Preparation method of high-purity hydrogel polypeptide

Abstract

The invention discloses a preparation method of high-purity hydrogel polypeptide, belongs to the technical field of polypeptide preparation, and particularly relates to hydrogel polypeptide obtained by mixing a hydrogel polypeptide crude product with a solvent and eluting and purifying the mixture in a column separation system by adopting an eluent. The eluent is a low-water mobile phase, and the low-water mobile phase is composed of methanol, acetic acid and water, or composed of acetonitrile, acetic acid and water; and the volume ratio of water in the low water mobile phase is 45% or less and not equal to 0%. The volume ratio of methanol to acetic acid in the low water mobile phase is 1: (0.5-5). The hydrogel polypeptide is prepared from Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly or palmitoyl tetrapeptide-20 (Ph-Val-Gln-Trp-Leu-Ile-Ala-Gly).

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide preparation, and particularly relates to a method for preparing a high-purity hydrogel polypeptide. Background Art

[0002] Hydrogel polypeptides are polymers with three-dimensional network structures formed by non-covalent interactions of polypeptide molecules in an aqueous solution system. Due to their porous and highly absorbent properties, hydrogel polypeptides have broad application prospects in the fields of beauty and medicine, especially in the medical field. In the field of wound repair, hydrogel polypeptides can provide a humid environment to keep the wound surface moist, allow gas to pass through and absorb exudate, and provide an ideal environment for wound healing. The three-dimensional network of the hydrogel not only makes it elastic like soft tissue, but also facilitates tissue regeneration and repair. In addition, drugs such as growth factors can be encapsulated in hydrogels to achieve sustained release of growth factors at the wound site, increase the half-life of growth factors, and promote angiogenesis, anti-inflammation and wound healing. In the field of medicine, antibacterial agents are wrapped in gels to selectively release them to the site of infection, thereby killing bacteria safely and efficiently; anti-tumor, cardiovascular and other drugs can be finely delivered through enzyme-responsive hydrogel polypeptides for targeted therapy.

[0003] At present, the application of hydrogel peptides is mostly concentrated in the field of cosmetics for external use. The peptide segment is extremely short, and the peptide raw materials are mostly obtained by solid phase synthesis, direct washing and drying after cutting, or by direct washing and drying after liquid phase chemical synthesis. The purity is low, such as palmitoyl tetrapeptide-20, the highest commercially available purity is 98%; such as oligopeptide-104, the commercially available purity is 95-99%. With the increasing demand for the application of hydrogel peptides in the medical field, the demand for high-purity hydrogel peptides is also increasing, but the characteristic of hydrogel peptides forming gels in aqueous solutions makes it impossible to purify through conventional reverse phase chromatography, ion chromatography, affinity chromatography and other chromatographic systems with water-based mobile phases to obtain peptide products with a purity greater than 99-100%. This technical bottleneck limits the application of hydrogel peptides in the medical field, especially in the field of in vivo drugs.

[0004] Therefore, developing a purification method for obtaining high-purity hydrogel polypeptides to obtain hydrogel polypeptides with low impurity content and high safety has great application prospects and economic and practical value. Summary of the invention

[0005] The purpose of the present invention is to provide a low-water mobile phase and a macroporous resin that can be used to purify a hydrogel polypeptide, which is a method for preparing a high-purity hydrogel polypeptide. The method of the present invention has a good purification effect on the hydrogel polypeptide, and can purify more hydrogel polypeptides, thereby increasing the yield of the hydrogel polypeptide.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A method for preparing a high-purity hydrogel polypeptide comprises: mixing a crude hydrogel polypeptide with a solvent, and eluting and purifying the hydrogel polypeptide with an eluent in a column separation system to obtain the hydrogel polypeptide; The eluent is a low water mobile phase, which consists of methanol, acetic acid and water, or the low water mobile phase consists of acetonitrile, acetic acid and water; The volume proportion of water in the low-water mobile phase is less than 45% and not equal to 0%. The present invention can avoid the hydrogel polypeptide from being in a gel state in a conventional methanol-water and acetonitrile-water based mobile phase by reducing the water content in the solution, thereby improving the solubility of the hydrogel polypeptide in the mobile phase.

