CN106699847B - Method for purifying hexapeptide at low cost - Google Patents
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Abstract
The invention discloses a method for purifying hexapeptide, which comprises the steps of adopting a reversed-phase polymer chromatographic column, loading a hexapeptide crude product water solution, washing with an alkaline mobile phase to remove trifluoroacetic acid in a large batch of hexapeptide crude products, eluting with an acidic mobile phase to obtain the hexapeptide acetate, and removing impurities in the hexapeptide. The method only needs one-step purification, reduces the production cost, not only can obtain the hexapeptide with the purity of more than 95 percent, but also can meet the requirements of low cost, high yield and industrialization of purifying the hexapeptide.
Description
Technical Field
The invention belongs to the field of polypeptide purification, and particularly relates to a purification method of hexapeptide.
Background
The hexapeptide is widely distributed in various parts of a human body, is an active substance existing and necessary in the human body, has all functions of the botulinum toxin, and avoids the defects that the botulinum toxin must be applied in an injection mode and the use cost is high.
The hexapeptide is used as a raw material of anti-wrinkle cosmetics in the world, and a plurality of high-end cosmetics apply the hexapeptide component for resisting wrinkles. The MAGIC CARE magic care hexapeptide wrinkle smoothing and tightening stock solution developed by the swiss legend group skin care laboratory in 2010 is a top-end representative of hexapeptide cosmetic products. When the hexapeptide is used as a cosmetic raw material, the purity requirement is more than or equal to 95 percent, but the synthesized hexapeptide contains a large amount of impurities and can be used as the cosmetic raw material only by further purification.
In the chinese patent application No. 201310593375.9, "a liquid phase fractional synthesis method of hexapeptide", a purification method of hexapeptide is disclosed, in which after crude hexapeptide is synthesized, the crude hexapeptide is purified by reverse phase high performance liquid chromatography to obtain hexapeptide. The method has the disadvantages of high organic reagent dosage and high cost, and can obtain qualified hexapeptide acetate by further salt conversion by using a weak anion exchange column.
Disclosure of Invention
The invention aims to overcome the defects of the conventional hexapeptide purification method and provide the hexapeptide acetate purification method which is low in cost, high in purity and suitable for industrialization.
The invention provides a method for purifying hexapeptide, which adopts a polymer reversed phase chromatographic column to purify a crude hexapeptide aqueous solution through a reversed phase high performance liquid chromatography, wherein after the crude hexapeptide aqueous solution is loaded, an alkaline mobile phase is firstly used for washing, and then an acidic mobile phase containing acetic acid is used for gradient elution to obtain the hexapeptide acetate solution.
In one embodiment of the invention, the packing of the polymeric reverse phase chromatography column is type F SBC MCI GEL reverse phase chromatography packing. Preferably, the particle size of the F type SBC MCI GEL reversed phase chromatographic packing is 30-50 μm.
In one embodiment of the present invention, the crude hexapeptide aqueous solution is prepared by dissolving crude hexapeptide in an aqueous solution, adjusting the pH to 6-9, filtering with a filter membrane and collecting the filtrate. Preferably, the mass percentage of the crude hexapeptide to water is 1:5-1:30, and more preferably 1: 20. Preferably, the pH of the solution obtained after dissolution is adjusted to 8.5, more preferably the pH of the solution obtained after dissolution is adjusted with ammonia.
In one embodiment of the invention, the crude hexapeptide is obtained by a solid phase synthesis method.
In one embodiment of the invention, the alkaline mobile phase consists of alkaline mobile phase a and alkaline mobile phase B, the alkaline mobile phase a being an aqueous solution with a pH value of 7.6-8.5 and the alkaline mobile phase B being a methanol solution of 7.6-8.5. In the alkaline loading, the ratio of the alkaline mobile phase A to the alkaline mobile phase B is (99-98): 1-2, and preferably the ratio of the alkaline mobile phase A to the alkaline mobile phase B is 99:1 (the ratios used in examples 1 and 2). Preferably, the flow rate of the alkaline mobile phase is from 10 to 300 ml/min, more preferably from 50 to 150 ml/min.
