CN113952944A - Method for purifying nonapeptide-1 - Google Patents
Method for purifying nonapeptide-1 Download PDFInfo
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Abstract
The invention provides a method for purifying nonapeptide-1, which belongs to the technical field of polypeptide purification, and particularly relates to a method for purifying a nonapeptide-1 crude product by combining gel chromatography column separation, reversed-phase high performance liquid chromatography and anion exchange chromatography, wherein a filler for the reversed-phase high performance liquid chromatography is a modified silica gel chromatography filler, the modified silica gel chromatography filler has good acid resistance and alkali resistance stability, cannot collapse in the separation process, and prolongs the service life of the nonapeptide-1.
Description
Technical Field
The invention belongs to the technical field of polypeptide purification, and particularly relates to a method for purifying nonapeptide-1.
Background
Nonapeptide-1 is a small molecule skin-brightening active peptide containing nine amino acids, is a bionic peptide of natural alpha-MSH, can competitively inhibit the combination of the alpha-MSH and a receptor MCI-R, and prevents the further activation of tyrosinase, thereby blocking the generation of melanin, reducing skin blackening and preventing the generation of brown spots. Nonapeptide-1 has become a very popular cosmetic material in recent years and is often added to skin-lightening creams, skin-lightening foundations, pressed powders, and spot-removing creams.
When the nonapeptide-1 is used as a cosmetic raw material, the chromatographic purity is required to be more than or equal to 99 percent, but because various side reactions and incomplete removal of a protective agent exist in the synthesis process of the nonapeptide-1, the nonapeptide-1 crude product contains impurity peptides with various molecular weights and properties, and the impurity peptides can be added into the cosmetics after being separated and purified. The commonly used method for separating and purifying the polypeptide comprises the following steps: salting out, gel filtration, ion exchange chromatography, affinity chromatography ultrafiltration, adsorption chromatography, isoelectric precipitation, and enzymolysis. The traditional method for purifying the nonapeptide-1 has high cost and low purification efficiency, and the obtained nonapeptide-1 has low purity. In order to improve the purity of nonapeptide-1, increase the purification efficiency and reduce the cost, extensive researchers have conducted improved studies on the purification of nonapeptide-1, for example, patent CN106749526A discloses a method for purifying nonapeptide-1 at low cost, which comprises purifying a large batch of crude nonapeptide-1 products by using a reverse phase polymer column to remove impurities in the crude nonapeptide-1 products, and then converting trifluoroacetic acid type nonapeptide-1 into acetic acid type nonapeptide-1 by using a weak anion exchange column, wherein the purity of the obtained nonapeptide-1 is more than 99%, the production cost is reduced, but the yield is only 79%, and therefore, it is still necessary to provide a method for purifying nonapeptide-1 with high yield and high purity.
Disclosure of Invention
In order to solve the problems in the prior art, the invention firstly provides a modified silica gel chromatographic packing which is spherical, uniform in particle size, free of agglomeration and excellent in acid and alkali resistance, and the nonapeptide-1 is purified by the packing, so that the cost is low, and the purity and the yield of the nonapeptide-1 are high.
In order to achieve the purpose, the invention adopts the technical scheme that:
a modified silica gel chromatography packing having the following structure:
silica gel microspheres grafted with a modified silane coupling agent; and
a hydrophilic epoxy resin coating layer formed on the surface of the silica gel microsphere;
wherein the modified silane coupling agent is 2-aminophenol-4-sulfonanilide modified silane coupling agent.
Specifically, the average pore diameter of the modified silica gel chromatographic packing is 6-10 nm, and the specific surface area is 230-290 m2/g。
Specifically, the modified silane coupling agent is prepared by the following method:
mixing vinyl triethoxysilane and 2-aminophenol-4-sulfonanilide, stirring and reacting for 1-4 h at the temperature of 28-35 ℃, and carrying out reduced pressure distillation to obtain the catalyst.
The addition mass ratio of the vinyl triethoxysilane to the 2-aminophenol-4-sulfonanilide is 1: 1-2.5.
The silica gel chromatographic packing has good mechanical tension, is not deformed under long-term high-pressure operation, has long service life, but has abundant high-reaction-activity silicon hydroxyl on the surface, is unstable under alkaline conditions, can keep and increase the retention, widen the peak and drag the alkaline compound when separating the alkaline compound, and reduces the service life due to the collapse of a silica gel packing matrix; according to the invention, the grafted modified silane coupling agent and the high-hydrophilicity epoxy resin are bonded on the surface of the silica gel, so that the silica gel is protected, and the acid resistance and the alkali resistance are improved.
The preparation method of the modified silica gel chromatographic packing comprises the following steps:
1) carrying out surface activation on the silica gel microspheres by using an acid solution to obtain surface-activated silica gel microspheres;
2) bonding and grafting a modified silane coupling agent on the surface of the surface-activated silica gel microsphere to obtain a silane coupling agent bonded silica gel microsphere;
3) introducing hydrophilic epoxy resin on the surface of the silane coupling agent bonded silica gel microsphere to form a coating layer, and obtaining the modified silica gel chromatographic packing.
