Purification method of high-purity RGD cyclic peptide
Through the step -type purification method, the proper preparation column fillers and the buffer salt system are used to perform multi -step pillar division purification and salt transfer treatment. Circle peptide preparation is suitable for industrial production.
Patent Information
- Application Number
- CN202411776358.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-05
- Publication Date
- 2025-01-21
AI Technical Summary
It is difficult to remove impurities in crude RGD cyclic peptides, especially impurities produced during reduction, and it is difficult to remove them effectively by ordinary purification methods.
Adopting the step -in -type purification method, by selecting the appropriate preparation column fillers and adjusting the pH value of the flowing salt salt to perform multi -step column division purification and salt treatment, the purity of the RGD cycle peptide is gradually increased.
Effectively control the impurities in RGD peptide rough products to below 0.1 %, reaching high purity of more than 99.5 %, high purification income, and suitable for industrialized production.
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Figure CN119331051A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of drug synthesis, and in particular to a method for purifying a high-purity RGD cyclic peptide. Background Art
[0002] Studies have found that RGD sequence peptides have a wide range of biological activities and can be used to treat cardiovascular diseases, osteoporosis, inflammation and other diseases. They can also prevent and treat tumors caused by abnormal cell adhesion, especially the metastasis of progressive tumors. On the other hand, RGD sequence peptides can be used as stimulants to promote the regeneration of damaged organs and tissues, wound healing, etc. RGD is a receptor for some integrins, and its selectivity depends in part on the conformation of RGD and the amino acid residues around RGD. RGD sequence peptides can specifically bind to 11 integrins and are receptor antagonists of integrins, which are divided into linear peptides and cyclic peptides. αvβ3 in the integrin family is highly expressed on the surface of many solid cancer tumor cells, and it can recognize the RGD peptide sequence. RGD peptides can prevent tumor cells from adhering to the extracellular matrix or directly induce tumor cell apoptosis by competitively binding to integrin receptors, ultimately achieving the purpose of inhibiting tumor cell proliferation, invasion and metastasis. They can be used to develop anti-angiogenic drugs and biological targets for molecular imaging probes for early tumor diagnosis.
[0003] RGD linear peptides have low stability, poor clinical performance, and poor selectivity for specific integrin receptors. Compared with RGD linear peptides, RGD cyclic peptides have more stable structures and stronger binding affinity and selectivity for integrin receptors. One of the RGD cyclic peptides (cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine [c(RGDfk)]) binds to integrin αvβ3 on the cell surface, enhancing the affinity of receptor-ligand binding, improving tumor uptake of labeled probes, and prolonging the retention time of molecular probes in tumors.
[0004] The purification of crude RGD cyclic peptide is quite difficult, especially for the impurities produced during the reduction of RGD cyclic peptide. Because they are similar to the main peak structure and have very small retention time difference in the chromatographic system, they are difficult to remove by ordinary purification methods. Summary of the invention
[0005] The purpose of the present invention is to provide a method for purifying high-purity RGD cyclic peptide in view of the problem that impurities in RGD cyclic peptide are difficult to remove.
[0006] To achieve the above purpose, the technical solution provided by the present invention is:
[0007] A method for purifying a high-purity RGD cyclic peptide comprises: step-by-step purification of a crude RGD cyclic peptide by a preparative column, step-by-step purification of the crude RGD cyclic peptide by a preparative column, and salt conversion to obtain a high-purity RGD cyclic peptide;
[0008] Wherein, step-by-step purification is performed by selecting the preparation column filler of each step and adjusting the mobile phase buffer salt of each step.
[0009] The purification method of high-purity RGD cyclic peptide specifically comprises the following steps:
[0010] (1) The crude RGD cyclic peptide was purified by a preparative column to obtain a pure sample of RGD cyclic peptide with a purity of 93-96%;
[0011] (2) performing a second purification operation on the RGD cyclic peptide pure sample through a preparation column to obtain a second pure sample of RGD cyclic peptide with a purity of 98% or more;
[0012] (3) The RGD cyclic peptide dipure sample is purified, separated and converted to salt by a preparative column, and then freeze-dried to obtain a high-purity RGD cyclic peptide sample with a purity of more than 99.5%.
[0013] In order to optimize the above technical solutions, the specific limitations adopted also include:
[0014] In step (1) and step (2), the preparative columns are all C18 filler preparative columns, the particle size of the filler is 5-20 μm, and the theoretical plate number of the column efficiency test of the preparative columns is 25000-50000.
