A method for preparing high-purity hydrogel polypeptide

Abstract

The present invention relates to a method for preparing a high-purity hydrogel polypeptide, belonging to the technical field of polypeptide preparation. Specifically, the method involves mixing a crude hydrogel polypeptide with a solvent and eluting and purifying the hydrogel polypeptide using an eluent in a column separation system to obtain the hydrogel polypeptide; the eluent is a low-water mobile phase consisting of methanol, acetic acid, and water, or 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 volume ratio of methanol to acetic acid in the low-water mobile phase is 1:0.5-5. The hydrogel polypeptide includes Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly or palmitoyl tetrapeptide-20.

Description

Technical Field

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

[0002] Hydrogel peptides are polymers with a three-dimensional network structure formed by non-covalent interactions of polypeptide molecules in an aqueous solution. Due to their porous and highly absorbent properties, hydrogel peptides have broad application prospects in the cosmetic and pharmaceutical fields, particularly in medicine. In wound repair, hydrogel peptides can provide a moist environment to keep the wound surface moist, allowing air to pass through and absorbing exudate, creating an ideal environment for wound healing. The hydrogel's three-dimensional network not only imparts elasticity similar to soft tissue, facilitating tissue regeneration and repair, but also allows the encapsulation of drugs such as growth factors within the hydrogel, enabling sustained release at the wound site and increasing their half-life, promoting angiogenesis, anti-inflammatory effects, and wound healing. In the pharmaceutical field, antimicrobial agents can be encapsulated within the hydrogel for selective release to the site of infection, effectively and safely killing bacteria. Anti-tumor and cardiovascular drugs can also be precisely delivered via enzyme-responsive hydrogel peptides for targeted therapy.

[0003] Currently, the application of hydrogel peptides is mostly concentrated in the field of topical cosmetics. The peptide segments are extremely short, and the peptide raw materials are mostly obtained by solid-phase synthesis, direct washing and drying after cutting, or by liquid-phase chemical synthesis followed by direct washing and drying. The purity is low. For example, the highest commercially available purity of palmitoyl tetrapeptide-20 is 98%; for example, the commercially available purity of oligopeptide-104 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. However, the characteristic of hydrogel peptides forming gels in aqueous solutions makes it impossible to purify them 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 present invention aims to provide a low-water mobile phase and macroporous resin for purifying hydrogel polypeptides, thereby providing a method for preparing high-purity hydrogel polypeptides. The method of the present invention is effective in purifying hydrogel polypeptides and can purify more hydrogel polypeptides, thereby increasing the yield of hydrogel polypeptides.

[0006] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0007] 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 using an eluent in a column separation system to obtain the hydrogel polypeptide;

[0008] The eluent is a low-water mobile phase consisting of methanol, acetic acid and water, or a low-water mobile phase consisting of acetonitrile, acetic acid and water;

[0009] The volume percentage of water in the low-water mobile phase is less than 45% and not equal to 0%. By reducing the water content in the solution, the present invention can prevent the hydrogel polypeptide from forming a gel in conventional methanol-water and acetonitrile-water based mobile phases, thereby improving the solubility of the hydrogel polypeptide in the mobile phase.

[0010] Preferably, the volume ratio of methanol to acetic acid in the low water mobile phase is 1:0.5-5.

[0011] Preferably, the hydrogel polypeptide comprises Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly or palmitoyl tetrapeptide-20.

[0012] Preferably, the column separation system includes a C18 column or a macroporous resin column, the macroporous resin column is filled with a macroporous resin, the macroporous resin includes a macroporous cross-linked resin, and the macroporous cross-linked resin is made by reacting p-styrene, p-chloromethylstyrene and N-hydroxymethylacrylamide. The present invention is prepared by reacting p-styrene, p-chloromethylstyrene and N-hydroxymethylacrylamide to form a macroporous cross-linked resin. 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 the 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.

[0013] Preferably, in the preparation of the macroporous cross-linked resin, a monomer solution and a porogen solution are mixed and reacted, and the porogen is removed to prepare the macroporous cross-linked resin; the monomer solution contains a polymerizable monomer and an initiator, the polymerizable monomer includes p-styrene, p-chloromethylstyrene and N-hydroxymethylacrylamide, and the porogen is PVA.

[0014] Preferably, the amount of p-chloromethylstyrene used is 10-30 wt % of the p-styrene, and the amount of N-hydroxymethylacrylamide used is 30-60 wt % of the p-styrene.

[0015] 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.

