CN109705206B - Purification method and application of thymalfasin - Google Patents
Purification method and application of thymalfasin Download PDFInfo
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Abstract
The invention relates to a purification method of thymalfasin, belonging to the technical field of purification of polypeptide compounds. The method comprises the step of adding ammonia water and perchloric acid into a product to be purified of thymalfasin for purification. Is particularly suitable for removing impurity D-Asn28Thymalfasin and Des-Ala3Adjusting the pH of the solution to be purified to 8-10 by using ammonia water, and adjusting the pH of the solution to be purified to 2-5 by using perchloric acid; purification was performed by liquid chromatography. The invention also discloses the application of ammonia water and perchloric acid in purifying thymalfasin. According to the method, the ammonia water and the perchloric acid are added into the thymalfasin to-be-purified product in sequence, so that the impurities in the thymalfasin crude product can be remarkably reduced, and particularly, the impurity D-Asn is remarkably reduced28Thymalfasin and Des-Ala3The content of thymalfasin can effectively improve the yield and purity of thymalfasin. And the method has convenient and quick operation.
Description
Technical Field
The invention relates to the technical field of purification of polypeptide compounds, in particular to a purification method of thymalfasin; the invention also relates to the use of ammonia and perchloric acid in the purification of thymalfasin.
Technical Field
Thymalfasin, english name: thymalfasin;
peptide sequence:
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH
thymalfasin is a vaccine immune response enhancer developed by Italian Saishen pharmacy and used as an immune injury patient, and is mainly used for treating chronic hepatitis B. At present, the medicine has a large market at home.
Thymalfasin is sold in the market of a plurality of domestic enterprises at present, and a qualified sample is obtained by adopting a solid-phase synthesis crude product and then adopting a reverse-phase purification mode. Purification plays an important role in the production process of thymalfasin.
Regarding purification of thymalfasin, a plurality of patents are reported in China, and a method for purifying thymalfasin by adopting activated carbon adsorption and using neutral alumina chromatography is disclosed in Chinese published patent document CN 201210052504; chinese published patent document CN201110186259 discloses a purification method of performing two-time purification and one-time salt conversion by using C18 reversed phase filler; chinese published patent document CN201010566609 discloses a purification method using ion exchange chromatography and then reverse phase chromatography; chinese published patent document CN103467593B discloses a purification method using 0.3% acetic acid aqueous solution and acetonitrile as mobile phase and polymer reversed phase filler as purification medium.
Because a large amount of deletion impurities, racemization impurities and hydrolysis impurities are inevitably generated in the solid-phase synthesis process, particularly D-Asn detected by the method of pharmacopoeia of 2015 edition28Thymalfasin and Des-Ala3Thymalfasin, which is difficult to control effectively with current methods.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a novel thymalfasin purification method which can improve the yield of thymalfasin and obviously reduce the impurity D-Asn28Thymalfasin and Des-Ala3The amount of thymalfasin present.
It is another object of the present invention to provide the use of ammonia and perchloric acid in the purification of thymalfasin.
The object of the present invention is achieved by the following means. The invention relates to a purification method of thymalfasin, which is characterized in that: the method comprises the step of adding ammonia water and perchloric acid into a product to be purified of thymalfasin for purification.
The invention relates to a purification method of thymalfasin, which adopts a further preferable technical scheme that: in the purification process: firstly, ammonia water is used for adjusting the pH value of the solution to be purified to 8-10, and then perchloric acid is used for adjusting the pH value of the solution to be purified to 2-5.
The invention relates to a purification method of thymalfasin, which adopts a further preferable technical scheme that: purifying by liquid chromatography with mobile phase of 0.05% -0.1% phosphoric acid water solution; the chromatographic column for purification is styrene divinylbenzene polymer reversed phase filler. The following steps are preferably used for pretreatment before purification: pretreatment with a 0.1% TFA/acetonitrile mobile phase system, column C18 reverse phase packing, removal of residual ether or other highly retained materials from the crude solution, and removal of acetonitrile by distillation at room temperature and low pressure.