In one embodiment of the invention, the acidic mobile phase containing acetic acid consists of an acidic mobile phase A and an acidic mobile phase B, wherein the acidic mobile phase A is an aqueous solution containing 0.1-5% (volume percent) of acetic acid, and the acidic mobile phase B is a methanol solution containing 0.1-5% (volume percent) of acetic acid. Preferably, the elution is carried out in a gradient of acidic mobile phase A and acidic mobile phase B, the elution gradient being such that the ratio of acidic mobile phase A to acidic mobile phase B is from (98.5-99.5) to (0.5-1.5) to (93-95): (5-7). Preferably, the elution gradient is from 99:1 to 95:5, or from 99:1 to 93:7 of acidic mobile phase a to acidic mobile phase B. Preferably, the flow rate of the acidic mobile phase is from 10 to 300 ml/min, more preferably from 50 to 150 ml/min.
In one embodiment of the present invention, the elution gradient of the acidic mobile phase is such that the ratio of acidic mobile phase A to acidic mobile phase B in 0- (50-150) min, preferably 0- (100-130) min is from (98.5-99.5) to (0.5-1.5) to (93-95): (5-7). Preferably, the ratio of acidic mobile phase A to acidic mobile phase B is from (98.5-99.5) to (0.5-1.5) to (93-95) in 0-100 minutes: (5-7).
In one embodiment of the invention, after gradient elution with an acidic mobile phase, a constant flow of methanol solution containing 5% (volume percent) acetic acid is used. Preferably, the flow rate for constant flow elution is 50 ml/min.
In one embodiment of the invention, the method further comprises the step of concentrating and drying the hexapeptide acetate solution to obtain hexapeptide acetate with the purity of more than 95%.
In an embodiment of the invention, wherein the column temperature for alkaline loading and acidic elution is 35-45 ℃, preferably 40 ℃.
The invention has the beneficial effects that:
1. the method breaks through the traditional polypeptide purification method with a plurality of columns used alternately, can obtain the hexapeptide with the purity of more than 95 percent only by using the reversed-phase polymer chromatographic column, and simultaneously removes trifluoroacetic acid brought by the polypeptide cutting process, thereby greatly shortening the purification time and reducing the purification difficulty.
2. The method saves the cost of the mobile phase in the whole purification process, and the purification process is more environment-friendly. The mobile phase acetonitrile used in the traditional purification process is large in dosage and high in price, while the method mainly uses an aqueous solution with the pH value of 7.6-8.5 as the mobile phase in the purification process, and only uses a small amount of methanol solution to obtain the qualified hexapeptide, so that the method is low in price and low in environmental pollution.
3. The method has simple steps, effectively overcomes the defect that impurities with different structures and different chemical properties in the crude peptide can be completely separated only by alternately using a plurality of columns, and obtains the hexapeptide with high purity (more than 95 percent) and high yield.
4. The method only needs one-step purification, namely alkaline sampling and acidic elution, so that the qualified acetic acid hexapeptide can be obtained, time is saved, cost is low, and the method is suitable for large-scale industrial production.
Drawings
FIG. 1 is the mass spectrum of hexapeptide acetate after purification in example 1.
FIG. 2 is a chromatogram of hexapeptide acetate after purification in example 1, wherein the hexapeptide acetate has a purity of 97.32%.
Detailed Description
The present invention will be described in further detail with reference to the following drawings and examples, but the present invention is not limited to these examples. Unless otherwise indicated, the materials and equipment used in the examples are commercially available and are those conventionally used in the art, provided they meet the experimental requirements.
