phase, and trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying, the peptide solution of collection purpose peak value;
2) resulting peptide solution is carried out vacuum rotary steam and concentrate, stay liquid concentrator subsequent use;
3) peptide solution after vacuum rotary steam is concentrated adopts the anionite-exchange resin exchange to change into acetate;
4) the vacuum rotary steam concentrated frozen is dry once more with final high purity acetic acid peptide solution, gets Powdered finished product.
Preferred scheme is: the pH of the phosphoric acid triethylamine damping fluid of said A phase is 2.0 ~ 3.5.
More preferred scheme is: the pH of the phosphoric acid triethylamine damping fluid of said A phase is 2.5 ~ 3.0.
Preferred scheme is: said trifluoroacetic acid aqueous solution is that the mass percentage concentration of Mobile phase B is 12-65%, and the detection wavelength is 220nm.
More preferred scheme is: said trifluoroacetic acid aqueous solution is that the mass percentage concentration of Mobile phase B is 15-55%.
Described reverse phase silica gel post is a kind of in tetraalkyl silane group silica gel, eight alkyl silane bonded silica gels or the octadecyl silane.
Described anionite-exchange resin is German bright Sheng negative resin (Lewatit) MP60 or Mitsubishi chemistry negative resin (Diaion) WA-30.
The invention has the beneficial effects as follows: one of proposition of the present invention is suitable for the pure not process method of Rayleigh of replacing of industrialization, uses the reversed-phased high performace liquid chromatographic purifying to replace not Rayleigh, and change salt with the anionite-exchange resin exchange; Can not only obtain HPLC purity greater than 98.0% smart peptide; And can mass production, reaching the purity height, yield is high; Cost is low, the requirement that efficient is fast.
Embodiment
Embodiment 1
1. sample preparation: will synthesize gained for Rayleigh not with deionized water dissolving (concentration of ordinary dissolution is about 50mg/ml), collect filtrate for later use after using the aperture as the 0.45um membrane filtration;
2. purifying
Purification condition: chromatographic column: with the silica gel bonded silica gel of tetraalkyl is the chromatographic column of stationary phase, and the pillar diameter with length is: 5cm * 25cm. moving phase: A mutually: phosphate aqueous solution is transferred pH to 2.3 with analytically pure triethylamine solution; B phase: trifluoroacetic acid aqueous solution.Flow velocity: 40-55ml/min.Detect wavelength: 220nm.Gradient: the mass percentage concentration of Mobile phase B: 12-60%, gradient treatment time 30-50min.Sample size is 1.05g;
Purge process: rinse chromatographic column well back with the acetonitrile of mass percentage concentration more than 60% and go up appearance, applied sample amount is a sample solution.Linear gradient elution is collected the purpose peak, places in the receiving flask subsequent use with collecting good peptide solution;
3. the peptide solution of collecting behind the purifying is carried out vacuum rotary steam and concentrate, thickening temperature is no more than 37 ℃, mainly is the concentration that reduces organic solvent (mainly being acetonitrile) in the peptide solution, stays liquid concentrator subsequent use;
4. commentaries on classics salt: get 50g anionite-exchange resin Lewatit MP60 and place sizeable commentaries on classics salt glass column; Use ultrapure water, ethanol, alkali cleaning; Series of steps such as pickling are washed to neutrality and are gone up the appearance use again; Peptide solution after concentrating poured into to be changeed in the salt glass column, and the flow velocity through the controlled liq sample reaches the purpose of changeing salt, collects the peptide solution that changes after the salt simultaneously;
5. will change all peptide solutions of gained after the salt, and be evaporated to 1g/50ml, thickening temperature is no more than 37 ℃, then lyophilize get purity greater than 98.0% for Rayleigh not, purification yield can get more than 40%.