The invention relates to a purification method of thymalfasin, which adopts a further preferable technical scheme that: the method comprises the following specific steps:
(1) preparing a crude thymalfasin product into a crude product solution;
(2) adjusting the pH value of the solution to be purified to be alkaline by using ammonia water, and adjusting the pH value to be acidic by using perchloric acid;
(3) purifying the sample with a 0.1% phosphoric acid/acetonitrile mobile phase system; and freeze-drying the obtained sample to obtain a thymalfasin pure solid.
The invention relates to a purification method of thymalfasin, which adopts a further preferable technical scheme that: adding a pretreatment step between the steps (1) and (2); the pretreatment step is as follows: pretreating with 0.1% TFA/acetonitrile mobile phase system and chromatographic column C18 reversed phase filler to remove residual diethyl ether or other high retention substances in the crude product solution, and distilling at room temperature under low pressure to remove acetonitrile; the pretreatment is preferably carried out using a 50 μm large particle size C18 reverse phase packing for the chromatography column.
The invention relates to a purification method of thymalfasin, which adopts a further preferable technical scheme that: in the step (1), dissolving the thymalfasin crude product by using purified water to obtain a crude product solution, and then filtering; filtration is preferably performed using a 0.45 μm filter.
The invention relates to a purification method of thymalfasin, which adopts a further preferable technical scheme that: in step (3), when the sample is purified by using a 0.1% phosphoric acid/acetonitrile mobile phase system, the acetonitrile gradient change takes 40min from 7% to 20%, changes from 20% to 50% within 5min and maintains for 3min, and changes from 50% to 7% within 2min and maintains for 10 min.
The invention relates to a purification method of thymalfasin, which adopts a further preferable technical scheme that: in the purification of the step (3): the detection wavelength is 220 nm; the model of the reversed phase packing of the chromatographic column styrene divinylbenzene polymer is UniPs10-300, 30mm multiplied by 250 mm.
The invention also discloses an application of ammonia water and perchloric acid in purification of thymalfasin, which is characterized in that: the application is that in the process of purifying thymalfasin by liquid chromatography, ammonia water and perchloric acid are added into thymalfasin solution to remove impurity D-Asn28Thymalfasin and/or Des-Ala impurity3Thymalfasin (Thymalfasin). The amounts of ammonia and perchloric acid used are preferably: firstly, ammonia water is used for adjusting the pH value of the solution to be purified to 8-10, and then perchloric acid is used for adjusting the pH value of the solution to be purified to 2-5.
In the method, the purified raw material thymalfasin crude product can be synthesized or prepared by any method disclosed in the prior art, and particularly contains impurity D-Asn28Thymalfasin and Des-Ala3A crude thymalfasin product of thymalfasin.
Compared with the prior art, the method has the following beneficial effects:
according to the method, the ammonia water and the perchloric acid are added into the thymalfasin to-be-purified product in sequence, so that the impurities in the thymalfasin crude product can be remarkably reduced, and particularly, the impurity D-Asn is remarkably reduced28Thymalfasin and Des-Ala3The content of thymalfasin can effectively improve the yield and purity of thymalfasin. And the method has convenient and quick operation.
Detailed Description
The technical solutions of the present invention are further described below to enable those skilled in the art to further understand the present invention, but not to limit the rights of the present invention.
Example 1, a purification method for thymalfasin: the method comprises the step of adding ammonia water and perchloric acid into a product to be purified of thymalfasin for purification. In the purification process: firstly, ammonia water is used for adjusting the pH value of the solution to be purified to 8, and then perchloric acid is used for adjusting the pH value of the solution to be purified to 2. Purifying by liquid chromatography with 0.05% phosphoric acid water solution as mobile phase; the chromatographic column for purification is styrene divinylbenzene polymer reversed phase filler.