Example 1
1. Sample dissolution
Dissolving 100g of crude hexapeptide obtained by solid phase synthesis in 1000ml of aqueous solution, adjusting the pH value to 8.5 by ammonia water, performing ultrasonic dispersion, filtering by a No. 4 sand core funnel after the solution is completely clarified, and collecting filtrate.
2. Purification of
Purifying the filtrate by using a reversed-phase high performance liquid chromatography, wherein the filler is an F-type reversed-phase polymer column of SBC MCIGEL reversed-phase chromatographic filler (provided by Chengdu scientific biological limited) with the particle size of 30-50 mu m, and the column packing volume is 3000 mL. Before sample introduction, the reversed phase polymer chromatographic column is firstly balanced by using an aqueous solution (pH is adjusted by ammonia water) with pH of 8.5 until the conductivity of a detector is constant, sample introduction is carried out, the sample introduction amount is 100g, then the reversed phase polymer chromatographic column is washed by an alkaline mobile phase for 100min, the ratio of an alkaline mobile phase A to an alkaline mobile phase B is 99:1 when the basic mobile phase A is washed, the alkaline mobile phase A is the aqueous solution with the pH value of 8.5, the alkaline mobile phase B is a methanol solution with the pH of 8.5, the flow rate is 50ml/min, the column temperature is 40 ℃, after the washing of the alkaline mobile phase is finished, the acidic mobile phase is used for carrying out acid gradient elution to obtain the hexapeptide acetate so as to remove impurities in a hexapeptide crude product, the acidic mobile phase A is the aqueous solution containing 1% (volume percentage) acetic acid, the acidic mobile phase B is the methanol solution containing 1% (volume percentage) acetic acid, the flow rate is 50ml/min, the acidic mobile phase A is selected for 0-100 min, then eluting with an acidic mobile phase A and an acidic mobile phase B at constant current (the ratio of the acidic mobile phase A to the acidic mobile phase B is 95:5), the column temperature is 40 ℃, the detection wavelength is 215nm, collecting the purified hexapeptide acetate solution, carrying out reduced pressure rotary evaporation and concentration on the purified hexapeptide acetate solution at 40 ℃, and freeze-drying to obtain hexapeptide acetate with the purity of more than 95%, wherein the purification yield of the hexapeptide is 72%. The mass spectrum characterization result is shown in figure 1, and the chromatogram is shown in figure 2.
Example 2
1. Sample dissolution
Dissolving 50g of crude hexapeptide obtained by solid phase synthesis in 1000ml of aqueous solution, adjusting the pH value to 8.5 by using ammonia water, performing ultrasonic dispersion, filtering by using a No. 4 sand core funnel after the solution is completely clarified, and collecting filtrate.
2. Purification of
Purifying the filtrate by using a reversed-phase high performance liquid chromatography, wherein the filler is an F-type reversed-phase polymer column of SBC MCIGEL reversed-phase chromatographic filler (provided by Chengdu scientific biological limited) with the particle size of 30-50 mu m, and the column packing volume is 3000 mL. Before sample injection, the reversed phase polymer chromatographic column is firstly balanced by using an aqueous solution (pH is adjusted by ammonia water) with pH of 8.5 until the conductivity of the detector is constant, the sample is loaded, the sample loading amount is 50g, then the reversed phase polymer chromatographic column is washed by using an alkaline mobile phase for 100min, the ratio of the alkaline mobile phase A to the alkaline mobile phase B is 99:1, the alkaline mobile phase A is the aqueous solution with the pH value of 8.5, and the alkaline mobile phase B is a methanol solution with the pH value of 8.5. The flow rate is 50ml/min, and the column temperature is 40 ℃; after the washing is finished, performing acid gradient elution by using an acid mobile phase to obtain the hexapeptide acetate so as to remove impurities in the crude hexapeptide product, wherein the acid mobile phase A is an aqueous solution containing 1% (volume percentage) of acetic acid, the acid mobile phase B is a methanol solution containing 1% (volume percentage) of acetic acid, the flow rate is 50ml/min, and the acid mobile phase A is selected from 0 to 130 minutes in a mobile phase gradient way: the acid mobile phase B is prepared from 99:1 to 95:5, then eluting with an acidic mobile phase A and an acidic mobile phase B at constant current (the ratio of the acidic mobile phase A to the acidic mobile phase B is 95:5), the column temperature is 40 ℃, the detection wavelength is 215nm, collecting the purified hexapeptide acetate solution, carrying out reduced pressure rotary evaporation and concentration on the purified hexapeptide acetate solution at 40 ℃, and carrying out freeze drying to obtain the hexapeptide acetate with the purity of more than 95%, wherein the purification yield of the hexapeptide is 75%.