According to the method, silicon hydroxyl on the surface of the silica gel reacts with the silane coupling agent, the silane coupling agent containing phenolic hydroxyl is bonded and fixed on the surface of the silica gel microsphere, then the high-hydrophilicity epoxy resin is bonded and grafted on the surface of the silica gel microsphere, a high-hydrophilicity layer is formed on the surface of the silica gel microsphere, the obtained modified silica gel chromatographic packing has proper pore volume and high specific surface area, the silicon hydroxyl on the surface of the silica gel is protected, alkali liquor cannot easily corrode the silica gel to lose the bonded phase, the alkali resistance is greatly improved, the service life is prolonged, the acid resistance is also improved, and probably because the modified silane coupling agent and the epoxy resin on the surface contain benzene rings and other groups, the hydrolysis of the bonded phase of the silica gel under low pH is hindered by virtue of a stereoscopic effect, so that the stability of the silica gel chromatographic packing under an acidic condition is improved; in addition, the inner layer of the obtained modified silica gel chromatographic packing is a hydrophobic layer which has enough charge repulsion acting force and hydrophobicity, so that the modified silica gel chromatographic packing has a good separation effect and is beneficial to elution of non-target substances, thereby improving the separation efficiency and purity of polypeptide, and the outer layer is hydrophilic and can prevent adsorption of protein and the like, so that nonspecific adsorption of water-soluble polymers, protein, biological enzyme, polypeptide and other biological macromolecules is reduced, and the service life is prolonged; the modified silica gel chromatographic packing is used for separating and purifying nonapeptide-1, so that the steric effect in the tail sealing reaction process is reduced, the chromatographic peak shape symmetry is good, and the column efficiency and the separation degree are improved.
Specifically, the acidic solution in the step 1) is a mixed aqueous solution containing 5-15 wt% of hydrochloric acid and 0.5-1 wt% of methanesulfonic acid.
Specifically, the specific operation of step 1) is as follows:
adding the silica gel microspheres into an acid solution, stirring for 0.5-5 h at 30-50 ℃, carrying out suction filtration, washing to be neutral, and carrying out vacuum drying at 80-100 ℃ to obtain the surface-activated silica gel microspheres.
Specifically, the mass ratio of the surface-activated silica gel microspheres to the modified silane coupling agent in the step 2) is 1: 1.8-3.
Specifically, the specific operation of step 2) is:
adding the surface-activated silica gel microspheres and the modified silane coupling agent into toluene under the protection of nitrogen, stirring and reacting for 0.5-6 h at 70-100 ℃, filtering after the reaction is finished, washing filter residues with toluene, acetone and methanol, extracting with acetone after washing, washing with water, and drying in vacuum at 60-80 ℃ to obtain the silane coupling agent bonded silica gel microspheres.
According to the method, the vinyl triethoxysilane modified by grafting 2-aminophenol-4-sulfonanilide is bonded and grafted on the surface of the activated silica gel microsphere, a uniform organic thin layer is formed on the surface of the activated silica gel microsphere, so that the alkali liquor can be prevented from reacting with silicon hydroxyl on the surface of the silica gel microsphere, the acid resistance and the alkali resistance are improved, the service life is prolonged, and the method can be used for separating and purifying the nonapeptide-1 and can improve the separation and purification effects of the nonapeptide-1.
Specifically, the hydrophilic epoxy resin in the step 3) is prepared by reacting bisphenol A polyether polyol and epoxy chloropropane, wherein the bisphenol A polyether polyol is BEO-6, and the molar ratio of the bisphenol A polyether polyol to the epoxy chloropropane is 1: 0.8-1.2.
Specifically, the mass ratio of the silane coupling agent bonded silica gel microspheres to the hydrophilic epoxy resin in the step 3) is 1: 10-30.
Specifically, the specific operation of step 3) is:
adding bisphenol A polyether polyol into toluene, adding a catalyst CAT-1, stirring for 20-50 min at 60-70 ℃, dropwise adding epoxy chloropropane, adding sodium hydroxide after dropwise adding, stirring for reacting for 1-4 h, neutralizing, filtering, and distilling under reduced pressure to obtain hydrophilic epoxy resin; and (3) placing the silane coupling agent bonded silica gel microspheres in hydrophilic epoxy resin, heating to 100-110 ℃, stirring for 0.5-1 h, adding a curing agent diethylaminopropylamine, continuously stirring for 10-50 min, filtering, and drying to obtain the modified silica gel chromatographic packing.