Embodiment 2
1. sample preparation: will synthesize gained for Rayleigh not with deionized water dissolving (concentration of ordinary dissolution is about 50mg/ml), collect filtrate for later use after using the aperture as the 0.45um membrane filtration;
2. purifying
Purification condition: chromatographic column: with eight alkyl silica gel bonded silica gel is the chromatographic column of stationary phase, and the pillar diameter with length is: 5cm * 25cm. moving phase: A mutually: phosphate aqueous solution is transferred pH to 3.4, B phase: trifluoroacetic acid aqueous solution with analytically pure triethylamine solution.Flow velocity: 40-55ml/min.Detect wavelength: 220nm.Gradient: the mass percentage concentration of Mobile phase B: 12 ~ 55%, gradient treatment time 40-60min.Sample size is 1.10g;
Purge process: rinse chromatographic column well back with the acetonitrile of mass percentage concentration more than 60% and go up appearance, applied sample amount is a sample solution.Linear gradient elution is collected the purpose peak, places in the receiving flask subsequent use with collecting good peptide solution;
3. the peptide solution of collecting behind the purifying is carried out vacuum rotary steam and concentrate, thickening temperature is no more than 37 ℃, mainly is the concentration that reduces organic solvent (mainly being acetonitrile) in the peptide solution, stays liquid concentrator subsequent use;
4. commentaries on classics salt: get 50g anionite-exchange resin Diaion WA-30 and place sizeable commentaries on classics salt glass column; Use ultrapure water, ethanol, alkali cleaning; Series of steps such as pickling are washed to neutrality and are gone up the appearance use again; Peptide solution after concentrating poured into to be changeed in the salt glass column, and the flow velocity through the controlled liq sample reaches the purpose of changeing salt, collects the peptide solution that changes after the salt simultaneously;
5. will change all peptide solutions of gained after the salt, and be evaporated to 1g/50ml, thickening temperature is no more than 37 ℃, then lyophilize get purity greater than 98.0% for Rayleigh not, purification yield can get more than 43%.
Embodiment 3
1. sample preparation: will synthesize gained for Rayleigh not with deionized water dissolving (concentration of ordinary dissolution is about 50mg/ml), collect filtrate for later use after using the aperture as the 0.45um membrane filtration;
2. purifying
Purification condition: chromatographic column: with the 18 alkyl silica gel bonded silica gel is the chromatographic column of stationary phase, and the pillar diameter with length is: 5cm * 25cm. moving phase: A mutually: phosphate aqueous solution is transferred pH to 2.5 with analytically pure triethylamine solution; B phase: trifluoroacetic acid aqueous solution.Flow velocity: 40-55ml/min.Detect wavelength: 220nm.Gradient: the mass percentage concentration of Mobile phase B: 15-55%, gradient treatment time 40-60min.Sample size is 1.11g;
Purge process: rinse chromatographic column well back with the acetonitrile of mass percentage concentration more than 60% and go up appearance, applied sample amount is a sample solution.Linear gradient elution is collected the purpose peak, places in the receiving flask subsequent use with collecting good peptide solution;
3. the peptide solution of collecting behind the purifying is carried out vacuum rotary steam and concentrate, thickening temperature is no more than 37 ℃, mainly is the concentration that reduces organic solvent (mainly being acetonitrile) in the peptide solution, stays liquid concentrator subsequent use;
4. commentaries on classics salt: get 50g anionite-exchange resin Lewatit MP60 and place sizeable commentaries on classics salt glass column; Use ultrapure water, ethanol, alkali cleaning; Series of steps such as pickling are washed to neutrality and are gone up the appearance use again; Peptide solution after concentrating poured into to be changeed in the salt glass column, and the flow velocity through the controlled liq sample reaches the purpose of changeing salt, collects the peptide solution that changes after the salt simultaneously;
5. will change all peptide solutions of gained after the salt, and be evaporated to 1g/50ml, thickening temperature is no more than 37 ℃, then lyophilize get purity greater than 98.0% for Rayleigh not, purification yield can get more than 48%.
Embodiment 4
1. sample preparation: will synthesize gained for Rayleigh not with deionized water dissolving (concentration of ordinary dissolution is about 50mg/ml), collect filtrate for later use after using the aperture as the 0.45um membrane filtration;
2. purifying
Purification condition: chromatographic column: with the 18 alkyl silica gel bonded silica gel is the chromatographic column of stationary phase, and the pillar diameter with length is: 10cm * 100cm. moving phase: A mutually: phosphate aqueous solution is transferred pH to 3.0 with analytically pure triethylamine solution; B phase: trifluoroacetic acid aqueous solution.Flow velocity: 80-120ml/min.Detect wavelength: 220nm.Gradient: the mass percentage concentration of Mobile phase B: 12-65%, gradient treatment time 60-100min.Sample size is 11.18g;
Purge process: rinse chromatographic column well back with the acetonitrile of mass percentage concentration more than 60% and go up appearance, applied sample amount is a sample solution.Linear gradient elution is collected the purpose peak, places in the receiving flask subsequent use with collecting good peptide solution;
3. the peptide solution of collecting behind the purifying is carried out vacuum rotary steam and concentrate, thickening temperature is no more than 37 ℃, mainly is the concentration that reduces organic solvent (mainly being acetonitrile) in the peptide solution, stays liquid concentrator subsequent use;
4. commentaries on classics salt: get 50g anionite-exchange resin Lewatit MP60 and place sizeable commentaries on classics salt glass column; Use ultrapure water, ethanol, alkali cleaning; Series of steps such as pickling are washed to neutrality and are gone up the appearance use again; Peptide solution after concentrating poured into to be changeed in the salt glass column, and the flow velocity through the controlled liq sample reaches the purpose of changeing salt, collects the peptide solution that changes after the salt simultaneously;
5. will change all peptide solutions of gained after the salt, and be evaporated to 1g/50ml, thickening temperature is no more than 37 ℃, then lyophilize get purity greater than 98.0% for Rayleigh not, purification yield can get more than 55%.