[0015] Preferably, in step (1) and step (2), the number of theoretical plates in the column efficiency test of the preparation column is 30,000-50,000.
[0016] Preferably, in step (1) and step (2), the preparation column is prepared using Daiso SP-100-8-ODS-P filler preparation column.
[0017] In step (1), the mobile phase is a phosphate buffer-acetonitrile system with a pH value of 4.5-7.5.
[0018] Preferably, the pH value of the mobile phase in step (1) is 6.0-7.5.
[0019] In step (1), the crude RGD cyclic peptide is dissolved in purified water, filtered with a filter membrane, the filtrate is collected, purified and separated by a C18 filler preparative column, and eluted with a phosphate buffer-acetonitrile system to obtain a pure sample of RGD cyclic peptide.
[0020] In step (2), the mobile phase is a phosphate buffer-acetonitrile system with a pH value of 2.0-5.0.
[0021] Preferably, the pH value of the mobile phase in step (2) is 3.0-5.0.
[0022] In step (2), the first pure sample of RGD cyclic peptide is purified and separated by a C18 filler preparative column and eluted with a phosphate buffer-acetonitrile system to obtain a second pure sample of RGD cyclic peptide.
[0023] The phosphate buffer in the present application can be any one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.
[0024] In step (3), the preparation column is selected to be a polymer reverse phase filler with a filler particle size of 10-50 μm.
[0025] Preferably, in step (1) and step (2), the preparation column is 30-300 preparative column.
[0026] In step (3), the mobile phase is an alkali solution-acetonitrile water / system.
[0027] The alkali solution in the present application can be any one or more of potassium carbonate, sodium carbonate, potassium hydroxide, and sodium hydroxide.
[0028] Preferably, the alkali solution is a mixture of sodium carbonate and sodium hydroxide in a volume ratio of 1:2-4.
[0029] In step (3), the pure sample of RGD cyclic peptide is purified and separated by a polymer reverse phase filler preparation column, eluted with an alkali solution-acetonitrile water / system, and freeze-dried to obtain a refined RGD cyclic peptide.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] The invention solves the problem that the crude RGD cyclic peptide obtained by synthesis has many impurities and is difficult to purify, especially the impurities generated during the reduction of the RGD cyclic peptide, which can be effectively controlled to be below 0.1%.
[0032] The invention selects the preparation column filler of each step and adjusts the mobile phase buffer salt of each step to perform a step-by-step purification scheme to gradually purify and convert the RGD cyclic peptide crude product into salt, thereby obtaining a high-purity RGD cyclic peptide with a high purification yield.
[0033] The method has the advantages of high product quality, good process stability, mild reaction conditions, simple operation and low production cost, and is very suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 : The crude product HPLC figure of embodiment 6 of the present invention.
[0035] Figure 2 : HPLC chart of a pure sample solution of the RGD cyclic peptide of Example 6 of the present invention.
[0036] Figure 3: HPLC diagram of the pure sample solution of RGD cyclic peptide of Example 6 of the present invention.
[0037] Figure 4 : HPLC chart of the refined RGD cyclic peptide solution of Example 6 of the present invention. DETAILED DESCRIPTION
[0038] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.
[0039] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.
[0040] For the sake of simplicity, this document only specifically discloses some numerical values and optional ranges. However, any lower limit can be combined with any upper limit to form an unambiguous range; and any lower limit can be combined with other lower limits to form an unambiguous range. Similarly, any upper limit can be combined with any other upper limit to form an unambiguous range; the optional items in the optional range can also be combined arbitrarily.
[0041] Unless otherwise specified, the terms used in this application have the commonly known meanings generally understood by those skilled in the art. Unless otherwise specified, the numerical values of the parameters mentioned in this application can be measured using various measurement methods commonly used in the art.
[0042] The present invention provides a method for purifying a high-purity RGD cyclic peptide, comprising:
[0043] The first pure method: After the crude RGD cyclic peptide is dissolved in purified water, it is filtered with a filter membrane, the filtrate is collected, and purified and separated by a C18 filler preparative column, and eluted with a phosphate buffer-acetonitrile system to obtain a pure sample of RGD cyclic peptide.
[0044] Second pure method: The first pure sample of RGD cyclic peptide was purified and separated by C18 filler preparation column, and eluted with phosphate buffer-acetonitrile system to obtain the second pure sample of RGD cyclic peptide.