By the method, the epoxy resin and the silicon hydroxyl in the silica gel act on the surface of the silica gel microsphere to form a hydrophilic layer, the 2-aminophenol-4-sulfonanilide modified silane coupling agent bonded on the surface of the silica gel microsphere contains phenolic hydroxyl, can react with epoxy groups in epoxy resin to increase bonding strength and increase the uniformity of a hydrophilic epoxy resin layer, so that the obtained silica gel chromatographic packing has a uniform spherical structure, good dispersibility, proper pore diameter and high specific surface area, and after the silica gel chromatographic packing is bonded by the epoxy resin, the alkali liquor can be further prevented from reacting with silicon hydroxyl on the surface of the silica gel microsphere, the acid resistance and the alkalinity are improved, the alkali liquor is used for separating and purifying the nonapeptide-1, the steric effect in the tail sealing reaction process is reduced, the chromatographic peak shape symmetry is good, and the column effect and the separation degree are greatly improved; in addition, due to the existence of ether bonds in the bisphenol A polyether polyol, the bisphenol A polyether polyol has good hydrophilicity, so that the nonspecific adsorption of water-soluble polymers, proteins, biological enzymes, polypeptides and other biological macromolecules is reduced, and the service life is prolonged.
The application of the modified silica gel chromatographic packing in purifying the nonapeptide-1 comprises the step of purifying the nonapeptide-1 by using the modified silica gel chromatographic packing in reversed-phase high performance liquid chromatography.
A method of purifying nonapeptide-1, the method comprising:
dissolving a purified nonapeptide-1 crude product by using double distilled water, centrifuging, and performing gel chromatography column chromatography separation;
purifying by adopting a reversed-phase high performance liquid chromatography to obtain a nonapeptide-1 purified solution; the filler is the modified silica gel chromatographic filler;
and step three, carrying out fine purification by using anion exchange chromatography to obtain the high-purity nonapeptide-1.
Specifically, the feed-liquid ratio of the nonapeptide-1 crude product to the double distilled water in the step one is 1 g: 20-50 mL.
Specifically, the filler used for the gel chromatography column separation in the first step is sephadex G-25, and the filling volume is 20-50 mL.
Specifically, the conditions for the gel chromatography column separation in the first step are as follows: mobile phase: double distilled water, detection wavelength: 215 nm; column temperature: 30-45 ℃; sample introduction amount: 0.5-2 mL; flow rate: 1-5 mL/min.
Specifically, the conditions of the reversed-phase high performance liquid chromatography in the second step are as follows: mobile phase: the phase A is trifluoroacetic acid aqueous solution with the volume percent of 0.05-1, and the phase B is acetonitrile solution containing trifluoroacetic acid with the volume percent of 0.05-0.5; elution gradient: eluting 35-55% of phase B for 20-40 min; detection wavelength: 215 nm; column temperature: 30-45 ℃; feeding amount: 4-20 mu L; flow rate: 10-30 mL/min.
Specifically, the filling volume in the second step is 20-50 mL.
Specifically, the filler of the anion exchange chromatography in the third step is DEAE high-flow rate agarose microspheres, and the filling volume is 20-100 mL.
Specifically, the conditions of the anion exchange chromatography in the third step are as follows: mobile phase: a mixed aqueous solution containing 0.1 to 2 vol% of acetic acid and 0.05 to 0.5 vol% of sodium acetate; detection wavelength: 215 nm; and (3) elution: 100% acetic acid/sodium acetate mixed solution; flow rate: 4-10 mL/min.
According to the invention, through the steps, the gel chromatography is firstly utilized to carry out coarse purification on the nonapeptide-1 crude product, then the reversed-phase high performance liquid chromatography is utilized to carry out separation and purification, the modified silica gel chromatography packing is utilized to purify the nonapeptide-1, the steric effect in the tail sealing reaction process is reduced, the chromatographic peak shape symmetry is good, the column efficiency and the separation degree are greatly improved, most of impurities are removed, the loss of the nonapeptide-1 is reduced, the yield and the purity are improved, the activity is not damaged, finally, the weak anion exchange column is utilized to carry out desalination, the trifluoroacetic acid type nonapeptide-1 is converted into the acetic acid type nonapeptide-1, the nonapeptide-1 is further purified, the purification efficiency is greatly improved, and the purity and the yield of the obtained nonapeptide-1 are high.
The nonapeptide-1 obtained by the method has the yield higher than 85 percent and the purity higher than 99 percent.
The application of the method for purifying the nonapeptide-1 in preparing the nonapeptide-1.
The method adopts the combination of gel chromatographic column separation, reversed-phase high performance liquid chromatography and anion exchange chromatography to purify the nonapeptide-1 crude product, thereby having the following beneficial effects: the separation and purification method is simple, can quickly remove impurities in the crude product of the nonapeptide-1, efficiently separates the nonapeptide-1, and has high yield, low loss, high purity and undamaged activity of the obtained nonapeptide-1; the filler for the reversed-phase high-performance liquid chromatography is a modified silica gel chromatographic filler which has good acid resistance and alkali resistance stability, can not collapse in the separation process and prolongs the service life, the modified silica gel filler is used for separating and purifying the nonapeptide-1, the peak shape can be improved, the problems of peak broadening and tailing can be solved, the nonapeptide-1 can be separated quickly and efficiently, the peak emergence time is short, the yield and the purity of the nonapeptide-1 are improved, and the reduction of activity is reduced.
