CN101798334B - Purification method of human parathyroid hormone (1-34) - Google Patents
Purification method of human parathyroid hormone (1-34) Download PDFInfo
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- CN101798334B CN101798334B CN 201010138985 CN201010138985A CN101798334B CN 101798334 B CN101798334 B CN 101798334B CN 201010138985 CN201010138985 CN 201010138985 CN 201010138985 A CN201010138985 A CN 201010138985A CN 101798334 B CN101798334 B CN 101798334B
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Abstract
The invention provides a purification method of human parathyroid hormone (1-34), which is suitable for industrialization and comprises the following steps: 1) taking octadecylsilane chemically bonded silica as a stationary phase, taking 0.05-0.2% trifluoroacetic acid aqueous solution as A phase and acetonitrile as B phase with the gradient of 20-40% of B%, and carrying out gradient elution to rough peptide solution; and 2) adopting the opposite-phase high performance liquid chromatography to convert the trifluoroacetic acid salt of human parathyroid hormone (1-34) into acetate. The purification method of human parathyroid hormone (1-34), which is provided by the invention, has simple and feasible operation, high purity and high yield, and produced human parathyroid hormone (1-34) can reach the industrialization requirement.
Description
Technical field
The present invention relates to the purification process of a peptide species, relate in particular to the purification process of human parathyroid hormone (1-34).
Background technology
Human parathyroid hormone (1-34) is also referred to as pTH (1-34), is the polypeptide that N-terminal 1-34 amino acid of human parathyroid hormone (Parathyroid Hormone, human) forms.Recombinant human parathyroid hormone (1-34) [rh pTH (1-34)] claim again teriparatide (teriparatide), by the research and development of U.S. Eli Lilly company, belongs to the gene recombination medicine, gone on the market by drugs approved by FDA in 2002.It is the 1st bone formation-promoter that is approved for the treatment serious osteoporosis, and more widely market application foreground is arranged.
Summary of the invention
The present invention proposes the method for a kind of purifying pTH (1-34), purity is high and yield good, reaches industrialized requirement.
For achieving the above object, technical scheme provided by the invention may further comprise the steps:
Step 1): take octadecylsilane chemically bonded silica as stationary phase, the trifluoroacetic acid aqueous solution take mass concentration as 0.05%-0.2% is the A phase, and acetonitrile is the B phase, and gradient: B%:20%～40% carries out gradient elution with thick peptide solution;
Described " thick peptide " refers to adopt liquid phase synthesizing method or solid-phase synthesis to obtain, and not yet passes through the human parathyroid hormone (1-34) that the thick peptide of human parathyroid hormone (1-34) of refinement treatment or purity can not fulfilling medicinals." thick peptide solution " refers to that thick peptide is dissolved in the resulting solution of acetonitrile solution, and employed water is pure water, and meets the water for injection standard, preferred ultrapure water, and concrete layoutprocedure sees sample preparation for details.The preferred chromatographically pure of the purity grade of " acetonitrile " of the present invention.A phase and B ratio mutually were greater than 20: 80, and less than 95: 5, the A phase can reach the purpose of thick peptide being carried out sharp separation with B in aforementioned proportion.
Step 1) hereinafter also is expressed as " purifying ".
Step 2) adopt reversed-phased high performace liquid chromatographic that the trifluoroacetate of human parathyroid hormone (1-34) is changed into acetate.Step 2) hereinafter also is expressed as " turning salt ".
The stationary phase of described " RPLC " is eight alkyl silane bonded silica gels, the method that turns salt is preferably: with the acetum flushing 15-30min that contains the 3%--8% acetonitrile, then use the acetonitrile wash-out, collect hPTH(1-34) solution; The concentration of acetum is 0.05%--0.2%, is preferably 0.1%.The acetonitrile concentration that elution step adopts is 30-60%, preferred 50%.