Claims (7)
1. a purifying replaces the not method of Rayleigh, it is characterized in that comprising the steps:
1) the solid phase synthesis gained use deionized water dissolving for the thick peptide in Rayleigh not, stationary phase is the reverse phase silica gel post, and moving phase has two phases, and phosphoric acid triethylamine buffered soln is the A phase, and trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying, the peptide solution of collection purpose peak value;
2) resulting peptide solution is carried out vacuum rotary steam and concentrate, stay liquid concentrator subsequent use;
3) peptide solution after vacuum rotary steam is concentrated adopts the anionite-exchange resin exchange to change into acetate;
4) the vacuum rotary steam concentrated frozen is dry once more with final high purity acetic acid peptide solution, gets Powdered finished product.
2. a kind of purifying according to claim 1 replaces the not method of Rayleigh, and it is characterized in that: the pH of said A phase phosphoric acid triethylamine damping fluid is 2.0 ~ 3.5.
3. a kind of purifying according to claim 2 replaces the not method of Rayleigh, and it is characterized in that: the pH of said A phase phosphoric acid triethylamine damping fluid is 2.5 ~ 3.0.
4. replace the not method of Rayleigh according to the said a kind of purifying of claim 1, it is characterized in that: the mass percentage concentration of said B phase trifluoroacetic acid aqueous solution is 12-65%, and the detection wavelength is 220nm.
5. a kind of purifying according to claim 4 replaces the not method of Rayleigh, and it is characterized in that: the mass percentage concentration of said B phase trifluoroacetic acid aqueous solution is 15-55%.
6. a kind of purifying according to claim 1 is characterized in that for the method for Rayleigh not: described reverse phase silica gel post is a kind of in tetraalkyl silane group silica gel, eight alkyl silane bonded silica gels or the octadecyl silane.
7. a kind of purifying according to claim 1 replaces the not method of Rayleigh, it is characterized in that: described anionite-exchange resin model is bright Sheng MP60 of Germany or Mitsubishi chemistry WA-30.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2012101946454A CN102702344A (en) | 2012-06-14 | 2012-06-14 | Method for purifying tesamorelin |
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| CN2012101946454A CN102702344A (en) | 2012-06-14 | 2012-06-14 | Method for purifying tesamorelin |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827258A (en) * | 2012-10-08 | 2012-12-19 | 吉尔生化（上海）有限公司 | Method for purifying Enfuvirtide |
| CN104829695A (en) * | 2015-05-04 | 2015-08-12 | 吉尔生化（上海）有限公司 | Purifying method for alarelin |
| CN105949284A (en) * | 2016-05-26 | 2016-09-21 | 吉尔生化（上海）有限公司 | Method for purifying sinapultide |
| CN110818790A (en) * | 2019-10-31 | 2020-02-21 | 成都圣诺生物制药有限公司 | Preparation method of temeprelin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1320354C (en) * | 2005-07-14 | 2007-06-06 | 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 |
| CN102268073A (en) * | 2011-07-19 | 2011-12-07 | 南昌佰泰生物科技有限公司 | Method for preparing somatostatin |
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2012
- 2012-06-14 CN CN2012101946454A patent/CN102702344A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1320354C (en) * | 2005-07-14 | 2007-06-06 | 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 |
| CN102268073A (en) * | 2011-07-19 | 2011-12-07 | 南昌佰泰生物科技有限公司 | Method for preparing somatostatin |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827258A (en) * | 2012-10-08 | 2012-12-19 | 吉尔生化（上海）有限公司 | Method for purifying Enfuvirtide |
| CN104829695A (en) * | 2015-05-04 | 2015-08-12 | 吉尔生化（上海）有限公司 | Purifying method for alarelin |
| CN104829695B (en) * | 2015-05-04 | 2018-09-07 | 吉尔生化（上海）有限公司 | A method of purifying alarelin |
| CN105949284A (en) * | 2016-05-26 | 2016-09-21 | 吉尔生化（上海）有限公司 | Method for purifying sinapultide |
| CN110818790A (en) * | 2019-10-31 | 2020-02-21 | 成都圣诺生物制药有限公司 | Preparation method of temeprelin |
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Application publication date: 20121003 |