Example 2, a purification method for thymalfasin: the method comprises the step of adding ammonia water and perchloric acid into a product to be purified of thymalfasin for purification. In the purification process: firstly, ammonia water is used for adjusting the pH value of the solution to be purified to 10, and then perchloric acid is used for adjusting the pH value of the solution to be purified to 5. Purifying by liquid chromatography with 0.1% phosphoric acid water solution as mobile phase; the chromatographic column for purification is styrene divinylbenzene polymer reversed phase filler.
Example 3, a purification method for thymalfasin: the method comprises the step of adding ammonia water and perchloric acid into a product to be purified of thymalfasin for purification. In the purification process: firstly, ammonia water is used for adjusting the pH value of the solution to be purified to 8.5, and then perchloric acid is used for adjusting the pH value of the solution to be purified to 3. Purifying by liquid chromatography with 0.1% phosphoric acid water solution as mobile phase; the chromatographic column for purification is styrene divinylbenzene polymer reversed phase filler. Pretreatment is carried out before purification: pretreatment with a 0.1% TFA/acetonitrile mobile phase system, column C18 reverse phase packing, removal of residual ether or other highly retained materials from the crude solution, and removal of acetonitrile by distillation at room temperature and low pressure.
Example 4, a method for purifying thymalfasin, comprising the steps of:
(1) preparing a crude thymalfasin product into a crude product solution;
(2) adjusting the pH value of the solution to be purified to 9 by using ammonia water, and adjusting the pH value to 4 by using perchloric acid;
(3) the purification was performed by liquid chromatography, using a 0.1% phosphoric acid/acetonitrile mobile phase system to purify the sample; and freeze-drying the obtained sample to obtain a thymalfasin pure solid.
Adding a pretreatment step between the steps (1) and (2); the pretreatment step is as follows: pretreating with 0.1% TFA/acetonitrile mobile phase system and chromatographic column C18 reversed phase filler to remove residual diethyl ether or other high retention substances in the crude product solution, and distilling at room temperature under low pressure to remove acetonitrile; the pretreatment can be carried out by using a chromatographic column with 50 μm large-particle size C18 reversed-phase packing.
In the step (1), the crude thymalfasin product can be dissolved by purified water to obtain a crude product solution, and then filtration treatment is carried out; filtration can be performed using a 0.45 μm filter.
In step (3), when the sample is purified by using a 0.1% phosphoric acid/acetonitrile mobile phase system, the acetonitrile gradient change takes 40min from 7% to 20%, changes from 20% to 50% within 5min and maintains for 3min, and changes from 50% to 7% within 2min and maintains for 10 min. In the purification: the detection wavelength is 220 nm; the model of the reversed phase packing of the chromatographic column styrene divinylbenzene polymer is UniPs10-300, 30mm multiplied by 250 mm.
Example 5 use of aqueous Ammonia and perchloric acid in purification of thymalfasin by liquid chromatography with addition of aqueous Ammonia and perchloric acid to the thymalfasin solution to remove the impurity D-Asn28Thymalfasin and/or Des-Ala impurity3Thymalfasin (Thymalfasin). The amounts of ammonia and perchloric acid used were: firstly, ammonia water is used for adjusting the pH value of the solution to be purified to 8, and then perchloric acid is used for adjusting the pH value of the solution to be purified to 5.
Example 6 use of aqueous Ammonia and perchloric acid in purification of thymalfasin by liquid chromatography with addition of aqueous Ammonia and perchloric acid to the thymalfasin solution to remove the impurity D-Asn28Thymalfasin and/or Des-Ala impurity3Thymalfasin (Thymalfasin). The amounts of ammonia and perchloric acid used were: firstly, ammonia water is used for adjusting the pH value of the solution to be purified to 10, and then perchloric acid is used for adjusting the pH value of the solution to be purified to 2.