Operation is simple and feasible, purity is high, yield is high for purifying pTH provided by the invention (1-34) method, reaches industrialized requirement.Adopt purification process product purity of the present invention can reach more than 98% yield 25%.
The purifying scale comprises following specification chromatographic column (pillar diameter * length): 5cm * 25cm, 15cm * 25cm, 30cm * 25cm.
Embodiment
Embodiment one:
1. sample preparation: thick peptide is joined ultrapure water, add 20% acetonitrile, the ultrasonic sample that makes dissolves the rear membrane filtration of using fully, collects filtrate for later use.
2. purifying:
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm * 25cm.Moving phase: A phase: 0.05% trifluoroacetic acid aqueous solution; The B phase: acetonitrile, flow velocity: 50-80ml/min, gradient: B%:20%～40% detects wavelength: 280nm.Sample size is 1.5-3g.
Purge process: rinse chromatographic column well the back balance loading with the acetonitrile more than 50%, applied sample amount is 1.5-3g.Linear gradient elution 60min collects the purpose peak, human parathyroid hormone (1-34) solution of collecting is no more than under 35 ℃ the condition vacuum rotary steam in water temperature and is concentrated into approximately behind the 10-20mg/mL for subsequent use.
3. turn salt: chromatographic column: the chromatographic column take eight alkyl silane bonded silica gels as stationary phase, pillar diameter and length are: 5cm * 25cm.Rinse chromatographic column well rear loading with the acetonitrile acetum more than 50%, applied sample amount 1.5-3g, with 0.05% the acetum flushing 15-30min that contains 3% acetonitrile, then use 50% acetonitrile wash-out, collect the purpose peak, human parathyroid hormone (1-34) solution collected is no more than under 35 ℃ of conditions vacuum rotary steam in water temperature is concentrated into approximately and goes to suitable big or small cillin bottle behind the 150-200mg/mL.Can obtain purity after the lyophilize greater than 98% pTH (1-34).
Embodiment two:
1. sample preparation: thick peptide is joined ultrapure water, add 20% acetonitrile, the ultrasonic sample that makes dissolves the rear membrane filtration of using fully, collects filtrate for later use.
2. purifying:
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15cm * 25cm.Moving phase: A phase: 0.1% trifluoroacetic acid aqueous solution; The B phase: acetonitrile, flow velocity: 400-450ml/min, gradient: B%:20%～40% detects wavelength: 280nm.Sample size is 10-20g.
Purge process: rinse chromatographic column well the back balance loading with the acetonitrile more than 50%, applied sample amount is 10-20g.Linear gradient elution 60min collects the purpose peak, human parathyroid hormone (1-34) solution of collecting is no more than under 35 ℃ the condition vacuum rotary steam in water temperature and is concentrated into approximately behind the 10-20mg/mL for subsequent use.
3. turn salt: chromatographic column: the chromatographic column take eight alkyl silane bonded silica gels as stationary phase, pillar diameter and length are: 15cm * 25cm.Rinse chromatographic column well rear loading with the acetonitrile acetum more than 50%, applied sample amount 10-20g, with 0.1% the acetum flushing 15-30min that contains 5% acetonitrile, then use 50% acetonitrile wash-out, collect the purpose peak, human parathyroid hormone (1-34) solution collected is no more than under 35 ℃ of conditions vacuum rotary steam in water temperature is concentrated into approximately and goes to suitable big or small cillin bottle behind the 150-200mg/mL.Can obtain purity after the lyophilize greater than 98% pTH (1-34).
Embodiment three:
1. sample preparation: thick peptide is joined ultrapure water, add 20% acetonitrile, the ultrasonic sample that makes dissolves the rear membrane filtration of using fully, collects filtrate for later use.
2. purifying:
Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm * 25cm.Moving phase: A phase: 0.2% trifluoroacetic acid aqueous solution; The B phase: acetonitrile, flow velocity: 1800-2000ml/min, gradient: B%:20%～40% detects wavelength: 280nm.Sample size is 55-75g.
