CN101781367A - Method for purifying human parathyroid hormone (1 to 34) - Google Patents
Method for purifying human parathyroid hormone (1 to 34) Download PDFInfo
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- CN101781367A CN101781367A CN201010116391A CN201010116391A CN101781367A CN 101781367 A CN101781367 A CN 101781367A CN 201010116391 A CN201010116391 A CN 201010116391A CN 201010116391 A CN201010116391 A CN 201010116391A CN 101781367 A CN101781367 A CN 101781367A
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- lepirudin
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Abstract
The invention relates to a method for purifying human parathyroid hormone (1 to 34). The invention provides a method suitable to industrially purify lepirudin, which comprises the following steps: (1) using octadecylsilane chemically bonded silica as a solid phase, using a phosphate buffered solution the pH value of which is between 2.5 and 3.5 as an A phase, using acetonitrile as a B phase and carrying out gradient elution on crude peptide solution, wherein the gradient B percent is between 17 and 32 percent; (2) and adopting the reversed phase high performance liquid chromatography to convert lepirudin into acetate. The method for purifying lepirudin, which is provided by the invention, has simple operation, high product purity and good yield and reaches the industrialization requirements.
Description
Technical field
The present invention relates to the purification process of a peptide species, relate in particular to the purification process of Lepirudin.
Background technology
Lepirudin (Lepirudin) is a kind of polypeptide drugs that are used for the treatment of illnesss such as thrombotic disease, cephalophyma, subcutaneous hematoma, and it is effective and side effect is little, has good market outlook.In disclosed document and patent, do not have and report the purification process that is applicable to scale operation and has high yield.
Summary of the invention
The present invention proposes a kind of purification process that is suitable for the Lepirudin of industrialization, purity height and yield are good.
For achieving the above object, technical scheme provided by the invention may further comprise the steps:
1) with the octadecylsilane chemically bonded silica being stationary phase, is that the phosphate buffer soln of 2.5-3.5 is the A phase with the pH value, and acetonitrile is the B phase, and gradient: B%:17%～32% carries out gradient elution with thick peptide solution; The phosphate buffer soln amount concentration of A phase is less than 0.2%mol/L.
Described " thick peptide " be meant and adopt liquid phase synthesizing method or solid-phase synthesis to obtain, and do not pass through the Lepirudin that the thick peptide of Lepirudin of refinement treatment or purity can not fulfilling medicinals as yet." thick peptide solution " is meant the water-soluble resulting solution of thick peptide, and water preferably meets water for injection standard, more preferably ultrapure water.The preferred chromatographically pure of the purity grade of " acetonitrile ".Step 1) hereinafter also is expressed as purifying.
2) adopt reversed-phased high performace liquid chromatographic that Lepirudin is changed into acetate.Step 2) hereinafter also is expressed as " commentaries on classics salt ".
The method of changeing salt is preferably: the stationary phase of described " RPLC " is an octadecylsilane chemically bonded silica, is the A phase with mass concentration 0.05%--0.2% aqueous acetic acid; Acetonitrile is the B phase, and gradient: B%:6%～36% carries out gradient elution.The concentration preferred 0.1% of A phase aqueous acetic acid.The preferred chromatographically pure of the purity grade of " acetonitrile ".
Lepirudin peptide purification method provided by the invention, simple to operate, purity is high, yield good, reaches industrialized requirement.
The purifying scale comprises following specification chromatographic column (pillar diameter * length): 50mm * 300mm, 150mm * 300mm, 300mm * 300mm.
Embodiment
Embodiment one:
1. sample preparation: thick peptide water dissolution makes sample dissolve the back membrane filtration fully, the collection filtrate for later use.
2. purifying:
Purification condition: chromatographic column: with the octadecylsilane chemically bonded silica is the chromatographic column of stationary phase, and pillar diameter and length are: 50mm * 300mm.Moving phase: A phase: amount concentration is that the 0.2%mol/L phosphoric acid solution is transferred pH to 2.5～3.5 with triethylamine; The B phase: acetonitrile, flow velocity: 70-80ml/min, gradient: B%:17%～32% detects wavelength: 230nm.Sample size is 1.3-1.5g.