Example 7 purification of thymalfasin comparative experiments:
experiment 1:
taking 1g of thymalfasin crude product, wherein the crude product is derived from synthesis. Dissolving in 100ml purified water, filtering with 0.45 μm water system filter membrane, and collecting the crude solution to obtain about 100 ml. The filtered crude product is pretreated, and the pretreatment method comprises the following steps: and (3) loading the crude product into a chromatographic column, wherein the chromatographic column is 50-micron large-particle-size C18 reversed-phase filler, the loading flow rate of 30mm multiplied by 250mm is 25ml/min, and after the loading is finished, eluting by using a 0.1% TFA/acetonitrile mobile phase system, wherein the elution gradient program is that the initial acetonitrile concentration is 5%, keeping for 5min, increasing to 30% within 2min, keeping for 20min, and ending. The flow rate was 25ml/min and the absorption wavelength was 220 nm. Collecting all fractions eluted, distilling off acetonitrile at 32 ℃ by using a rotary evaporation evaporator under low pressure, and continuing to purify the sample by the following purification methods: loading the pretreated sample without acetonitrile into a polymer reversed phase chromatographic column with the model of UniPs10-300 and the model of 30mm multiplied by 250mm, and eluting by using a 0.1% phosphoric acid/acetonitrile mobile phase system after loading, wherein the elution gradient is as follows: the initial acetonitrile concentration was 7%, the change from 7% to 20% took 40min, the change from 20% to 50% was maintained for 3min within 5min, the change from 50% to 7% was maintained for 10min within 2min, and the elution was completed. The flow rate was 25ml/min and the dilution wavelength was 220 nm. Collecting the elution fraction by stages, detecting by using an analytical chromatograph, and taking qualified fractions. Distilling the qualified fraction with a rotary evaporator at 32 deg.C under low pressure to remove acetonitrile, and lyophilizing to obtain solid product.
Experiment 2:
taking 1g of thymalfasin crude product, wherein the crude product is derived from synthesis. Dissolving in 100ml purified water, filtering with 0.45 μm water system filter membrane, and collecting the crude solution to obtain about 100 ml. The filtered crude product is pretreated, and the pretreatment method comprises the following steps: and (3) loading the crude product into a chromatographic column, wherein the chromatographic column is 50-micron large-particle-size C18 reversed-phase filler, the loading flow rate of 30mm multiplied by 250mm is 25ml/min, and after the loading is finished, eluting by using a 0.1% TFA/acetonitrile mobile phase system, wherein the elution gradient program is that the initial acetonitrile concentration is 5%, keeping for 5min, increasing to 30% within 2min, keeping for 20min, and ending. The flow rate was 25ml/min and the absorption wavelength was 220 nm. Collecting all fractions eluted, distilling off acetonitrile at 32 ℃ by using a rotary evaporation evaporator under low pressure, and continuing to purify the sample by the following purification methods: the sample from which acetonitrile was removed after the pretreatment was adjusted to pH 8.0 with ammonia water and adjusted to pH 5.0 with perchloric acid. Loading the sample with the adjusted pH value into a polymer reversed phase chromatographic column, wherein the model of the chromatographic column is UniPs10-300 and 30mm multiplied by 250mm, and after loading, eluting by using a 0.1 percent phosphoric acid/acetonitrile mobile phase system, and the elution gradient is as follows: the initial acetonitrile concentration was 7%, the change from 7% to 20% took 40min, the change from 20% to 50% was maintained for 3min within 5min, the change from 50% to 7% was maintained for 10min within 2min, and the elution was completed. The flow rate was 25ml/min and the dilution wavelength was 220 nm. Collecting the elution fraction by stages, detecting by using an analytical chromatograph, and taking qualified fractions. Distilling the qualified fraction with a rotary evaporator at 32 deg.C under low pressure to remove acetonitrile, and lyophilizing to obtain solid product.