Example 3
In purification step 2 of this example, the mobile phase gradient was selected from 0 to 130 minutes A: B from 99:1 to 93: 7. The other steps are the same as the example 2, the hexapeptide acetate with the purity of more than 95 percent is obtained, and the purification yield is 65 percent.
Example 4
1. Sample dissolution
Dissolving 300g of crude hexapeptide obtained by solid phase synthesis in 1500ml of aqueous solution, adjusting the pH value to 8.5 by ammonia water, performing ultrasonic dispersion, filtering by a No. 4 sand core funnel after the solution is completely clarified, and collecting filtrate.
2. Purification of
Purifying the filtrate by using a reversed-phase high performance liquid chromatography, wherein the filler is an F-type reversed-phase polymer column of SBC MCIGEL reversed-phase chromatographic filler (provided by Chengdu scientific biological limited) with the particle size of 30-50 mu m, and the column packing volume is 3000 mL. Before sample introduction, the reversed phase polymer chromatographic column is firstly balanced by using an aqueous solution (pH is adjusted by ammonia water) with pH of 8.5 until the conductivity of a detector is constant, the sample is loaded, the sample loading amount is 300g, then the reversed phase polymer chromatographic column is washed by an alkaline mobile phase for 100min, the ratio of an alkaline mobile phase A to an alkaline mobile phase B is 99:1, the alkaline mobile phase A is the aqueous solution with the pH value of 8.5, the flow rate of a methanol solution with the pH of 8.5 of the alkaline mobile phase B is 150ml/min, and the column temperature is 40 ℃; after washing, acid mobile phase acid gradient elution is carried out to obtain the acetic acid hexapeptide so as to remove impurities in the hexapeptide crude product, wherein the acid mobile phase A is an aqueous solution containing 1% acetic acid, the acid mobile phase B is a methanol solution containing 1% acetic acid, the flow rate is 150ml/min, the acid mobile phase A is selected for 0-130 minutes in a mobile phase gradient mode, the acid mobile phase B is eluted from 99:1 to 95:5 in a constant current mode by using the acid mobile phase A and the acid mobile phase (the ratio of the acid mobile phase A to the acid mobile phase B is 95:5), the column temperature is 40 ℃, the detection wavelength is 215nm, the purified acetic acid hexapeptide solution is collected, the purified acetic acid hexapeptide solution is reduced pressure and rotary evaporated and concentrated at the temperature of 40 ℃, freeze drying is carried out, the acetic acid hexapeptide with the purity of more than 95% is obtained, and the purification yield of the hexapeptide is 70%.