Purge process: rinse chromatographic column well in back balance sample with the acetonitrile more than 50%, applied sample amount is 1.3-1.5g.Linear gradient elution 45min collects the purpose peak, is no more than 30 ℃ conditions under vacuum rotary steam be concentrated into about 100～120mg/ml after standby in water temperature the Lepirudin solution collected.
3, change salt: with the octadecylsilane chemically bonded silica is the chromatographic column of stationary phase, and pillar diameter and length are: 50mm * 300mm.Moving phase: A phase: 0.1% aqueous acetic acid; The B phase: trifluoroacetic acid aqueous solution, flow velocity: 70-80ml/min detects wavelength: 230nm, gradient: B%:6%～36%.Sample size is 1.6-2.0g.
Change the salt process: rinse chromatographic column well back with the acetonitrile more than 50% and go up sample, applied sample amount is the 150-200ml sample solution.Linear gradient elution 30min collects the purpose peak, the Lepirudin solution of collecting is merged to revolve to steam go to suitable big or small cillin bottle after being concentrated into about 50-80mg/ml, carries out lyophilize then, can obtain purity greater than 98% Lepirudin.
Embodiment two:
1. sample preparation: thick peptide water dissolution makes sample dissolve the back membrane filtration fully, the collection filtrate for later use.
2. purifying for the first time: purification condition: chromatographic column: with the octadecylsilane chemically bonded silica is the chromatographic column of stationary phase, and pillar diameter and length are: 150mm * 300mm.Moving phase: A phase: the 0.2%mol/L phosphoric acid solution is transferred pH to 2.5～3.5 with triethylamine; The B phase: acetonitrile, flow velocity: 450-550ml/min, gradient: B%:17%～32% detects wavelength: 230nm.Sample size is 13-15g.
Purge process: rinse chromatographic column well in back balance sample with the acetonitrile more than 50%, applied sample amount is 13-15g.Linear gradient elution 60min collects the purpose peak, is no more than 30 ℃ conditions under vacuum rotary steam be concentrated into about 70-80mg/ml after standby in water temperature the Lepirudin solution collected.
3, change salt: with the octadecylsilane chemically bonded silica is the chromatographic column of stationary phase, and pillar diameter and length are: 150mm * 300mm.Moving phase: A phase: 0.1% aqueous acetic acid; The B phase: trifluoroacetic acid aqueous solution, flow velocity: 450-550ml/min detects wavelength: 230nm, gradient: B%:6%～36%.Sample size is 15-20g.
Change the salt process: rinse chromatographic column well back with the acetonitrile more than 50% and go up sample, applied sample amount is the 1200-1600ml sample solution.Linear gradient elution 60min collects the purpose peak, the Lepirudin solution of collecting is merged to revolve to steam go to suitable big or small cillin bottle after being concentrated into about 50-80mg/ml, carries out lyophilize then, can obtain purity greater than 98% Lepirudin.
Embodiment three:
1. sample preparation: thick peptide water dissolution makes sample dissolve the back membrane filtration fully, the collection filtrate for later use.
2. purifying for the first time: purification condition: chromatographic column: with the octadecylsilane chemically bonded silica is the chromatographic column of stationary phase, and pillar diameter and length are: 300mm * 300mm.Moving phase: A phase: the 0.2%mol/L phosphoric acid solution is transferred pH to 2.5～3.5 with triethylamine; The B phase: acetonitrile, flow velocity: 1900-2200ml/min, gradient: B%:17%～32% detects wavelength: 230nm.Sample size is 55-75g.
Purge process: rinse chromatographic column well in back balance sample with the acetonitrile more than 50%, applied sample amount is 55-75g.Linear gradient elution 120min collects the purpose peak, is no more than 30 ℃ conditions under vacuum rotary steam be concentrated into about 70-80mg/ml after standby in water temperature the Lepirudin solution collected.
3, change salt: with the octadecylsilane chemically bonded silica is the chromatographic column of stationary phase, and pillar diameter and length are: 300mm * 300mm.Moving phase: A phase: 0.1% aqueous acetic acid; The B phase: trifluoroacetic acid aqueous solution, flow velocity: 1900-2200ml/min detects wavelength: 230nm.Gradient: B%:6%～36%.Sample size is 55-75g.