Experiment 3:
taking 1g of thymalfasin crude product, wherein the crude product is derived from synthesis. Dissolving in 100ml purified water, filtering with 0.45 μm water system filter membrane, and collecting the crude solution to obtain about 100 ml. The filtered crude product is pretreated, and the pretreatment method comprises the following steps: and (3) loading the crude product into a chromatographic column, wherein the chromatographic column is 50-micron large-particle-size C18 reversed-phase filler, the loading flow rate of 30mm multiplied by 250mm is 25ml/min, and after the loading is finished, eluting by using a 0.1% TFA/acetonitrile mobile phase system, wherein the elution gradient program is that the initial acetonitrile concentration is 5%, keeping for 5min, increasing to 30% within 2min, keeping for 20min, and ending. The flow rate was 25ml/min and the absorption wavelength was 220 nm. Collecting all fractions eluted, distilling off acetonitrile at 32 ℃ by using a rotary evaporation evaporator under low pressure, and continuing to purify the sample by the following purification methods: the sample from which acetonitrile was removed after the pretreatment was adjusted to pH 10.0 with ammonia water and adjusted to pH 2.0 with perchloric acid. Loading the sample with the adjusted pH value into a polymer reversed phase chromatographic column, wherein the model of the chromatographic column is UniPs10-300 and 30mm multiplied by 250mm, and after loading, eluting by using a 0.1 percent phosphoric acid/acetonitrile mobile phase system, and the elution gradient is as follows: the initial acetonitrile concentration was 7%, the change from 7% to 20% took 40min, the change from 20% to 50% was maintained for 3min within 5min, the change from 50% to 7% was maintained for 10min within 2min, and the elution was completed. The flow rate was 25ml/min and the dilution wavelength was 220 nm. Collecting the elution fraction by stages, detecting by using an analytical chromatograph, and taking qualified fractions. Distilling the qualified fraction with a rotary evaporator at 32 deg.C under low pressure to remove acetonitrile, and lyophilizing to obtain solid product.
Experiment 4:
taking 1g of thymalfasin crude product, wherein the crude product is derived from synthesis. Dissolving in 100ml purified water, filtering with 0.45 μm water system filter membrane, and collecting the crude solution to obtain about 100 ml. The filtered crude product is pretreated, and the pretreatment method comprises the following steps: and (3) loading the crude product into a chromatographic column, wherein the chromatographic column is 50-micron large-particle-size C18 reversed-phase filler, the loading flow rate of 30mm multiplied by 250mm is 25ml/min, and after the loading is finished, eluting by using a 0.1% TFA/acetonitrile mobile phase system, wherein the elution gradient program is that the initial acetonitrile concentration is 5%, keeping for 5min, increasing to 30% within 2min, keeping for 20min, and ending. The flow rate was 25ml/min and the absorption wavelength was 220 nm. Collecting all fractions eluted, distilling off acetonitrile at 32 ℃ by using a rotary evaporation evaporator under low pressure, and continuing to purify the sample by the following purification methods: the sample from which acetonitrile was removed after the pretreatment was adjusted to pH 10.0 with ammonia water and adjusted to pH 5.0 with perchloric acid. Loading the sample with the adjusted pH value into a polymer reversed phase chromatographic column, wherein the model of the chromatographic column is UniPs10-300 and 30mm multiplied by 250mm, and after loading, eluting by using a 0.1 percent phosphoric acid/acetonitrile mobile phase system, and the elution gradient is as follows: the initial acetonitrile concentration was 7%, the change from 7% to 20% took 40min, the change from 20% to 50% was maintained for 3min within 5min, the change from 50% to 7% was maintained for 10min within 2min, and the elution was completed. The flow rate was 25ml/min and the dilution wavelength was 220 nm. Collecting the elution fraction by stages, detecting by using an analytical chromatograph, and taking qualified fractions. Distilling the qualified fraction with a rotary evaporator at 32 deg.C under low pressure to remove acetonitrile, and lyophilizing to obtain solid product.