[0007] Preferably, the volume ratio of methanol to acetic acid in the low water mobile phase is 1:0.5-5.

[0008] Preferably, the hydrogel polypeptide comprises Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly or palmitoyl tetrapeptide-20.

[0009] Preferably, the column separation system comprises a C18 column or a macroporous resin column, the macroporous resin column is filled with a macroporous resin, the macroporous resin comprises a macroporous cross-linked resin, and the macroporous cross-linked resin is made by reacting p-styrene, p-chloromethylstyrene and N-hydroxymethyl acrylamide. The present invention prepares a macroporous cross-linked resin by reacting p-styrene, p-chloromethylstyrene and N-hydroxymethyl acrylamide, and the macroporous cross-linked resin in the present invention has a chloromethyl structure, an N-hydroxymethyl structure and an amide structure. When a low-water mobile phase and a macroporous cross-linked resin are used in combination, Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly or palmitoyl tetrapeptide-20 can be better purified and separated, wherein the separation effect of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly is better than the separation effect of palmitoyl tetrapeptide-20.

[0010] Preferably, in the preparation of the macroporous cross-linked resin, a monomer solution and a porogen solution are mixed and reacted, and the macroporous cross-linked resin is prepared after the porogen is removed; the monomer solution contains a polymerizable monomer and an initiator, the polymerizable monomer includes p-styrene, p-chloromethylstyrene and N-hydroxymethyl acrylamide, and the porogen is PVA.

[0011] Preferably, the amount of p-chloromethylstyrene used is 10-30wt% of p-styrene, and the amount of N-hydroxymethylacrylamide used is 30-60wt% of p-styrene.

[0012] Preferably, the solvents of the monomer solution are toluene and methyl isobutyl carbinol, and the amount of methyl isobutyl carbinol used is 80-120 wt % of toluene.

[0013] Preferably, the porogen is PVA, and the porogen solution contains 0.2-1 wt % of the porogen.

[0014] Preferably, the amount of the porogen solution used is 200-400 wt % of the monomer solution.

[0015] Preferably, the initiator is BPO, and the amount of the initiator used is 0.5-2 wt % of the polymerization monomer.

[0016] Preferably, in the preparation of the macroporous cross-linked resin, a porogen is added to deionized water and mixed to obtain a porogen solution, and then the polymerized monomer is mixed with a solvent and an initiator to obtain a monomer solution, the monomer solution is mixed with the porogen solution, and the mixture is reacted at a temperature of 60-90° C. for 5-15 hours. After the reaction is completed, the reaction solution is removed, the porogen is removed by washing with hot water, and the mixture is dried to obtain a macroporous cross-linked resin.

[0017] More preferably, in the preparation of the macroporous cross-linked resin, the porogen is PVA, the porogen solution contains 0.2-1 wt % of the porogen, and the amount of the porogen solution used is 200-400 wt % of the monomer solution.

[0018] More preferably, in the preparation of the macroporous cross-linked resin, the polymerizable monomers include p-styrene, p-chloromethylstyrene, and N-hydroxymethylacrylamide, the amount of p-chloromethylstyrene used is 10-30wt% of p-styrene, the amount of N-hydroxymethylacrylamide used is 30-60wt% of p-styrene, and the amount of the polymerizable monomers used is 20-40wt% of the solvent.

[0019] More preferably, in the preparation of the macroporous cross-linked resin, the solvents are toluene and methyl isobutyl carbinol, and the amount of methyl isobutyl carbinol used is 80-120 wt % of toluene.

[0020] More preferably, in the preparation of the macroporous cross-linked resin, the initiator is BPO, and the amount of the initiator used is 0.5-2 wt % of the polymerization monomer.

[0021] Preferably, the macroporous resin also includes an amine-modified resin. The amine-modified resin is prepared by reacting a macroporous cross-linked resin with di-n-butyrimide. The amine-modified resin prepared by the present invention has a macroporous cross-linked structure and an aminated structure. Under the action of the macroporous cross-linked structure and the aminated structure, the purification effect of the hydrogel polypeptide is further improved.

