CN101798335A - Purification method of thymosin extrasin alpha 1 - Google Patents
Purification method of thymosin extrasin alpha 1 Download PDFInfo
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- CN101798335A CN101798335A CN201010138995A CN201010138995A CN101798335A CN 101798335 A CN101798335 A CN 101798335A CN 201010138995 A CN201010138995 A CN 201010138995A CN 201010138995 A CN201010138995 A CN 201010138995A CN 101798335 A CN101798335 A CN 101798335A
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Abstract
The invention provides a technical method applicable to the industrialized purification of thymosin extrasin alpha 1, which comprises the following steps: using octadecylsilane chemically bonded silica as a fixed phase; using a trifluoroacetic acid water solution as a phase A, using acetonitrile as a phase B; controlling the gradient (B%) between 10 and 60 percent; carrying out gradient elution on a crude peptide solution; and carrying out the step of salt conversion, wherein the thymosin extrasin with trifluoroacetic acid salt is converted into salt-free thymosin extrasin by a reverse-phase efficient liquid phase chromatography method. The invention adopts the one-step reverse-phase efficient liquid phase chromatography method for purification, and uses the reverse-phase efficient liquid phase chromatography method for converting the salt, so the purified thymosin extrasin alpha 1 has high purification and high yield, and the industrialization requirement can be met.
Description
Technical field
The present invention relates to the purification process of a peptide species, relate in particular to the purification process of thymosin.
Background technology
Thymosin (Thymosin α 1) is to obtain a kind of small molecule bioactive polypeptide by separation and purification in the thymosin fraction 5 (TF-5), form by the arrangement of 28 seed amino acids, molecular weight 3108.37, its content accounts for 0.6% of TF-5, has higher immune-enhancing activity.The Thymosin alpha 1 mechanism of action mainly is to act on the differentiation of T lymphocyte, growth and sophisticated each stage, thereby regulates cellular immune function, and enhancing body diseases prevention and resistance against diseases are the good polypeptide drugs of a kind of market application foreground.
Summary of the invention
The present invention proposes a processing method that is suitable for industrialization purifying thymosin, use reversed-phased high performace liquid chromatographic purifying thymosin, purity height and yield are good, reach industrialized requirement.
For achieving the above object, the technical scheme taked of the present invention may further comprise the steps into:
1) with the octadecylsilane chemically bonded silica being stationary phase, is the A phase with the aqueous solution of trifluoroacetic acid, and acetonitrile is the B phase, and gradient B%:10%-60% carries out gradient elution with thick peptide solution;
The concentration of described " aqueous solution of trifluoroacetic acid " is 0.01%-1%, is preferably 0.05%-0.3%.Described " thick peptide " be meant and adopt liquid phase synthesizing method or solid-phase synthesis to obtain, and do not pass through the thymosin that the thick peptide of thymosin of refinement treatment or purity can not fulfilling medicinals as yet." thick peptide solution " is meant that thick peptide is dissolved in the resulting solution of pure water, and pure water meets the water for injection standard, preferred ultrapure water.The preferred preferred chromatographically pure of the purity grade of " acetonitrile ".Step 1) hereinafter also is expressed as purifying.
2) change salt: adopt reversed-phased high performace liquid chromatographic trifluoroacetate to be changed into salt-free.
The stationary phase of described " RPLC " is eight alkyl silane bonded silica gels.Described " commentaries on classics salt " refers to 50% acetonitrile wash-out the thymosin trifluoroacetate be changed into salt-free thymosin again with the water flushing that contains 5% acetonitrile.
The purifying scale comprises following specification chromatographic column: 5cm * 25cm (pillar diameter * length), 15cm * 25cm, 30cm * 25cm.
Embodiment
Embodiment one:
1. sample preparation: thick peptide dissolves with ultrapure water, 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: 5cm * 25cm.Moving phase: A phase: 0.05% trifluoroacetic acid aqueous solution, B phase: acetonitrile, flow velocity: 50-80ml/min.Detect wavelength: 230nm.Gradient: B%:10%-60% (elution time 50min).Sample size is 1.5-2g.
Purge process: rinse chromatographic column well in back balance sample with the acetonitrile more than 50%, applied sample amount is 1.5-2g.Linear gradient elution 50min collects the purpose peak, and the thymosin solution of collecting is standby after water temperature is no more than 35 ℃ of following vacuum rotary steams to be concentrated into about 10-20mg/mL.