Drawings
FIG. 1 is a scanning electron micrograph of a chromatographic packing of modified silica gel obtained in example 1;
FIG. 2 is a graph showing the results of pore size and specific surface area tests on a modified silica gel chromatographic packing; in the figure, B represents pore diameter, and C represents specific surface area;
FIG. 3 is a graphical representation of the results of the acid and base resistance tests of the modified silica gel chromatographic packing; in the figure, B represents alkali resistance, and C represents acid resistance;
FIG. 4 is a chromatogram of nonapeptide-1 after purification in example 7;
FIG. 5 is a schematic representation of the results of the purity test for nonapeptide-1;
FIG. 6 is a graph showing the results of the yield test of nonapeptide-1.
Detailed Description
The following examples are presented to further illustrate the essence of the present invention, and it should be noted that these examples are only intended to specifically describe the present invention and should not be construed as limiting the present invention.
In the invention, in order to further improve the yield of nonapeptide-1, in the purification process of anion exchange chromatography, 0.02-0.08 wt% of sodium succinate and 0.01-0.03 vol% of 1-methyl-4-piperidinol are also contained in the mobile phase; specifically, the phase B is a mixed aqueous solution containing 0.1-2 vol% of acetic acid, 0.05-0.5 vol% of sodium acetate, 0.02-0.08 wt% of sodium succinate and 0.01-0.03 vol% of 1-methyl-4-piperidinol.
Example 1:
this example provides a modified silica gel chromatography packing prepared by the following method:
1) adding 1g of silica gel microspheres into 20 mL of mixed solution containing 8 wt% of hydrochloric acid and 0.6 wt% of methanesulfonic acid, stirring for 2 h at 40 ℃, carrying out suction filtration, washing to be neutral, and carrying out vacuum drying at 90 ℃ to obtain surface-activated silica gel microspheres;
2) mixing 2 g of vinyl triethoxysilane and 4.5 g of 2-aminophenol-4-sulfonanilide, stirring and reacting for 2 hours at the temperature of 30 ℃, and carrying out reduced pressure distillation to obtain a 2-aminophenol-4-sulfonanilide modified silane coupling agent; under the protection of nitrogen, adding 1g of surface-activated silica gel microspheres and 2.6 g of 2-aminophenol-4-sulfonanilide modified silane coupling agent into 30 g of toluene, stirring and reacting for 4 hours at 80 ℃, filtering after the reaction is finished, washing filter residues with toluene, acetone and methanol, extracting with acetone, washing with water, and drying in vacuum at 70 ℃ to obtain silane coupling agent bonded silica gel microspheres;
3) adding 25 g of BEO-6 into 200 g of toluene, adding 0.05 g of catalyst CAT-1, stirring for 40 min at 65 ℃, dropwise adding 4.6 g of epoxy chloropropane, adding sodium hydroxide after dropwise adding, stirring for reacting for 2.5 h, neutralizing, filtering, and distilling under reduced pressure to obtain hydrophilic epoxy resin; and (2) putting 1g of silane coupling agent bonded silica gel microspheres into 20 g of hydrophilic epoxy resin, adding 5 g of glycerol, heating to 105 ℃, stirring for 0.5 h, adding 6 g of curing agent diethylaminopropylamine, continuously stirring for 20 min, taking out, and drying to obtain the epoxy resin coated silica gel chromatographic packing.
The modified silica gel chromatographic packing obtained in the embodiment is characterized by a JSM-6360LV type scanning electron microscope, and the result is shown in figure 1, and the observation of figure 1 shows that the silica gel still has good spherical morphology and uniform particle size and has no agglomeration phenomenon after being bonded by a 2-aminophenol-4-sulfonanilide modified silane coupling agent and hydrophilic epoxy resin.
Example 2:
this example provides another modified silica gel chromatography packing material prepared substantially the same as example 1 except that in step 1), vinyltriethoxysilane and 2-aminophenol-4-sulfonanilide were added in amounts of 2 g and 1g, respectively.
Example 3:
this example provides another modified silica gel chromatography packing material prepared substantially the same as example 1 except that in step 1), vinyltriethoxysilane and 2-aminophenol-4-sulfonanilide were added in amounts of 2 g and 2 g, respectively.
Example 4:
this example provides another modified silica gel chromatography packing material prepared substantially the same as example 1 except that in step 1), vinyltriethoxysilane and 2-aminophenol-4-sulfonanilide were added in amounts of 2 g and 5 g, respectively.
Example 5:
this example provides another modified silica gel chromatography packing material prepared substantially the same as example 1 except that in step 1), vinyltriethoxysilane and 2-aminophenol-4-sulfonanilide were added in amounts of 2 g and 6 g, respectively.
Example 6:
this example provides another modified silica gel chromatographic packing prepared in substantially the same manner as example 1, except that in step 2) the modified silane coupling agent was replaced with vinyltriethoxysilane, i.e., the silane coupling agent was not modified with 2-aminophenol-4-sulfonanilide.