[0022] Preferably, in the preparation of the amine-modified resin, the macroporous cross-linked resin is added to DMF, and swelled at 20-40°C for 6-24 hours, then di-n-butyrimide and anhydrous potassium carbonate are added, and reacted at 30-50°C for 24-72 hours. After the reaction is completed, the resin is washed and dried to obtain the amine-modified resin.

[0023] More preferably, in the preparation of the amine-modified resin, the amount of the macroporous cross-linked resin used is 20-30 wt % of DMF.

[0024] More preferably, in the preparation of the amine-modified resin, the amount of di-n-butyrimide used is 20-40 wt % of the macroporous cross-linked resin.

[0025] More preferably, in the preparation of the amine-modified resin, the amount of anhydrous potassium carbonate used is 10-30 wt % of the macroporous cross-linked resin.

[0026] Preferably, in the preparation of the hydrogel polypeptide, the crude hydrogel polypeptide is mixed with a solvent, and an eluent is used in a column separation system for elution and purification to obtain the hydrogel polypeptide. The hydrogel polypeptide in the crude hydrogel polypeptide includes Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly or palmitoyl tetrapeptide-20, the solvent is DMSO, and the amount of the crude hydrogel polypeptide and the solvent used is 20-100 mg / mL.

[0027] More preferably, in the preparation of the hydrogel polypeptide, the column separation system comprises a C18 column or a macroporous resin column.

[0028] More preferably, in the preparation of the hydrogel polypeptide, the eluent comprises a low-water mobile phase, the low-water mobile phase consists of methanol, acetic acid and water, and the volume proportion of water in the low-water mobile phase is less than 45% and not equal to 0%.

[0029] More preferably, in the preparation of the hydrogel polypeptide, the volume ratio of methanol to acetic acid in the low water mobile phase is 1:0.5-5.

[0030] The invention adopts a low-water mobile phase to separate and purify the hydrogel polypeptide, and the low-water mobile phase greatly improves the solubility of the hydrogel polypeptide in the mobile phase. The hydrogel polypeptide is in a gel state in a conventional mobile phase with methanol-water and acetonitrile-water as the base, but is in a clear and transparent solution state in the low-water mobile phase of the invention; the low-water mobile phase greatly improves the sample loading of the hydrogel polypeptide in the reverse phase chromatography purification process. The sample loading of the hydrogel polypeptide in the conventional mobile phase with methanol-water and acetonitrile-water as the base is mg level, but the sample loading can reach g level in the low-water mobile phase of the technical solution; the low-water mobile phase greatly improves the peak shape of the hydrogel polypeptide in the reverse phase chromatography purification process. When the hydrogel polypeptide is prepared by using the conventional mobile phase with methanol-water and acetonitrile-water as the base, the peak shape is mostly broadened and tailed, but when the hydrogel polypeptide is prepared by using the low-water mobile phase of the invention, the peak shape is narrow and has no tailing.

[0031] The present invention also uses polymerization monomers including p-styrene, p-chloromethylstyrene, and N-hydroxymethyl acrylamide to mix with porogens including PVA, and polymerizes under the action of an initiator, and finally removes the porogen to obtain a macroporous cross-linked resin. The macroporous cross-linked resin prepared by the method can be used in the separation of hydrogel polypeptides, and can improve the separation effect of hydrogel polypeptides. The combined use of the macroporous cross-linked resin and a low-water mobile phase has a better effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a scanning electron micrograph of the macroporous cross-linked resin.

[0033] Figure 2 This is a graph showing the yield of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly.

[0034] Figure 3 This is the yield diagram of palmitoyl tetrapeptide-20. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0037] Example 1: A method for preparing a high-purity hydrogel polypeptide Preparation of hydrogel polypeptide: The crude hydrogel polypeptide is mixed with a solvent, and an eluent is used for elution and purification in a column separation system to obtain the hydrogel polypeptide. The hydrogel polypeptide in the crude hydrogel polypeptide is Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly, the solvent is DMSO, the amount of the crude hydrogel polypeptide and the solvent is 40 mg / mL, the column separation system is a C18 column, and the eluent is a low water mobile phase, which is composed of methanol, acetic acid and water, and the volume ratio of methanol, acetic acid and water in the low water mobile phase is 2:9:9.