3, change salt: chromatographic column: with eight alkyl silane bonded silica gels is the chromatographic column of stationary phase, and pillar diameter and length are: 5cm * 25cm.Rinse chromatographic column well back with the acetonitrile solution more than 50% and go up sample, applied sample amount 1.5-2g, with the water flushing 15-30min that contains 5% acetonitrile, use 50% acetonitrile wash-out at last, collect the purpose peak, the thymosin solution of collecting is no more than in water temperature goes to suitable big small vessels after 35 ℃ of following vacuum rotary steams are concentrated into about 150-200mg/mL.Can obtain purity after the lyophilize greater than 98% thymosin.
Embodiment two:
1. sample preparation: thick peptide dissolves with ultrapure water, 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: 15cm * 25cm.Moving phase: A phase: 0.3% trifluoroacetic acid aqueous solution; B phase: acetonitrile, flow velocity: 450-550ml/min.Detect wavelength: 230nm.Gradient: B%:10%-60% (elution time 50min).Sample size is 10-15g.
Purge process: rinse chromatographic column well in back balance sample with the acetonitrile more than 50%, applied sample amount is 10-15g.Linear gradient elution 50min collects the purpose peak, and the thymosin solution of collecting is standby after water temperature is no more than 35 ℃ of following vacuum rotary steams to be concentrated into about 10-20mg/mL.
3, change salt: chromatographic column: with eight alkyl silane bonded silica gels is the chromatographic column of stationary phase, and pillar diameter and length are: 15cm * 25cm.Rinse chromatographic column well back with the acetonitrile solution more than 50% and go up sample, applied sample amount 10-15g, with the water flushing 15-30min that contains 5% acetonitrile, use 50% acetonitrile wash-out at last, collect the purpose peak, the thymosin solution of collecting is no more than in water temperature goes to suitable big small vessels after 35 ℃ of following vacuum rotary steams are concentrated into about 150-200mg/mL.Can obtain purity after the lyophilize greater than 98% thymosin.
Embodiment three:
1. sample preparation: thick peptide dissolves with ultrapure water, 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: 30cm * 25cm.Moving phase: A phase: 0.1% trifluoroacetic acid aqueous solution; B phase: acetonitrile, flow velocity: 1900-2200ml/min.Detect wavelength: 230nm.Gradient: B%:10%-60% (elution time 50min).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 50min collects the purpose peak, and the thymosin solution of collecting is standby after water temperature is no more than 35 ℃ of following vacuum rotary steams to be concentrated into about 10-20mg/mL.
3, change salt: chromatographic column: with eight alkyl silane bonded silica gels is the chromatographic column of stationary phase, and pillar diameter and length are: 30cm * 25cm.Rinse chromatographic column well back with the acetonitrile solution more than 50% and go up sample, applied sample amount 55-75g, with the water flushing 15-30mi n that contains 5% acetonitrile, use 50% acetonitrile wash-out at last, collect the purpose peak, the thymosin solution of collecting is no more than in water temperature goes to suitable big small vessels after 35 ℃ of following vacuum rotary steams are concentrated into about 150-200mg/mL.Can obtain the thymosin that purity requires greater than 98% conformance with standard after the lyophilize.
In sum, operation is simple and feasible, elaboration purity is higher than 98%, ideal yield coefficient, purifying cost are lower in order to last method purifying thymosin, reaches industrialized requirement.
Claims (5)
1. the purification process of a thymosin may further comprise the steps:
1) with the octadecylsilane chemically bonded silica being stationary phase, is the A phase with the aqueous solution of trifluoroacetic acid, and acetonitrile is the B phase, and gradient B%:10%-60% carries out gradient elution with thick peptide solution;
2) change salt: adopt reversed-phased high performace liquid chromatographic trifluoroacetate to be changed into salt-free.
2. according to the described method of claim 1, it is characterized in that: the trifluoroacetic acid concentration of the trifluoroacetic acid aqueous solution of the A phase of step 1) is 0.05%-0.3%.
3. according to any described method of claim 1 to 2, it is characterized in that: step 2) the stationary phase of RPLC be eight alkyl silane bonded silica gels.
4. according to any described method of claim 1 to 2, it is characterized in that: step 2) with the water flushing that contains 5% acetonitrile, with 50% acetonitrile wash-out the thymosin trifluoroacetate is changed into salt-free thymosin again.