[0016] Preferably, the porogen is PVA, and the porogen solution contains 0.2-1 wt % of the porogen.

[0017] Preferably, the amount of the porogen solution used is 200-400 wt % of the monomer solution.

[0018] Preferably, the initiator is BPO, and the amount of the initiator used is 0.5-2 wt % of the polymerization monomer.

[0019] 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 and the porogen solution are mixed and reacted at a temperature of 60-90° C. for 5-15 hours. After the reaction is completed, the reaction liquid is removed, the porogen is washed with hot water to remove the porogen, and the resin is dried to obtain the macroporous cross-linked resin.

[0020] 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.

[0021] More preferably, in the preparation of the macroporous cross-linked resin, the polymerization monomers include p-styrene, p-chloromethylstyrene, and N-hydroxymethylacrylamide, the usage amount of p-chloromethylstyrene is 10-30wt% of p-styrene, the usage amount of N-hydroxymethylacrylamide is 30-60wt% of p-styrene, and the usage amount of the polymerization monomers is 20-40wt% of the solvent.

[0022] 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.

[0023] 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.

[0024] 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. These macroporous cross-linked and aminated structures further enhance the purification efficiency of hydrogel polypeptides.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Preferably, in the preparation of the hydrogel polypeptide, a 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 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.

[0030] More preferably, in the preparation of the hydrogel polypeptide, the column separation system comprises a C18 column or a macroporous resin column.

[0031] 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%.

[0032] 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.

[0033] The present invention uses a low-water mobile phase to separate and purify the hydrogel polypeptide. 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 conventional mobile phases based on methanol-water and acetonitrile-water, but is in a clear and transparent solution state in the low-water mobile phase of the present invention. The low-water mobile phase greatly improves the sample loading capacity of the hydrogel polypeptide in the reverse-phase chromatography purification process. The sample loading capacity of the hydrogel polypeptide in conventional mobile phases based on methanol-water and acetonitrile-water is mg-level, but the sample loading capacity can reach g-level in the low-water mobile phase of the present 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 using conventional mobile phases based on methanol-water and acetonitrile-water, the peak shape is often broadened and tailed, but when the hydrogel polypeptide is prepared using the low-water mobile phase of the present invention, the peak shape is narrow and has no tailing.

[0034] The present invention further adopts a method of mixing polymerization monomers including p-styrene, p-chloromethylstyrene, and N-hydroxymethylacrylamide with a porogen including PVA, polymerizing the mixture under the action of an initiator, and finally removing the porogen to obtain a macroporous cross-linked resin. The macroporous cross-linked resin prepared by the above method can be used for the separation of hydrogel polypeptides, and can improve the separation effect of the 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

[0035] Figure 1 This is a scanning electron micrograph of the macroporous cross-linked resin.

[0036] Figure 2 This is a graph showing the yield of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly.

[0037] Figure 3 This is the yield diagram of palmitoyl tetrapeptide-20. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0039] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0040] Example 1: A method for preparing a high-purity hydrogel polypeptide

[0041] Preparation of hydrogel polypeptide: A 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 hydrogel polypeptide in the crude hydrogel polypeptide is Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly, the solvent is DMSO, the amount of crude hydrogel polypeptide and solvent used is 40 mg / mL, the column separation system is a C18 column, and the eluent is a low-water mobile phase composed of methanol, acetic acid, and water, with the volume ratio of methanol, acetic acid, and water in the low-water mobile phase being 2:9:9.

[0042] Example 2: A method for preparing a high-purity hydrogel polypeptide

[0043] Preparation of macroporous cross-linked resin: A porogen is added to deionized water to obtain a porogen solution. The polymerizable monomers are then mixed with a solvent and an initiator to obtain a monomer solution. The monomer solution and the porogen solution are mixed and reacted at 80°C for 10 hours. After the reaction is complete, the reaction solution is removed, washed with hot water to remove the porogen, and dried to obtain a macroporous cross-linked resin. The porogen is PVA, and the porogen solution contains 0.5 wt% of the porogen, with the amount of the porogen solution being 300 wt% of the monomer solution. The polymerizable monomers include p-styrene, p-chloromethylstyrene, and N-hydroxymethylacrylamide, with the amount of p-chloromethylstyrene being 15 wt% of the p-styrene and the amount of N-hydroxymethylacrylamide being 40 wt% of the p-styrene. The amount of the polymerizable monomers is 30 wt% of the solvent. The solvents are toluene and methyl isobutyl carbinol, with the amount of methyl isobutyl carbinol being 100 wt% of the toluene. The initiator is BPO, with the amount of the initiator being 1.2 wt% of the polymerizable monomers.