[0038] Example 2: A method for preparing a high-purity hydrogel polypeptide Preparation of macroporous cross-linked resin: adding porogen to deionized water to obtain porogen solution, then mixing polymerized monomer with solvent and initiator to obtain monomer solution, mixing monomer solution with porogen solution, reacting at 80°C for 10h, removing reaction solution after reaction, washing with hot water to remove porogen, drying to obtain macroporous cross-linked resin. The porogen is PVA, the porogen solution contains 0.5wt% porogen, and the amount of porogen solution is 300wt% of the monomer solution; polymerized monomers include p-styrene, p-chloromethylstyrene, and N-hydroxymethylacrylamide, the amount of p-chloromethylstyrene is 15wt% of p-styrene, the amount of N-hydroxymethylacrylamide is 40wt% of p-styrene, and the amount of polymerized monomer is 30wt% of solvent; the solvent is toluene and methyl isobutyl carbinol, the amount of methyl isobutyl carbinol is 100wt% of toluene, the initiator is BPO, and the amount of initiator is 1.2wt% of the polymerized monomer.

[0039] Preparation of hydrogel polypeptide: The crude hydrogel polypeptide is mixed with a solvent, and an eluent is used for elution and purification in a column separation system to obtain the hydrogel polypeptide. The hydrogel polypeptide in the crude hydrogel polypeptide is Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly, the solvent is DMSO, the amount of the crude hydrogel polypeptide and the solvent is 40 mg / mL, the column separation system is a macroporous resin column, the macroporous resin column is filled with a macroporous cross-linked resin, the eluent is a low water mobile phase, the low water mobile phase is composed of methanol, acetic acid and water, and the volume ratio of methanol, acetic acid and water in the low water mobile phase is 2:9:9.

[0040] Example 3: A method for preparing a high-purity hydrogel polypeptide The difference between this embodiment and embodiment 2 lies in the preparation of the macroporous cross-linked resin.

[0041] Preparation of macroporous cross-linked resin: adding porogen to deionized water to obtain porogen solution, then mixing polymerized monomer with solvent and initiator to obtain monomer solution, mixing monomer solution with porogen solution, reacting at 80°C for 10h, removing reaction solution after reaction, washing with hot water to remove porogen, drying to obtain macroporous cross-linked resin. The porogen is PVA, the porogen solution contains 0.5wt% porogen, and the amount of porogen solution is 300wt% of the monomer solution; the polymerized monomer includes p-styrene, p-chloromethylstyrene, N-hydroxymethylacrylamide, the amount of p-chloromethylstyrene is 25wt% of p-styrene, the amount of N-hydroxymethylacrylamide is 50wt% of p-styrene, and the amount of polymerized monomer is 30wt% of solvent; the solvent is toluene and methyl isobutyl carbinol, the amount of methyl isobutyl carbinol is 100wt% of toluene, the initiator is BPO, and the amount of initiator is 1.2wt% of the polymerized monomer.

[0042] Example 4: A method for preparing a high-purity hydrogel polypeptide Compared with Example 3, the difference between this example and Example 3 lies in the preparation of the hydrogel polypeptide, in which the macroporous cross-linked resin filled in the macroporous resin column is replaced with an amine-modified resin.

[0043] Preparation of amine-modified resin: Add macroporous cross-linked resin to DMF, swell for 12 hours at 30°C, then add di-n-butyrimide and anhydrous potassium carbonate, react for 48 hours at 40°C, wash and dry after the reaction is completed to obtain amine-modified resin. The amount of macroporous cross-linked resin used is 25wt% of DMF, the amount of di-n-butyrimide used is 24wt% of macroporous cross-linked resin, and the amount of anhydrous potassium carbonate used is 20wt% of macroporous cross-linked resin.

[0044] Example 5: A method for preparing a high-purity hydrogel polypeptide The difference between this embodiment and embodiment 4 lies in the preparation of the amine-modified resin.