Experiment 5:
taking 1g of thymalfasin crude product, wherein the crude product is derived from synthesis. Dissolving in 100ml purified water, filtering with 0.45 μm water system filter membrane, and collecting the crude solution to obtain about 100 ml. The filtered crude product is pretreated, and the pretreatment method comprises the following steps: and (3) loading the crude product into a chromatographic column, wherein the chromatographic column is 50-micron large-particle-size C18 reversed-phase filler, the loading flow rate of 30mm multiplied by 250mm is 25ml/min, and after the loading is finished, eluting by using a 0.1% TFA/acetonitrile mobile phase system, wherein the elution gradient program is that the initial acetonitrile concentration is 5%, keeping for 5min, increasing to 30% within 2min, keeping for 20min, and ending. The flow rate was 25ml/min and the absorption wavelength was 220 nm. Collecting all fractions eluted, distilling off acetonitrile at 32 ℃ by using a rotary evaporation evaporator under low pressure, and continuing to purify the sample by the following purification methods: the sample from which acetonitrile was removed after the pretreatment was adjusted to pH 8.0 with ammonia water and adjusted to pH 2.0 with perchloric acid. Loading the sample with the adjusted pH value into a polymer reversed phase chromatographic column, wherein the model of the chromatographic column is UniPs10-300 and 30mm multiplied by 250mm, and after loading, eluting by using a 0.1 percent phosphoric acid/acetonitrile mobile phase system, and the elution gradient is as follows: the initial acetonitrile concentration was 7%, the change from 7% to 20% took 40min, the change from 20% to 50% was maintained for 3min within 5min, the change from 50% to 7% was maintained for 10min within 2min, and the elution was completed. The flow rate was 25ml/min and the dilution wavelength was 220 nm. Collecting the elution fraction by stages, detecting by using an analytical chromatograph, and taking qualified fractions. Distilling the qualified fraction with a rotary evaporator at 32 deg.C under low pressure to remove acetonitrile, and lyophilizing to obtain solid product.
In experiments 1-5 above:
experiment 1: ammonia water and perchloric acid are not added into a sample to be purified;
experiment 2: adjusting the pH value of a sample to be purified to 8 by using ammonia water, and adjusting the pH value to 5 by using perchloric acid;
experiment 3: adjusting the pH value of a sample to be purified to 10 by using ammonia water, and adjusting the pH value to 2 by using perchloric acid;
experiment 4: adjusting the pH value of a sample to be purified to 10 by using ammonia water, and adjusting the pH value to 5 by using perchloric acid;
experiment 5: the sample to be purified was adjusted to pH 8 with ammonia and pH 2 with perchloric acid.
By inspection, the content of the main impurities in the purified finished product solid is shown in the following table:
the impurity D-Asn in the product obtained by purifying the thymalfasin crude product by the inventor by adopting the method of the prior art (the source of the thymalfasin crude product is the same as that of the experiment)28Thymalfasin and Des-Ala3Content of thymalfasin:
(1) purifying thymalfasin crude product by the method described in Chinese published patent document CN201210052504, and determining D-Asn in the purified product28The content of thymalfasin was: 1.28%, Des-Ala3-thymalfasin content: 0.92 percent;
(2) the method described in Chinese published patent document CN201110186259 is adopted to purify the thymalfasin crude product and measure D-Asn in the purified product28-thymalfasin content: 0.39%, Des-Ala3-thymalfasin content: 0.50 percent;
(3) adopts Chinese published patent document CN201010566609 the method of purifying thymalfasin crude product and determining D-Asn in the purified product28The content of thymalfasin was: 0.59%, Des-Ala3-thymalfasin content: 0.46 percent;
(4) the method described in Chinese published patent document CN103467593B is adopted to purify the thymalfasin crude product and measure D-Asn in the purified product28The content of thymalfasin was: 0.25%, Des-Ala3-thymalfasin content: 0.43 percent;
the above experimental results prove that: the impurity D-Asn can be obviously reduced by adding ammonia water and perchloric acid into a sample to be purified28Thymalfasin and Des-Ala3The amount of thymalfasin present.