Test example 1:
pore diameter and specific surface area test:
the pore size and specific surface area of the modified silica gel chromatographic packing obtained in examples 1 to 6 were measured by using a V-Sorb 2800P specific surface and pore size analyzer of Beijing Jineeangstrom science and technology Limited, and the measurement results are shown in FIG. 2.
FIG. 2 shows the results of pore size and specific surface area tests of modified silica gel chromatographic packing, wherein B represents pore size and C represents specific surface area, and it can be seen from FIG. 2 that the average pore size of the silica gel chromatographic packing obtained in preferred embodiments 1, 3 and 4 is 6-10 nm, and the specific surface area is 238-285 m2The data of comparative examples 1 to 5 show that 2-aminophenol-4-sulfonic acid is added in the preparation process of the modified silica gel chromatographic packingThe increase of the addition amount of the anilide increases the uniform coating of the silica gel microspheres, the average pore diameter is reduced, and the specific surface area is correspondingly reduced, while the silane coupling agent bonded on the surfaces of the silica gel microspheres in the embodiment 6 is not modified, the coating layers of the silica gel microspheres are fewer and the uniformity is poorer, so that the average pore diameter and the specific surface area of the obtained silica gel chromatographic packing are the largest. In conclusion, the aperture of the silica gel chromatographic packing is controlled to be 6-10 nm, the specific surface area is controlled to be 230-280 m and the addition amount of 2-aminophenol-4-sulfonanilide in the modification process of the silane coupling agent is regulated and controlled2Between/g, the reduction of the separation and purification efficiency of the nonapeptide-1 caused by the undersize or oversize pore diameter and the specific surface area is avoided.
Test example 2:
and (3) acid and alkali resistance test:
alkali resistance: the packing materials of examples 1 to 6 were packed in a column, and the retention loss rate of methyl p-hydroxybenzoate and the detection wavelength of 254 nm were measured after 20 hours by using 200 mmol/L acetonitrile/triethylamine aqueous solution (acetonitrile/triethylamine volume ratio 3: 7, pH = 11.0) as a mobile phase while maintaining the flow rate at 1.0 mL/min and the column temperature at 60 ℃.
Acid resistance: the filler of the embodiment 1 to 6 is filled into a column, the flow rate is kept at 1.0 mL/min, the column temperature is kept at 60 ℃, 0.5 percent trifluoroacetic acid is used as a mobile phase, the retention loss rate of the methyl p-hydroxybenzoate is measured after 20 hours, and the wavelength is 254 nm.
Fig. 3 is a result of testing acid and alkali resistance (retention loss rate of methyl p-hydroxybenzoate) of the modified silica gel chromatographic packing, wherein B represents alkali resistance, and C represents acid resistance observation fig. 3 shows that after six columns are washed at pH =11.0 and 60 ℃ for 20 h, the retention loss rates of methyl p-hydroxybenzoate of the columns of examples 1, 3 and 4 are small, the retention loss rates of examples 2 and 5 are large, and the retention loss rate of example 6 is the highest, which may be caused by that the silane coupling agent is modified to help form a uniform coating layer on the surface of the silica gel microspheres, so that the silica gel microspheres are well wrapped, contact of the alkaline mobile phase with the silica hydroxyl on the surface of the silica gel is reduced, the mobile phase is not easy to erode the silica gel to lose bonding phase, and the silica gel is well protected, so the loss rate is low, and the alkali resistance is improved; the above-mentioned effects are closely related to the degree of modification of the silane coupling agent. In conclusion, the silica gel microspheres are bonded and coated by the modified silane coupling agent and the epoxy resin, so that the alkali resistance of the silica gel chromatographic packing can be obviously improved, and the defects of the existing silica gel chromatographic packing are overcome.
It can also be seen from fig. 3 that after the six columns are washed with 0.5% trifluoroacetic acid at 60 ℃ for 20 hours, the difference of the retention loss rates of methyl paraben is significant, the retention loss rates of examples 1, 3 and 4 are not higher than 4.4%, while the retention loss rates of examples 2 and 5 are higher than 6.7%, and the retention loss rate of example 6 is 16.5%, which is significantly higher than example 1, which indicates that after the silica gel microspheres are bonded with the modified silane coupling agent and the epoxy resin, the silica gel microspheres can be well protected, and the disappearance of the silica gel bonding phase can be reduced, so that the loss rate is reduced, and the acid resistance is improved.