[0045] Preparation of amine-modified resin: Add macroporous cross-linked resin to DMF, swell for 12 hours at 30°C, then add di-n-butyrimide and anhydrous potassium carbonate, react for 48 hours at 40°C, wash and dry after the reaction is completed to obtain amine-modified resin. The amount of macroporous cross-linked resin used is 25wt% of DMF, the amount of di-n-butyrimide used is 36wt% of the macroporous cross-linked resin, and the amount of anhydrous potassium carbonate used is 20wt% of the macroporous cross-linked resin.

[0046] Comparative Example 1: A method for preparing high-purity hydrogel polypeptide The difference between this comparative example and Example 1 lies in the preparation of the hydrogel polypeptide.

[0047] Preparation of hydrogel polypeptide: The crude hydrogel polypeptide is mixed with a solvent, and an eluent is used for elution and purification in a column separation system to obtain a hydrogel polypeptide. The hydrogel polypeptide in the crude hydrogel polypeptide is Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly, the solvent is DMSO, the amount of the crude hydrogel polypeptide and the solvent is 40 mg / mL, the column separation system is a C18 column, the eluent is a mixture of acetonitrile and formic acid aqueous solution, the formic acid aqueous solution contains 0.1 wt% formic acid, and the amount of acetonitrile and formic acid aqueous solution in the eluent is gradually adjusted from a volume ratio of 1:4 to a volume ratio of 1:1.5 within 60 minutes.

[0048] Comparative Example 2: A method for preparing high-purity hydrogel polypeptide The difference between this comparative example and Example 2 lies in the preparation of the hydrogel polypeptide.

[0049] Preparation of hydrogel polypeptide: The crude hydrogel polypeptide is mixed with a solvent, and an eluent is used for elution and purification in a column separation system to obtain a hydrogel polypeptide. The hydrogel polypeptide in the crude hydrogel polypeptide is Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly, the solvent is DMSO, the amount of the crude hydrogel polypeptide and the solvent is 40 mg / mL, the column separation system is a macroporous resin column, the macroporous resin column is filled with a macroporous cross-linked resin, the eluent is a mixture of acetonitrile and formic acid aqueous solution, the formic acid aqueous solution contains 0.1wt% formic acid, and the amount of acetonitrile and formic acid aqueous solution in the eluent is gradually adjusted from a volume ratio of 1:4 to a volume ratio of 1:1.5 within 60 minutes.

[0050] Example 6: A method for preparing a high-purity hydrogel polypeptide Preparation of hydrogel polypeptide: The crude hydrogel polypeptide is mixed with a solvent, and an eluent is used for elution and purification in a column separation system to obtain a hydrogel polypeptide. The hydrogel polypeptide in the crude hydrogel polypeptide is palmitoyl tetrapeptide-20, the solvent is DMSO, the amount of the crude hydrogel polypeptide and the solvent is 30 mg / mL, the column separation system is a C18 column, and the eluent is a low water mobile phase, which is composed of methanol, acetic acid and water, and the volume ratio of methanol, acetic acid and water in the low water mobile phase is 9:6:5.

[0051] Example 7: A method for preparing a high-purity hydrogel polypeptide Preparation of macroporous cross-linked resin: adding porogen to deionized water to obtain porogen solution, then mixing polymerized monomer with solvent and initiator to obtain monomer solution, mixing monomer solution with porogen solution, reacting at 80°C for 10h, removing reaction solution after reaction, washing with hot water to remove porogen, drying to obtain macroporous cross-linked resin. The porogen is PVA, the porogen solution contains 0.5wt% porogen, and the amount of porogen solution is 300wt% of the monomer solution; polymerized monomers include p-styrene, p-chloromethylstyrene, and N-hydroxymethylacrylamide, the amount of p-chloromethylstyrene is 15wt% of p-styrene, the amount of N-hydroxymethylacrylamide is 40wt% of p-styrene, and the amount of polymerized monomer is 30wt% of solvent; the solvent is toluene and methyl isobutyl carbinol, the amount of methyl isobutyl carbinol is 100wt% of toluene, the initiator is BPO, and the amount of initiator is 1.2wt% of the polymerized monomer.