Change the salt process: rinse chromatographic column well back with the acetonitrile more than 50% and go up sample, applied sample amount is the 1200-1600ml sample solution.Linear gradient elution 90min collects the purpose peak, the Lepirudin solution of collecting is merged to revolve to steam go to suitable big or small cillin bottle after being concentrated into about 50-80ml/g, carries out lyophilize then, can obtain purity greater than 98% Lepirudin.
Claims (6)
1. the purification process of a Lepirudin may further comprise the steps:
1) with the octadecylsilane chemically bonded silica being stationary phase, is that the phosphate buffer soln of 2.5-3.5 is the A phase with the pH value, and acetonitrile is the B phase, and gradient: B%:17%～32% carries out gradient elution with thick peptide solution;
2) adopt reversed-phased high performace liquid chromatographic that Lepirudin is changed into acetate.
2. method according to claim 1 is characterized in that: the phosphate buffer soln of A phase adopts triethylamine to transfer pH to obtain by phosphoric acid solution in the step 1).
3. method according to claim 1 is characterized in that: the phosphate buffer soln amount concentration of A phase is less than 0.2%mol/L in the step 1).
4. method according to claim 2 is characterized in that: the phosphate buffer soln amount concentration of A phase is less than 0.2%mol/L in the step 1).
5. according to any described method of claim 1 to 4, it is characterized in that: method performing step 2) is that the stationary phase of described " RPLC " is an octadecylsilane chemically bonded silica, is the A phase with mass concentration 0.05%--0.2% aqueous acetic acid; Acetonitrile is the B phase, and gradient: B%:6%～36% carries out gradient elution.
6. method according to claim 5 is characterized in that: the aqueous acetic acid concentration of A phase is 0.1%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010116391 CN101781367B (en) | 2010-02-26 | 2010-02-26 | Method for purifying lepirudin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010116391 CN101781367B (en) | 2010-02-26 | 2010-02-26 | Method for purifying lepirudin |
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| CN101781367A true CN101781367A (en) | 2010-07-21 |
| CN101781367B CN101781367B (en) | 2013-08-21 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 201010116391 Expired - Fee Related CN101781367B (en) | 2010-02-26 | 2010-02-26 | Method for purifying lepirudin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102477094A (en) * | 2010-11-25 | 2012-05-30 | 北京凯因科技股份有限公司 | Separation and purification process for synthetic thymosin alpha 1 |
| CN102993293A (en) * | 2012-12-05 | 2013-03-27 | 深圳翰宇药业股份有限公司 | Method for purifying teriparatide acetate |
-
2010
- 2010-02-26 CN CN 201010116391 patent/CN101781367B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 《Biochemistry》 19950731 Vincenzo De Filippis et al Core Domain of Hirudin from the Leech Hirudinaria manillensis: Chemical Synthesis, Purification, and Characterization of a Trp3 Analog of Fragment 1 -47 第9552-9564页 1-6 , * |
| 《Journal of Peptide Science》 20060228 SPYROS GOULAS et al Convergent solid-phase synthesis of hirudin 第116-123页 1-6 , * |
| SPYROS GOULAS ET AL: "Convergent solid-phase synthesis of hirudin", 《JOURNAL OF PEPTIDE SCIENCE》 * |
| VINCENZO DE FILIPPIS ET AL: "Core Domain of Hirudin from the Leech Hirudinaria manillensis: Chemical Synthesis, Purification, and Characterization of a Trp3 Analog of Fragment 1 -47", 《BIOCHEMISTRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102477094A (en) * | 2010-11-25 | 2012-05-30 | 北京凯因科技股份有限公司 | Separation and purification process for synthetic thymosin alpha 1 |
| CN102993293A (en) * | 2012-12-05 | 2013-03-27 | 深圳翰宇药业股份有限公司 | Method for purifying teriparatide acetate |
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| Publication number | Publication date |
|---|---|
| CN101781367B (en) | 2013-08-21 |
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