[0044] Preparation of hydrogel polypeptide: A 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 hydrogel polypeptide in the crude hydrogel polypeptide is Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly, the solvent is DMSO, the amount of crude hydrogel polypeptide and solvent used is 40 mg / mL, the column separation system is a macroporous resin column filled with a macroporous cross-linked resin, and the eluent is a low-water mobile phase composed of methanol, acetic acid, and water, with the volume ratio of methanol, acetic acid, and water in the low-water mobile phase being 2:9:9.

[0045] Example 3: A method for preparing a high-purity hydrogel polypeptide

[0046] Compared with Example 2, this example differs in the preparation of the macroporous cross-linked resin.

[0047] Preparation of macroporous cross-linked resin: A porogen is added to deionized water to obtain a porogen solution. The polymerizable monomers are then mixed with a solvent and an initiator to obtain a monomer solution. The monomer solution and the porogen solution are mixed and reacted at 80°C for 10 hours. After the reaction is complete, the reaction solution is removed, washed with hot water to remove the porogen, and dried to obtain a macroporous cross-linked resin. The porogen is PVA, and the porogen solution contains 0.5 wt% of the porogen, with the amount of the porogen solution being 300 wt% of the monomer solution. The polymerizable monomers include p-styrene, p-chloromethylstyrene, and N-hydroxymethylacrylamide, with the amount of p-chloromethylstyrene being 25 wt% of the p-styrene and the amount of N-hydroxymethylacrylamide being 50 wt% of the p-styrene. The amount of the polymerizable monomers is 30 wt% of the solvent. The solvents are toluene and methyl isobutyl carbinol, with the amount of methyl isobutyl carbinol being 100 wt% of the toluene. The initiator is BPO, with the amount of the initiator being 1.2 wt% of the polymerizable monomers.

[0048] Example 4: A method for preparing a high-purity hydrogel polypeptide

[0049] Compared with Example 3, this example differs in the preparation of the hydrogel polypeptide, in that the macroporous cross-linked resin filled in the macroporous resin column is replaced with an amine-modified resin.

[0050] Preparation of amine-modified resin: Macroporous cross-linked resin was added to DMF and swelled at 30°C for 12 hours. Di-n-butyrimide and anhydrous potassium carbonate were then added and reacted at 40°C for 48 hours. After completion of the reaction, the resin was washed and dried to obtain the amine-modified resin. The amount of macroporous cross-linked resin used was 25wt% of the DMF, the amount of di-n-butyrimide was 24wt% of the macroporous cross-linked resin, and the amount of anhydrous potassium carbonate was 20wt% of the macroporous cross-linked resin.

[0051] Example 5: A method for preparing a high-purity hydrogel polypeptide

[0052] Compared with Example 4, this example differs in the preparation of the amine-modified resin.

[0053] Preparation of amine-modified resin: Macroporous cross-linked resin was added to DMF and swelled at 30°C for 12 hours. Di-n-butyrimide and anhydrous potassium carbonate were then added and reacted at 40°C for 48 hours. After completion of the reaction, the resin was washed and dried to obtain the amine-modified resin. The amount of macroporous cross-linked resin used was 25wt% of the DMF, the amount of di-n-butyrimide was 36wt% of the macroporous cross-linked resin, and the amount of anhydrous potassium carbonate was 20wt% of the macroporous cross-linked resin.

[0054] Comparative Example 1: A method for preparing high-purity hydrogel polypeptide

[0055] Compared with Example 1, this comparative example differs in the preparation of the hydrogel polypeptide.

[0056] Preparation of hydrogel polypeptide: A 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 hydrogel polypeptide in the crude hydrogel polypeptide is Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly, the solvent is DMSO, the amount of crude hydrogel polypeptide and solvent used is 40 mg / mL, the column separation system is a C18 column, and the eluent is a mixture of acetonitrile and an aqueous formic acid solution containing 0.1 wt% formic acid. The volume ratio of acetonitrile to formic acid in the eluent is gradually adjusted from a volume ratio of 1:4 to a volume ratio of 1:1.5 over 60 minutes.

[0057] Comparative Example 2: A method for preparing high-purity hydrogel polypeptide

[0058] Compared with Example 2, this comparative example differs in the preparation of the hydrogel polypeptide.