[0052] Preparation of hydrogel polypeptide: The crude hydrogel polypeptide is mixed with a solvent, and an eluent is used for elution and purification in a column separation system to obtain the hydrogel polypeptide. The hydrogel polypeptide in the crude hydrogel polypeptide is palmitoyl tetrapeptide-20, the solvent is DMSO, the amount of the crude hydrogel polypeptide and the solvent is 30 mg / mL, the column separation system is a macroporous resin column, the macroporous resin column is filled with a macroporous cross-linked resin, the eluent is a low water mobile phase, the low water mobile phase is composed of methanol, acetic acid and water, and the volume ratio of methanol, acetic acid and water in the low water mobile phase is 9:6:5.

[0053] Example 8: A method for preparing a high-purity hydrogel polypeptide The difference between this embodiment and embodiment 7 lies in the preparation of the macroporous cross-linked resin.

[0054] Preparation of macroporous cross-linked resin: adding porogen to deionized water to obtain porogen solution, then mixing polymerized monomer with solvent and initiator to obtain monomer solution, mixing monomer solution with porogen solution, reacting at 80°C for 10h, removing reaction solution after reaction, washing with hot water to remove porogen, drying to obtain macroporous cross-linked resin. The porogen is PVA, the porogen solution contains 0.5wt% porogen, and the amount of porogen solution is 300wt% of the monomer solution; the polymerized monomer includes p-styrene, p-chloromethylstyrene, N-hydroxymethylacrylamide, the amount of p-chloromethylstyrene is 25wt% of p-styrene, the amount of N-hydroxymethylacrylamide is 50wt% of p-styrene, and the amount of polymerized monomer is 30wt% of solvent; the solvent is toluene and methyl isobutyl carbinol, the amount of methyl isobutyl carbinol is 100wt% of toluene, the initiator is BPO, and the amount of initiator is 1.2wt% of the polymerized monomer.

[0055] Example 9: A method for preparing a high-purity hydrogel polypeptide The difference between this example and Example 8 lies in the preparation of the hydrogel polypeptide, in which the macroporous cross-linked resin filled in the macroporous resin column is replaced with an amine-modified resin.

[0056] Preparation of amine-modified resin: Add macroporous cross-linked resin to DMF, swell for 12 hours at 30°C, then add di-n-butyrimide and anhydrous potassium carbonate, react for 48 hours at 40°C, wash and dry after the reaction is completed to obtain amine-modified resin. The amount of macroporous cross-linked resin used is 25wt% of DMF, the amount of di-n-butyrimide used is 24wt% of macroporous cross-linked resin, and the amount of anhydrous potassium carbonate used is 20wt% of macroporous cross-linked resin.

[0057] Example 10: A method for preparing a high-purity hydrogel polypeptide The difference between this embodiment and embodiment 9 lies in the preparation of the amine-modified resin.

[0058] Preparation of amine-modified resin: Add macroporous cross-linked resin to DMF, swell for 12 hours at 30°C, then add di-n-butyrimide and anhydrous potassium carbonate, react for 48 hours at 40°C, wash and dry after the reaction is completed to obtain amine-modified resin. The amount of macroporous cross-linked resin used is 25wt% of DMF, the amount of di-n-butyrimide used is 36wt% of the macroporous cross-linked resin, and the amount of anhydrous potassium carbonate used is 20wt% of the macroporous cross-linked resin.

[0059] Comparative Example 3: A method for preparing high-purity hydrogel polypeptide The difference between this comparative example and Example 5 lies in the preparation of the hydrogel polypeptide.

[0060] Preparation of hydrogel polypeptide: The crude hydrogel polypeptide is mixed with a solvent, and an eluent is used for elution and purification in a column separation system to obtain a hydrogel polypeptide. The hydrogel polypeptide in the crude hydrogel polypeptide is palmitoyl tetrapeptide-20, the solvent is DMSO, the amount of the crude hydrogel polypeptide and the solvent is 30 mg / mL, the column separation system is a C18 column, the eluent is a mixture of acetonitrile and formic acid aqueous solution, the formic acid aqueous solution contains 0.1 wt% formic acid, and the amount of acetonitrile and formic acid aqueous solution in the eluent is gradually adjusted from a volume ratio of 1:4 to a volume ratio of 1:1.5 within 60 minutes.