5. according to the described method of claim 3, it is characterized in that: step 2) with the water flushing that contains 5% acetonitrile, with 50% acetonitrile wash-out the thymosin trifluoroacetate is changed into salt-free thymosin again.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010138995 CN101798335B (en) | 2010-03-30 | 2010-03-30 | Purification method of thymosin extrasin alpha 1 |
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| CN 201010138995 CN101798335B (en) | 2010-03-30 | 2010-03-30 | Purification method of thymosin extrasin alpha 1 |
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| CN101798335A true CN101798335A (en) | 2010-08-11 |
| CN101798335B CN101798335B (en) | 2013-02-13 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102675450A (en) * | 2012-05-14 | 2012-09-19 | 滨海吉尔多肽有限公司 | Method for purifying thymosin beta4 |
| CN103163234A (en) * | 2012-07-12 | 2013-06-19 | 海南合瑞制药股份有限公司 | Deletion peptide, non-peptide impurities and detection method for peptide phase 11 of solid-phase synthesis thymulin alpha1 |
| CN103467593A (en) * | 2013-09-05 | 2013-12-25 | 杭州诺泰制药技术有限公司 | Purification method of thymalfasin |
| CN104672308A (en) * | 2014-12-23 | 2015-06-03 | 青岛康原药业有限公司 | Method for preparing vasopressin tannate |
| CN105254746A (en) * | 2015-10-19 | 2016-01-20 | 吉尔生化（上海）有限公司 | Method for desalinating thymopeptide alpha 1 |
-
2010
- 2010-03-30 CN CN 201010138995 patent/CN101798335B/en not_active Expired - Fee Related
Non-Patent Citations (8)
| Title |
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| 《中国优秀博硕学位论文全文数据库（博士） 基础科学辑》 20030615 修朝阳 I_人甲状旁腺素1_34肽的制备II_人胸腺素_1的制备及其与干扰素_1b的融合 第50页第5部分和第53页第5部分 1-5 , * |
| 《南京工业大学学报》 20040331 程虎等 固相合成胸腺素alpha1 第79页第2.1-2.31部分 1-5 , * |
| 《南京工业大学学报》 20040331 程虎等 固相合成胸腺素alpha1 第79页第2.1-2.31部分 1-5 , 2 * |
| 《现代仪器》 20091231 赵玉兰等 化学合成胸腺素alpha1 反相色谱分离影响因素的研究 第28页第2.3部分 3,5 , * |
| 《现代仪器》 20091231 赵玉兰等 化学合成胸腺素alpha1 反相色谱分离影响因素的研究 第28页第2.3部分 3,5 , 2 * |
| 修朝阳: "I_人甲状旁腺素1_34肽的制备II_人胸腺素_1的制备及其与干扰素_1b的融合", 《中国优秀博硕学位论文全文数据库（博士） 基础科学辑》 * |
| 程虎等: "固相合成胸腺素α1", 《南京工业大学学报》 * |
| 赵玉兰等: "化学合成胸腺素α1 反相色谱分离影响因素的研究", 《现代仪器》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102675450A (en) * | 2012-05-14 | 2012-09-19 | 滨海吉尔多肽有限公司 | Method for purifying thymosin beta4 |
| CN103163234A (en) * | 2012-07-12 | 2013-06-19 | 海南合瑞制药股份有限公司 | Deletion peptide, non-peptide impurities and detection method for peptide phase 11 of solid-phase synthesis thymulin alpha1 |
| CN103163234B (en) * | 2012-07-12 | 2014-09-24 | 海南合瑞制药股份有限公司 | Deletion peptide, non-peptide impurities and detection method for peptide phase 11 of solid-phase synthesis thymulin alpha1 |
| CN103467593A (en) * | 2013-09-05 | 2013-12-25 | 杭州诺泰制药技术有限公司 | Purification method of thymalfasin |
| CN103467593B (en) * | 2013-09-05 | 2015-05-13 | 杭州阿德莱诺泰制药技术有限公司 | Purification method of thymalfasin |
| CN104672308A (en) * | 2014-12-23 | 2015-06-03 | 青岛康原药业有限公司 | Method for preparing vasopressin tannate |
| CN105254746A (en) * | 2015-10-19 | 2016-01-20 | 吉尔生化（上海）有限公司 | Method for desalinating thymopeptide alpha 1 |
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| Publication number | Publication date |
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| CN101798335B (en) | 2013-02-13 |
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