[0059] Preparation of hydrogel polypeptide: A 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 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 filled with a macroporous cross-linked resin, and the eluent is a mixture of acetonitrile and an aqueous formic acid solution containing 0.1 wt% formic acid. The volume ratio of acetonitrile to formic acid in the eluent is gradually adjusted from a volume ratio of 1:4 to a volume ratio of 1:1.5 over 60 minutes.

[0060] Example 6: A method for preparing a high-purity hydrogel polypeptide

[0061] Preparation of hydrogel polypeptide: 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 hydrogel polypeptide in the crude hydrogel polypeptide is palmitoyl tetrapeptide-20, the solvent is DMSO, the amount of crude hydrogel polypeptide and solvent used 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. The volume ratio of methanol, acetic acid, and water in the low-water mobile phase is 9:6:5.

[0062] Example 7: A method for preparing a high-purity hydrogel polypeptide

[0063] Preparation of macroporous cross-linked resin: A porogen is added to deionized water to obtain a porogen solution. The polymerizable monomers are then mixed with a solvent and an initiator to obtain a monomer solution. The monomer solution and the porogen solution are mixed and reacted at 80°C for 10 hours. After the reaction is complete, the reaction solution is removed, washed with hot water to remove the porogen, and dried to obtain a macroporous cross-linked resin. The porogen is PVA, and the porogen solution contains 0.5 wt% of the porogen, with the amount of the porogen solution being 300 wt% of the monomer solution. The polymerizable monomers include p-styrene, p-chloromethylstyrene, and N-hydroxymethylacrylamide, with the amount of p-chloromethylstyrene being 15 wt% of the p-styrene and the amount of N-hydroxymethylacrylamide being 40 wt% of the p-styrene. The amount of the polymerizable monomers is 30 wt% of the solvent. The solvents are toluene and methyl isobutyl carbinol, with the amount of methyl isobutyl carbinol being 100 wt% of the toluene. The initiator is BPO, with the amount of the initiator being 1.2 wt% of the polymerizable monomers.

[0064] Preparation of hydrogel polypeptide: 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 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 used is 30 mg / mL, the column separation system is a macroporous resin column filled with a macroporous cross-linked resin, and the eluent is a low-water mobile phase composed of methanol, acetic acid, and water, with the volume ratio of methanol, acetic acid, and water in the low-water mobile phase being 9:6:5.

[0065] Example 8: A method for preparing a high-purity hydrogel polypeptide

[0066] Compared with Example 7, this example differs in the preparation of the macroporous cross-linked resin.

[0067] Preparation of macroporous cross-linked resin: A porogen is added to deionized water to obtain a porogen solution. The polymerizable monomers are then mixed with a solvent and an initiator to obtain a monomer solution. The monomer solution and the porogen solution are mixed and reacted at 80°C for 10 hours. After the reaction is complete, the reaction solution is removed, washed with hot water to remove the porogen, and dried to obtain a macroporous cross-linked resin. The porogen is PVA, and the porogen solution contains 0.5 wt% of the porogen, with the amount of the porogen solution being 300 wt% of the monomer solution. The polymerizable monomers include p-styrene, p-chloromethylstyrene, and N-hydroxymethylacrylamide, with the amount of p-chloromethylstyrene being 25 wt% of the p-styrene and the amount of N-hydroxymethylacrylamide being 50 wt% of the p-styrene. The amount of the polymerizable monomers is 30 wt% of the solvent. The solvents are toluene and methyl isobutyl carbinol, with the amount of methyl isobutyl carbinol being 100 wt% of the toluene. The initiator is BPO, with the amount of the initiator being 1.2 wt% of the polymerizable monomers.

[0068] Example 9: A method for preparing a high-purity hydrogel polypeptide

[0069] Compared with Example 8, this example differs in the preparation of the hydrogel polypeptide, in that the macroporous cross-linked resin filled in the macroporous resin column is replaced with an amine-modified resin.

[0070] Preparation of amine-modified resin: Macroporous cross-linked resin was added to DMF and swelled at 30°C for 12 hours. Di-n-butyrimide and anhydrous potassium carbonate were then added and reacted at 40°C for 48 hours. After completion of the reaction, the resin was washed and dried to obtain the amine-modified resin. The amount of macroporous cross-linked resin used was 25wt% of the DMF, the amount of di-n-butyrimide was 24wt% of the macroporous cross-linked resin, and the amount of anhydrous potassium carbonate was 20wt% of the macroporous cross-linked resin.

[0071] Example 10: A method for preparing a high-purity hydrogel polypeptide

[0072] The difference between this embodiment and embodiment 9 lies in the preparation of the amine-modified resin.