[0061] Comparative Example 4: A method for preparing a high-purity hydrogel polypeptide The difference between this comparative example and Example 5 lies in the preparation of the hydrogel polypeptide.

[0062] Preparation of hydrogel polypeptide: The crude hydrogel polypeptide is mixed with a solvent, and an eluent is used for elution and purification in a column separation system to obtain the hydrogel polypeptide. The hydrogel polypeptide in the crude hydrogel polypeptide is palmitoyl tetrapeptide-20, the solvent is DMSO, the amount of the crude hydrogel polypeptide and the solvent is 30 mg / mL, the column separation system is a macroporous resin column, the macroporous resin column is filled with a macroporous cross-linked resin, the eluent is a mixture of acetonitrile and formic acid aqueous solution, the formic acid aqueous solution contains 0.1wt% formic acid, and the amount of acetonitrile and formic acid aqueous solution in the eluent is gradually adjusted from a volume ratio of 1:4 to a volume ratio of 1:1.5 within 60 minutes.

[0063] Test example: The present invention characterizes the surface morphology of the macroporous cross-linked resin prepared in Example 2. The scanning electron microscopy results are as follows: Figure 1 As shown, there are a large number of pores in the macroporous cross-linked resin.

[0064] The yield of the hydrogel polypeptide obtained by the methods of Examples 1-10 and Comparative Examples 1-2 was tested in the present invention, wherein the amount of the crude Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly used was 400 mg, and the amount of the crude palmitoyl tetrapeptide-20 used was 300 mg.

[0065] The yield of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly is as follows Figure 2 As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, D1 is Comparative Example 1, and D2 is Comparative Example 2. The present invention finds that the crude Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly is a colloidal insoluble substance in a mixed solution of methanol-water or acetonitrile-water and a conventional acid-base buffer. Therefore, in the purification method of Comparative Example 1, it is almost impossible to separate Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly from the crude product. The purification method of Comparative Example 1 can be used to purify Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly, while the yield after purification by the purification method of Comparative Example 1 is only 0.67%. If the water in the separation system is greatly reduced, the purification effect of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly can be greatly improved. Therefore, the purification method of Example 1 of the present invention can increase the yield to 72.41%. However, it is impossible to completely eliminate water from the low-water mobile phase. In order to further improve the purification effect of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly The invention discloses a macroporous cross-linked resin for purifying crude products. The macroporous cross-linked resin is prepared by polymerizing monomers including p-styrene, p-chloromethylstyrene and N-hydroxymethyl acrylamide. It is found that when a low-water mobile phase is used together with the macroporous cross-linked resin, the yield of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly obtained by using only a low-water mobile phase can be further improved. , indicating that the combined use of low-water mobile phase and macroporous cross-linked resin has a better effect, and when the amount of p-chloromethylstyrene and N-hydroxymethylacrylamide in the macroporous cross-linked resin is further increased, the purification effect of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly is better. The present invention also found that if the macroporous cross-linked resin containing p-chloromethylbenzene structure is further modified with amine, the yield of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly obtained is higher.