Claims (15)
1. A method for purifying hexapeptide is characterized in that a reversed-phase polymer chromatographic column is adopted to purify a crude hexapeptide aqueous solution through a reversed-phase high performance liquid chromatography, wherein an alkaline mobile phase is firstly used for sampling the crude hexapeptide aqueous solution, and then an acidic mobile phase containing acetic acid is used for gradient elution to obtain an acetic acid hexapeptide solution;
wherein, the filler of the reversed phase polymer chromatographic column is F type SBC MCI GEL reversed phase chromatographic filler;
wherein the alkaline mobile phase consists of an alkaline mobile phase A and an alkaline mobile phase B, the alkaline mobile phase A is an aqueous solution with the pH value of 7.6-8.5, the alkaline mobile phase B is a methanol solution with the pH value of 7.6-8.5, the flow rate of the alkaline mobile phase is 10-300 ml/min, and the ratio of the alkaline mobile phase A to the alkaline mobile phase B is (99-98): 1-2 during alkaline sample loading;
wherein the acidic mobile phase containing acetic acid consists of an acidic mobile phase A and an acidic mobile phase B, the acidic mobile phase A is an aqueous solution containing 0.1-5% (volume percent) of acetic acid, the acidic mobile phase B is a methanol solution containing 0.1-5% (volume percent) of acetic acid, gradient elution is carried out by using the acidic mobile phase A and the acidic mobile phase B, and the elution gradient is selected from the following steps that the ratio of the acidic mobile phase A to the acidic mobile phase B is (98.5-99.5) to (0.5-1.5) to (93-95) in 0- (50-150) minute: (5-7), wherein the flow rate of the acidic mobile phase is 10-300 ml/min.
2. The method of claim 1, wherein the F-type SBC MCI GEL reverse phase chromatography packing has a particle size of 30 to 50 μ ι η.
3. The process as claimed in claim 1, wherein the aqueous solution of crude hexapeptide is prepared by dissolving crude hexapeptide in an aqueous solution, adjusting pH to 6-9, filtering with a filter membrane and collecting the filtrate.
4. The method as claimed in claim 3, wherein the mass percentage of the crude hexapeptide to water is 1:5-1: 30.
5. The method of claim 4, wherein the mass percentage of crude hexapeptide to water is 1: 20.
6. The process according to claim 3, wherein the pH of the solution obtained after dissolution is adjusted to 8.5.
7. The method of claim 1, wherein the crude hexapeptide is obtained by solid phase synthesis.
8. The method of claim 1, wherein the flow rate of the alkaline mobile phase is 50-150 ml/min.
9. The method of claim 1, wherein the elution gradient is from 99:1 to 95:5, or from 99:1 to 93:7 of acidic mobile phase a to acidic mobile phase B.
10. The method of claim 1, wherein the flow rate of the acidic mobile phase is 50-150 ml/min.
11. The method of claim 1, wherein the ratio of acidic mobile phase a to acidic mobile phase B is from (98.5-99.5) to (0.5-1.5) to (93-95) in 0-100 minutes: (5-7).
12. The method of claim 1, wherein after performing the gradient elution, the elution is performed with a constant flow of methanol solution containing 5% acetic acid.
13. The method of claim 1, further comprising the step of concentrating and drying the solution of acetate hexapeptide to obtain acetate hexapeptide with a purity of greater than 95%.
14. The method of claim 1, wherein the column temperature at loading and elution is 35-45 ℃.
15. The method of claim 14, wherein the column temperature at loading and elution is 40 ℃.
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| KR102293250B1 (en) * | 2017-09-18 | 2021-08-26 | 애니젠 주식회사 | Active material-hexapeptide complexes and cosmetic composition comprising same |
| CN109748954B (en) * | 2017-11-06 | 2022-03-01 | 正大天晴药业集团股份有限公司 | Purification method of degarelix |
| CN108707182A (en) * | 2018-06-04 | 2018-10-26 | 江苏吉泰肽业科技有限公司 | A kind of method for preparing purified of slightly solubility lipopeptid |
| CN111057140B (en) * | 2018-10-17 | 2023-06-13 | 深圳市健元医药科技有限公司 | Selepress purification method |
| CN110426476A (en) * | 2019-08-15 | 2019-11-08 | 珠海伊斯佳科技股份有限公司 | The measuring method of Argireline content in a kind of cosmetics |
| CN111423490A (en) * | 2020-03-13 | 2020-07-17 | 江苏吉泰肽业科技有限公司 | Purification method of achirelin |
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