[0073] Preparation of amine-modified resin: Macroporous cross-linked resin was added to DMF and swelled at 30°C for 12 hours. Di-n-butyrimide and anhydrous potassium carbonate were then added and reacted at 40°C for 48 hours. After completion of the reaction, the resin was washed and dried to obtain the amine-modified resin. The amount of macroporous cross-linked resin used was 25wt% of the DMF, the amount of di-n-butyrimide was 36wt% of the macroporous cross-linked resin, and the amount of anhydrous potassium carbonate was 20wt% of the macroporous cross-linked resin.

[0074] Comparative Example 3: A method for preparing high-purity hydrogel polypeptide

[0075] Compared with Example 5, this comparative example differs in the preparation of the hydrogel polypeptide.

[0076] Preparation of hydrogel polypeptide: 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 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 mixture of acetonitrile and an aqueous formic acid solution, the aqueous formic acid solution containing 0.1 wt% formic acid. The volume ratio of acetonitrile to formic acid in the eluent is gradually adjusted from 1:4 to 1:1.5 over 60 minutes.

[0077] Comparative Example 4: Preparation method of high-purity hydrogel polypeptide

[0078] Compared with Example 5, this comparative example differs in the preparation of the hydrogel polypeptide.

[0079] Preparation of hydrogel polypeptide: A 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 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 filled with a macroporous cross-linked resin, and the eluent is a mixture of acetonitrile and an aqueous formic acid solution, the aqueous formic acid solution containing 0.1 wt% formic acid. The volume ratio of acetonitrile to formic acid in the eluent is gradually adjusted from a volume ratio of 1:4 to a volume ratio of 1:1.5 over 60 minutes.

[0080] Test example:

[0081] The surface morphology of the macroporous cross-linked resin prepared in Example 2 was characterized by scanning electron microscopy. Figure 1 As shown, there are a large number of pores in the macroporous cross-linked resin.

[0082] 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 crude Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly used was 400 mg, and the amount of crude palmitoyl tetrapeptide-20 used was 300 mg.

[0083] The yield of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly is as follows Figure 2As shown, wherein 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 found that the crude Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly product was 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 was almost impossible to separate the crude Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly from the crude product. The purification of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly was performed, while the yield after purification by the purification method of Comparative Example 1 was 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 in the low-water mobile phase. In order to further improve the purification efficiency of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly The present invention improves the purification effect of crude products. A macroporous cross-linked resin is prepared for the purification of crude Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly products. The macroporous cross-linked resin is obtained 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 using only the low-water mobile phase can be further improved. , indicating that the combined use of a low-water mobile phase and a 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 even better. The present invention also found that if the macroporous cross-linked resin containing a p-chloromethylbenzene structure is further modified with amines, the yield of Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly obtained is even higher.

[0084] The yield of palmitoyl tetrapeptide-20 is as follows Figure 3As shown, wherein 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 colloidal 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%. The low water mobile phase cannot completely use water. In order to further improve the purity of the crude palmitoyl tetrapeptide-20, The present invention prepares a macroporous cross-linked resin for the purification of 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 using only 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 present 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.

[0085] The present invention conducted purity tests 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%.

[0086] 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 do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art 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.

[0087] 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 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, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution 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 consists of methanol, acetic acid and water; The volume percentage of water in the low-water mobile phase is less than 45% and not equal to 0%; The column separation system includes a C18 column or a macroporous resin column, the macroporous resin column is filled with a macroporous resin, the macroporous resin includes a macroporous cross-linked resin, and the macroporous cross-linked resin is prepared by reacting p-styrene, p-chloromethylstyrene and N-hydroxymethyl acrylamide; In the preparation of the macroporous cross-linked resin, a monomer solution and a porogen solution are mixed and reacted, and the porogen is removed to prepare the macroporous cross-linked resin. The monomer solution contains a polymerizable monomer and an initiator, wherein the polymerizable monomers include p-styrene, p-chloromethylstyrene, and N-hydroxymethylacrylamide, and the porogen is PVA. The p-chloromethylstyrene is used in an amount of 10-30 wt% of the p-styrene, and the N-hydroxymethylacrylamide is used in an amount of 30-60 wt% of the p-styrene. 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.

2. The method for preparing a high-purity hydrogel polypeptide according to claim 1, wherein: 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, wherein: 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, wherein: 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.

5. The method for preparing a high-purity hydrogel polypeptide according to claim 1, wherein: The initiator is BPO, and the usage amount of the initiator is 0.5-2 wt % of the polymerization monomer.

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