Example 7:
this example provides a method for purifying nonapeptide-1 comprising the steps of:
dissolving 1G of a nonapeptide-1 crude product in 30 mL of double distilled water, centrifuging for 20 min at 8000 r/min, adding the double distilled water into a supernatant to prepare a solution of 50 mg/mL, standing overnight, performing gel chromatography separation by using sephadex G-25, wherein the column filling volume is 30 mL, the mobile phase is the double distilled water, the column temperature is 35 ℃, the sample injection amount is 1.5 mL, the flow rate is 4 mL/min, and eluting components are collected according to an absorbance curve at 215 nm to obtain a nonapeptide-1 crude solution;
step two, the modified silica gel chromatographic packing obtained in the example 1 is used for carrying out reversed-phase high performance liquid chromatography purification, the filling volume of the column is 30 mL, the mobile phase A is a trifluoroacetic acid aqueous solution with the volume percent of 0.08, the phase B is a trifluoroacetic acid acetonitrile solution with the volume percent of 0.2, the flow rate is 25 mL/min, the column temperature is 40 ℃, the feeding amount is 8 muL, the flow rate is 25 mL/min, the detection wavelength is 215 nm, the column is washed by the trifluoroacetic acid aqueous solution with the volume percent of 0.08 before sample injection, and the elution purification is carried out after the sample injection, wherein the elution conditions are as follows: eluting phase B with 48% for 40 min, collecting purified nonapeptide-1 solution, and concentrating under reduced pressure to 30 mg/mL;
and step three, carrying out anion exchange chromatography purification by using DEAE high-flow rate agarose microspheres, wherein the filling volume of a column is 30 mL, the mobile phase is a mixed solution containing 0.5 vol% of acetic acid and 0.2 vol% of sodium acetate, the flow rate is 8 mL/min, the column temperature is 40 ℃, the detection wavelength is 215 nm, the column is washed by 1 vol% of acetic acid water solution before sample injection, elution and purification are carried out after sample injection, a target peak is collected, reduced pressure concentration is carried out, and freeze drying is carried out to obtain the high-purity nonapeptide-1.
Example 8:
this example provides an alternative method for purifying nonapeptide-1, which comprises the same steps as in example 7, except that the packing used in step two is the modified silica gel chromatography packing obtained in example 2.
Example 9:
this example provides an alternative method for purifying nonapeptide-1, which comprises the same steps as in example 7, except that the packing used in step two is the modified silica gel chromatography packing obtained in example 3.
Example 10:
this example provides an alternative method for purifying nonapeptide-1, which comprises the same steps as in example 7, except that the packing used in step two is the modified silica gel chromatography packing obtained in example 4.
Example 11:
this example provides an alternative method for purifying nonapeptide-1, which comprises the same steps as in example 7, except that the packing used in step two is the modified silica gel chromatography packing obtained in example 5.
Example 12:
this example provides an alternative method for purifying nonapeptide-1, which comprises the same steps as in example 7, except that the packing used in step two is the modified silica gel chromatography packing obtained in example 6.
Example 13:
this example provides an alternative method for purifying nonapeptide-1, which comprises the same steps as in example 7, except that the filler in step two is an untreated silica gel chromatography filler.
Example 14:
this example provides another method for purifying nonapeptide-1, which comprises the steps substantially the same as those of example 7, except that the mobile phase of step three is a mixed aqueous solution comprising 0.5 vol% of acetic acid, 0.2 vol% of sodium acetate, 0.06 vol% of sodium succinate, and 0.02 vol% of 1-methyl-4-piperidinol.
Example 15:
this example provides an alternative method for purifying nonapeptide-1, which comprises essentially the same steps as in example 14, except that the mobile phase of step three does not contain sodium succinate.
Example 16:
this example provides an alternative method for purifying nonapeptide-1, which comprises essentially the same steps as in example 14, except that the mobile phase of step three does not comprise 1-methyl-4-piperidinol.
It should be clear that each of the crude nonapeptide-1 products of examples 7-16 above is derived from the crude nonapeptide-1 product obtained under the same experimental conditions.
Test example 3:
purity and yield test:
the high-purity nonapeptide-1 obtained in examples 7 to 16 was sufficiently dried, weighed, and subjected to high performance liquid chromatography to determine the purity.
FIG. 4 is a chromatogram of nonapeptide-1 purified in example 7, and it can be seen that the purified nonapeptide-1 has a high purity and the impurity peak is completely separated.
FIGS. 5 and 6 are the results of testing the purity and yield of the high-purity nonapeptide-1 obtained in examples 7 to 16, respectively, and it can be seen from FIGS. 5 and 6 that when the modified silica gel chromatography packing obtained in examples 1, 3 and 4 is reversed-phase high performance liquid chromatography packing, i.e., when the crude product is purified by the schemes described in examples 7, 9 and 10, the purity of the nonapeptide-1 obtained by combining gel chromatography column separation and anion exchange chromatography is high and reaches 99% or more, and the yield reaches 85% or more, while the purity of example 11 is still 99% or more, and the yield is lower than that of example 7, which indicates that the excessive modification of the silane coupling agent can reduce the purification yield and has little influence on the purity; compared with examples 7-13, the purity and yield of nonapeptide-1 obtained by purification have obvious difference, which shows that the modification treatment of the silane coupling agent has great influence on the separation and purification of nonapeptide-1, and the silica gel chromatographic packing with proper average pore diameter and specific surface area can be obtained by controlling the addition amount of 2-aminophenol-4-sulfonanilide in the modification process of the silane coupling agent, so that the separation efficiency and purity of nonapeptide-1 can be improved; it can also be seen from fig. 6 that the yield of nonapeptide-1 obtained by purification in example 14 is higher than that of example 7, and reaches 87.5%, and the yield of nonapeptide-1 obtained by purification in examples 15 and 16 is not significantly changed compared with example 7, which indicates that sodium succinate and 1-methyl-4-piperidinol are simultaneously added in the mobile phase during the separation process of anion exchange chromatography, so that certain synergistic effect is achieved, the ionic strength of the mobile phase is increased, the hydrophobic effect of nonapeptide-1 and the stationary phase in anion exchange chromatography can be inhibited, the separation selectivity is improved, and the yield of nonapeptide-1 is increased.
Conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (10)
1. The modified silica gel chromatographic packing is characterized by having the following structure:
silica gel microspheres grafted with a modified silane coupling agent; and
a hydrophilic epoxy resin coating layer formed on the surface of the silica gel microsphere;
wherein the modified silane coupling agent is 2-aminophenol-4-sulfonanilide modified silane coupling agent.
2. The modified silica gel chromatographic packing according to claim 1, wherein the modified silica gel chromatographic packing has an average pore diameter of 6 to 10 nm and a specific surface area of 230 to 290 m2/g。
3. The modified silica gel chromatographic packing of claim 1, wherein the modified silane coupling agent is prepared by a method comprising:
mixing vinyl triethoxysilane and 2-aminophenol-4-sulfonanilide, stirring and reacting for 1-4 hours at the temperature of 28-35 ℃, and carrying out reduced pressure distillation to obtain a silane coupling agent; the addition mass ratio of the vinyl triethoxysilane to the 2-aminophenol-4-sulfonanilide is 1: 1-2.5.
4. A method of preparing a modified silica gel chromatography packing material as claimed in any one of claims 1 to 3, comprising the steps of:
1) carrying out surface activation on the silica gel microspheres by using an acid solution to obtain surface-activated silica gel microspheres;
2) bonding and grafting a modified silane coupling agent on the surface of the surface-activated silica gel microsphere to obtain a silane coupling agent bonded silica gel microsphere;
3) introducing hydrophilic epoxy resin on the surface of the silane coupling agent bonded silica gel microsphere to form a coating layer, and obtaining the modified silica gel chromatographic packing.
5. The method according to claim 4, wherein the mass ratio of the surface-activated silica gel microspheres to the modified silane coupling agent in the step 2) is 1: 1.8-3.
6. The method according to claim 4, wherein the hydrophilic epoxy resin in step 3) is prepared by reacting bisphenol A polyether polyol and epichlorohydrin; the bisphenol A polyether polyol is BEO-6, and the molar ratio of the bisphenol A polyether polyol to the epoxy chloropropane is 1: 0.8-1.2.
7. The method according to claim 4 or 6, wherein the mass ratio of the silane coupling agent bonded silica gel microspheres to the hydrophilic epoxy resin in the step 3) is 1: 20-50.
8. Use of a modified silica gel chromatography packing material according to any one of claims 1 to 3 for the purification of nonapeptide-1, wherein the use comprises the use of the modified silica gel chromatography packing material in a reverse phase high performance liquid chromatography process for the purification of nonapeptide-1.
9. A method for purifying nonapeptide-1, comprising:
dissolving a purified nonapeptide-1 crude product by using double distilled water, centrifuging, and performing gel chromatography column chromatography separation;
purifying by adopting a reversed-phase high performance liquid chromatography to obtain a nonapeptide-1 purified solution; the filler is the modified silica gel chromatographic filler as defined in any one of claims 1-3, and the filling volume is 20-50 mL;
and step three, carrying out fine purification by using anion exchange chromatography to obtain the high-purity nonapeptide-1.