Claims (8)
1. A purification method of thymalfasin, which is characterized in that: the method comprises the steps of adding ammonia water and perchloric acid into a product to be purified of thymalfasin for purification;
in the purification process: firstly, ammonia water is used for adjusting the pH value of a solution to be purified to 8-10, and then perchloric acid is used for adjusting the pH value of the solution to be purified to 2-5;
the purification method comprises the following specific steps:
(1) preparing a crude thymalfasin product into a crude product solution;
(2) adjusting the pH value of the solution to be purified to be alkaline by using ammonia water, and adjusting the pH value to be acidic by using perchloric acid;
(3) purifying the sample with a 0.1% phosphoric acid/acetonitrile mobile phase system; and freeze-drying the obtained sample to obtain a thymalfasin pure solid.
2. The method of claim 1, wherein the thymalfasin is purified by: adding a pretreatment step between the steps (1) and (2); the pretreatment step is as follows: pretreatment with a 0.1% TFA/acetonitrile mobile phase system, column C18 reverse phase packing, removal of residual ether or other highly retained materials from the crude solution, and removal of acetonitrile by distillation at room temperature and low pressure.
3. The method of claim 2, wherein the thymalfasin is purified by: the pretreatment was carried out using a 50 μm large particle size C18 reverse phase packing on a chromatographic column.
4. The method of claim 1, wherein the thymalfasin is purified by: in the step (1), the crude thymalfasin product is dissolved by purified water to obtain a crude product solution, and then filtration treatment is carried out.
5. The method of claim 4, wherein the purification of thymalfasin comprises: filtration was performed using a 0.45 μm filter.
6. The method of claim 1, wherein the thymalfasin is purified by: in step (3), when the sample is purified by using a 0.1% phosphoric acid/acetonitrile mobile phase system, the acetonitrile gradient change takes 40min from 7% to 20%, changes from 20% to 50% within 5min and maintains for 3min, and changes from 50% to 7% within 2min and maintains for 10 min.
7. The method of claim 1, wherein the thymalfasin is purified by: in the purification of the step (3): the detection wavelength is 220 nm; the model of the reversed phase packing of the styrene-divinylbenzene polymer of the chromatographic column is UniPs10-300, 30mm multiplied by 250 mm.
8. The application of ammonia water and perchloric acid in purifying thymalfasin is characterized in that: the application is that in the process of purifying thymalfasin by liquid chromatography, ammonia water and perchloric acid are added into thymalfasin solution to remove impurity D-Asn28Thymalfasin and/or Des-Ala impurity3-thymalfasin; the amounts of ammonia and perchloric acid used were: firstly, ammonia water is used for adjusting the pH value of the solution to be purified to 8-10, and then perchloric acid is used for adjusting the pH value of the solution to be purified to 2-5.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286091A (en) * | 2011-07-05 | 2011-12-21 | 哈药集团生物工程有限公司 | Solid phase synthesis process of thymosin alpha1 |
| 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 |
| CN102617727A (en) * | 2012-03-02 | 2012-08-01 | 海南灵康制药有限公司 | Thymalfasin compound and novel preparation method thereof |
| CN103467593A (en) * | 2013-09-05 | 2013-12-25 | 杭州诺泰制药技术有限公司 | Purification method of thymalfasin |
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Patent Citations (4)
| 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 |
| CN102286091A (en) * | 2011-07-05 | 2011-12-21 | 哈药集团生物工程有限公司 | Solid phase synthesis process of thymosin alpha1 |
| CN102617727A (en) * | 2012-03-02 | 2012-08-01 | 海南灵康制药有限公司 | Thymalfasin compound and novel preparation method thereof |
| CN103467593A (en) * | 2013-09-05 | 2013-12-25 | 杭州诺泰制药技术有限公司 | Purification method of thymalfasin |
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