[0066] The yield of palmitoyl tetrapeptide-20 is as follows Figure 3As shown, S6 is Example 6, S7 is Example 7, S8 is Example 8, S9 is Example 9, S10 is Example 10, D3 is Comparative Example 3, and D4 is Comparative Example 4. The present invention finds that the crude palmitoyl tetrapeptide-20 is a colloid insoluble substance in a methanol-water solution with a methanol ratio of less than 90%. Therefore, in the purification method of Comparative Example 3, it is almost impossible to purify palmitoyl tetrapeptide-20, and the yield after purification by the purification method of Comparative Example 3 is only 0.28%. If the water in the separation system is greatly reduced, the purification effect of palmitoyl tetrapeptide-20 can be greatly improved. Therefore, the purification method of Example 6 of the present invention can increase the yield to 63.82%, and the low water mobile phase cannot completely use water. In order to further improve the purity of the crude palmitoyl tetrapeptide-20 The invention discloses a macroporous cross-linked resin for purifying a crude palmitoyl tetrapeptide-20. The macroporous cross-linked resin is obtained by polymerizing monomers including p-styrene, p-chloromethylstyrene and N-hydroxymethylacrylamide. It is found that when a low-water mobile phase is used together with the macroporous cross-linked resin, the yield of palmitoyl tetrapeptide-20 obtained only with the low-water mobile phase can be further improved, indicating that the use of the low-water mobile phase and the macroporous cross-linked resin together has a better effect. When the amount of p-chloromethylstyrene and N-hydroxymethylacrylamide in the macroporous cross-linked resin is further increased, the purification effect of palmitoyl tetrapeptide-20 is better. The invention also finds that if the macroporous cross-linked resin containing a p-chloromethylbenzene structure is further subjected to amine modification treatment, the yield of the obtained palmitoyl tetrapeptide-20 is higher.

[0067] The present invention conducted a purity test on the hydrogel polypeptides obtained by the methods of Examples 1-10 and Comparative Examples 1-2. The purity of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly prepared by the method of the present invention reached more than 99.9%, and the purity of palmitoyl tetrapeptide-20 prepared by the method of the present invention reached more than 99.99%.

[0068] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0069] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing a high-purity hydrogel polypeptide, comprising: The crude hydrogel polypeptide is mixed with a solvent, and purified by elution using an eluent in a column separation system to obtain the hydrogel polypeptide; The eluent is a low-water mobile phase, which is composed of methanol, acetic acid and water, or the low-water mobile phase is composed of acetonitrile, acetic acid and water; The volume percentage of water in the low-water mobile phase is less than 45% and not equal to 0%.

2. The method for preparing a high-purity hydrogel polypeptide according to claim 1, characterized in that: The volume ratio of methanol to acetic acid in the low water mobile phase is 1:0.5-5.

3. The method for preparing a high-purity hydrogel polypeptide according to claim 1, characterized in that: The hydrogel polypeptide includes Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly or palmitoyl tetrapeptide-20.

4. The method for preparing a high-purity hydrogel polypeptide according to claim 1, characterized in that: The column separation system comprises a C18 column or a macroporous resin column, the macroporous resin column is filled with a macroporous resin, the macroporous resin comprises a macroporous cross-linked resin, and the macroporous cross-linked resin is prepared by reacting p-styrene, p-chloromethylstyrene and N-hydroxymethyl acrylamide.

5. The method for preparing a high-purity hydrogel polypeptide according to claim 4, characterized in that: In the preparation of the macroporous cross-linked resin, a monomer solution and a porogen solution are mixed and reacted, and the macroporous cross-linked resin is prepared after the porogen is removed; the monomer solution contains a polymerizable monomer and an initiator, the polymerizable monomer includes p-styrene, p-chloromethylstyrene and N-hydroxymethyl acrylamide, and the porogen is PVA.

6. The method for preparing a high-purity hydrogel polypeptide according to claim 5, characterized in that: The usage amount of the p-chloromethylstyrene is 10-30wt% of the p-styrene, and the usage amount of the N-hydroxymethylacrylamide is 30-60wt% of the p-styrene.

7. The method for preparing a high-purity hydrogel polypeptide according to claim 5, characterized in that: The solvents of the monomer solution are toluene and methyl isobutyl carbinol, and the usage amount of methyl isobutyl carbinol is 80-120 wt % of toluene.

8. The method for preparing a high-purity hydrogel polypeptide according to claim 5, characterized in that: The porogen is PVA, and the porogen solution contains 0.2-1 wt % of the porogen.

9. The method for preparing a high-purity hydrogel polypeptide according to claim 5, characterized in that: The usage amount of the porogen solution is 200-400 wt % of the monomer solution.

10. The method for preparing a high-purity hydrogel polypeptide according to claim 5, characterized in that: The initiator is BPO, and the usage amount of the initiator is 0.5-2wt% of the polymerization monomer.

Images