10. Use of the method of purifying nonapeptide-1 of claim 9 for the preparation of nonapeptide-1.
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|---|---|---|---|---|
| CN116236819A (en) * | 2023-05-09 | 2023-06-09 | 成都佩德生物医药有限公司 | Method for purifying polypeptide toxins in batches and composite double-layer chromatographic column |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CS960887A1 (en) * | 1987-12-22 | 1988-08-16 | Vyskocil Frantisek | 3-amine-4-hydroxy-/n-phenyl/-benzensulphamide and process for preparing thereof |
| US20040101947A1 (en) * | 2002-11-01 | 2004-05-27 | Promega Corporation | Cell lysis composition, methods of use, apparatus and kit |
| JP2005515217A (en) * | 2001-12-29 | 2005-05-26 | ポリペプチド ラボラトリーズ アーエス | Peptide purification |
| EP2100923A1 (en) * | 2006-12-25 | 2009-09-16 | Kaneka Corporation | Curable composition |
| WO2009141384A2 (en) * | 2008-05-21 | 2009-11-26 | Novo Nordisk A/S | Process for the purification of factor vii polypeptides using affinity resins comprising specific ligands |
| US20110319592A1 (en) * | 2008-11-13 | 2011-12-29 | Novo Nordisk A/S | Process for the Purification of Antibodies Using Affinity Resins Comprising Specific Ligands |
| CN103055832A (en) * | 2012-12-31 | 2013-04-24 | 浙江月旭材料科技有限公司 | Chromatographic packing for separating water soluble polymer and protein and preparation method of same |
| CN104073149A (en) * | 2014-07-21 | 2014-10-01 | 朱蕾 | Preparation method for lightproof and waterproof polyurethane coating |
| CN104449325A (en) * | 2014-12-17 | 2015-03-25 | 袁福德 | Prepration method of nanometer material capable of improving fire resistance of waterborne polyurethane coating and adhesive |
| US20150086606A1 (en) * | 2012-03-01 | 2015-03-26 | Bioactive Tecnologia Em Polimeros Ltda-Me | Bioresorbable and bioactive three-dimensional porous material and method for the production thereof |
| CN104549183A (en) * | 2013-10-21 | 2015-04-29 | 天津汉荣生物技术有限公司 | Silica gel chromatography packing and preparation method thereof |
| CN106749526A (en) * | 2016-12-22 | 2017-05-31 | 陕西慧康生物科技有限责任公司 | A kind of method of nine victory peptides 1 of low cost purifying |
| CN107312072A (en) * | 2017-06-20 | 2017-11-03 | 浙江湃肽生物有限公司 | A kind of method of purifies and separates Atosiban |
| CN112625481A (en) * | 2020-12-16 | 2021-04-09 | 江苏德莱利建筑装饰工程有限公司 | Epoxy floor coating with formaldehyde absorption performance and preparation method thereof |
-
2021
- 2021-12-23 CN CN202111584870.4A patent/CN113952944B/en active Active
Patent Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CS960887A1 (en) * | 1987-12-22 | 1988-08-16 | Vyskocil Frantisek | 3-amine-4-hydroxy-/n-phenyl/-benzensulphamide and process for preparing thereof |
| JP2005515217A (en) * | 2001-12-29 | 2005-05-26 | ポリペプチド ラボラトリーズ アーエス | Peptide purification |
| US20060014694A1 (en) * | 2001-12-29 | 2006-01-19 | Rasmussen Jon H | Peptide purification |
| US20040101947A1 (en) * | 2002-11-01 | 2004-05-27 | Promega Corporation | Cell lysis composition, methods of use, apparatus and kit |
| EP2100923A1 (en) * | 2006-12-25 | 2009-09-16 | Kaneka Corporation | Curable composition |
| WO2009141384A2 (en) * | 2008-05-21 | 2009-11-26 | Novo Nordisk A/S | Process for the purification of factor vii polypeptides using affinity resins comprising specific ligands |
| US20110319592A1 (en) * | 2008-11-13 | 2011-12-29 | Novo Nordisk A/S | Process for the Purification of Antibodies Using Affinity Resins Comprising Specific Ligands |
| US20150086606A1 (en) * | 2012-03-01 | 2015-03-26 | Bioactive Tecnologia Em Polimeros Ltda-Me | Bioresorbable and bioactive three-dimensional porous material and method for the production thereof |
| CN103055832A (en) * | 2012-12-31 | 2013-04-24 | 浙江月旭材料科技有限公司 | Chromatographic packing for separating water soluble polymer and protein and preparation method of same |
| CN104549183A (en) * | 2013-10-21 | 2015-04-29 | 天津汉荣生物技术有限公司 | Silica gel chromatography packing and preparation method thereof |
| CN104073149A (en) * | 2014-07-21 | 2014-10-01 | 朱蕾 | Preparation method for lightproof and waterproof polyurethane coating |
| CN104449325A (en) * | 2014-12-17 | 2015-03-25 | 袁福德 | Prepration method of nanometer material capable of improving fire resistance of waterborne polyurethane coating and adhesive |
| CN106749526A (en) * | 2016-12-22 | 2017-05-31 | 陕西慧康生物科技有限责任公司 | A kind of method of nine victory peptides 1 of low cost purifying |
| CN107312072A (en) * | 2017-06-20 | 2017-11-03 | 浙江湃肽生物有限公司 | A kind of method of purifies and separates Atosiban |
| CN112625481A (en) * | 2020-12-16 | 2021-04-09 | 江苏德莱利建筑装饰工程有限公司 | Epoxy floor coating with formaldehyde absorption performance and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| KAMINI GOVENDER ET AL.: ""The development of a sub/supercritical fluid chromatography based purification method for peptides"", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
| 苏晨灿 等: ""九肽-1纯化方法研究"", 《山东工业技术》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116236819A (en) * | 2023-05-09 | 2023-06-09 | 成都佩德生物医药有限公司 | Method for purifying polypeptide toxins in batches and composite double-layer chromatographic column |
| CN116236819B (en) * | 2023-05-09 | 2023-08-04 | 成都佩德生物医药有限公司 | Method for purifying polypeptide toxins in batches and composite double-layer chromatographic column |
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