Purge process: rinse chromatographic column well the back balance loading with the acetonitrile more than 50%, applied sample amount is 55-75g.Linear gradient elution 60min collects the purpose peak, human parathyroid hormone (1-34) solution of collecting is no more than under 35 ℃ the condition vacuum rotary steam in water temperature and is concentrated into approximately behind the 10-20mg/mL for subsequent use.
3. turn salt: chromatographic column: the chromatographic column take eight alkyl silane bonded silica gels as stationary phase, pillar diameter and length are: 30cm * 25cm.Rinse chromatographic column well rear loading with the acetonitrile acetum more than 50%, applied sample amount 50-80g, with 0.2% the acetum flushing 15-30min that contains 8% acetonitrile, then use 50% acetonitrile wash-out, collect the purpose peak, human parathyroid hormone (1-34) solution collected is no more than under 35 ℃ of conditions vacuum rotary steam in water temperature is concentrated into approximately and goes to suitable big or small cillin bottle behind the 150-200mg/mL.Can obtain purity after the lyophilize greater than 98% pTH (1-34).
Claims (4)
1. the purification process of a human parathyroid hormone (1-34) is characterized in that may further comprise the steps:
Step 1): take octadecylsilane chemically bonded silica as stationary phase, the trifluoroacetic acid aqueous solution take 0.05%-0.2% is the A phase, and acetonitrile is the B phase, and gradient: B%:20%～40% carries out gradient elution with thick peptide solution,
Step 2): adopt reversed-phased high performace liquid chromatographic that the trifluoroacetate of human parathyroid hormone (1-34) is changed into acetate, this method that turns salt is: eight alkyl silane bonded silica gels, with the acetum flushing 15-30min that contains the 3%--8% acetonitrile, then use the acetonitrile wash-out of 30%--60%, collect hPTH(1-34) solution; Wherein the concentration of acetum is 0.05%--0.2%.
2. method according to claim 1 is characterized in that: A phase and B ratio mutually are greater than 20:80, less than 95:5 in the step 1).
3. method according to claim 1 and 2, it is characterized in that: the concentration of the trifluoroacetic acid aqueous solution of A phase is 0.1% in the step 1).
4. method according to claim 1, it is characterized in that: the concentration of acetum is 0.1%.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201010138985 CN101798334B (en) | 2010-03-30 | 2010-03-30 | Purification method of human parathyroid hormone (1-34) |
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| CN 201010138985 CN101798334B (en) | 2010-03-30 | 2010-03-30 | Purification method of human parathyroid hormone (1-34) |
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| CN101798334A CN101798334A (en) | 2010-08-11 |
| CN101798334B true CN101798334B (en) | 2013-02-13 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102584982B (en) * | 2012-02-10 | 2014-02-05 | 深圳翰宇药业股份有限公司 | Method for purifying solid-phase synthetic coarse liraglutide |
| CN102993274B (en) * | 2012-11-30 | 2014-08-20 | 深圳翰宇药业股份有限公司 | Purification method of ganirelix acetate |
| CN102993293B (en) * | 2012-12-05 | 2014-05-07 | 深圳翰宇药业股份有限公司 | Method for purifying teriparatide acetate |
| CN103122023B (en) * | 2013-03-08 | 2016-03-30 | 深圳翰宇药业股份有限公司 | A kind of purification process of triptorelin |
| CN111057141B (en) * | 2018-10-17 | 2023-11-14 | 深圳市健元医药科技有限公司 | Tripeptide refining process |
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Non-Patent Citations (3)
| Title |
|---|
| 王良友等.人甲状旁腺素（1-34）的合成与聚乙二醇化修饰.《中国生化药物杂志》.2005,第77页第2.1和2.2部分. * |
| 甲状旁腺素1-34肽的固相合成;白振锋;《合成化学》;20070620;第390页第1.4部分 * |
| 白振锋.甲状旁腺素1-34肽的固相合成.《合成化学》.2007,第390页第1.4部分. |
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