[0045] Salt conversion method: The pure sample of RGD cyclic peptide was purified and separated by polymer reverse phase preparation column, eluted with alkali solution-acetonitrile water / system, and freeze-dried to obtain the refined RGD cyclic peptide.
[0046] Example 1
[0047] Dissolution of crude peptide: 620 mg of crude RGD cyclic peptide was dissolved in 40 ml of purified water by stirring.
[0048] Filtration: Install a 0.45 μm microporous polyethersulfone filter membrane into the G4 sand core, fully wet it, add the crude peptide solution, connect the vacuum pump, open the control valve, and filter. After the filtration is completed, add 10 ml of purified water to the filter and filter it. The filtrate is combined with the crude peptide solution.
[0049] First purification: The crude peptide solution to be purified was loaded, and the column efficiency test of Daiso SP-100-8-ODS-P filler preparative column showed that the theoretical plate number was 38804 (EP);
[0050] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 7.00)
[0051] Mobile phase B: acetonitrile
[0052] Elution was performed according to the first purification method to obtain a pure solution with an HPLC purity of 96.746%.
[0053] Second pure: The first pure solution was concentrated under reduced pressure and loaded onto a Daiso SP-100-8-ODS-P filler preparative column;
[0054] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 4.00) Mobile phase B: acetonitrile
[0055] Elution was performed according to the second pure method to obtain a second pure solution with an HPLC purity of 99.136%.
[0056] Salt transfer: The pure solution is concentrated under reduced pressure and then loaded onto the sample. The product was purified and separated by 30-300 filler preparation column, and eluted with sodium hydroxide-acetonitrile-water system according to gradient to obtain a refined RGD cyclic peptide solution, which was concentrated under reduced pressure and then freeze-dried to obtain a powdered RGD cyclic peptide refined product with a weight of 402 mg, a yield of 64.8%, and a HPLC purity of 99.683%.
[0057] Example 2
[0058] Dissolution of crude peptide: Dissolve 1530 mg of crude RGD cyclic peptide in 40 ml of purified water by stirring.
[0059] Filtration: Install a 0.45 μm microporous polyethersulfone filter membrane into the G4 sand core, fully wet it, add the crude peptide solution, connect the vacuum pump, open the control valve, and filter. After the filtration is completed, add 10 ml of purified water to the filter and filter it. The filtrate is combined with the crude peptide solution.
[0060] First purification: the crude peptide solution to be purified was loaded, and the column efficiency test of Daiso SP-100-8-ODS-P filler preparative column showed the theoretical plate number to be 37750 (EP);
[0061] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 7.00)
[0062] Mobile phase B: acetonitrile
[0063] Gradient elution was performed according to the first purification method to obtain a pure solution with an HPLC purity of 95.377%.
[0064] Second pure: The first pure solution was concentrated under reduced pressure and loaded onto a Daiso SP-100-8-ODS-P filler preparative column;
[0065] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 4.01)
[0066] Mobile phase B: acetonitrile
[0067] Gradient elution was performed according to the second pure method to obtain a second pure solution with an HPLC purity of 99.317%.
[0068] Salt transfer: The pure solution is concentrated under reduced pressure and then loaded onto the sample. The product was purified and separated by using a 30-300 filler preparative column and eluted with a sodium hydroxide-acetonitrile-water system according to a gradient to obtain a refined RGD cyclic peptide solution, which was concentrated under reduced pressure and then freeze-dried to obtain a powdered RGD cyclic peptide refined product with a weight of 996 mg, a yield of 65.1%, and an HPLC purity of 99.602%.
[0069] Example 3
[0070] Dissolution of crude peptide: 1488 mg of crude RGD cyclic peptide was dissolved in 40 ml of purified water by stirring.
[0071] Filtration: Install a 0.45 μm microporous polyethersulfone filter membrane into the G4 sand core, fully wet it, add the crude peptide solution, connect the vacuum pump, open the control valve, and filter. After the filtration is completed, add 10 ml of purified water to the filter and filter it. The filtrate is combined with the crude peptide solution.
[0072] First purification: The crude peptide solution to be purified was loaded, and the column efficiency test of Daiso SP-100-8-ODS-P preparative column showed that the theoretical plate number was 37043 (EP);
[0073] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 6.98)
[0074] Mobile phase B: acetonitrile
[0075] Gradient elution was performed according to the first purification method to obtain a pure solution with an HPLC purity of 94.175%.
[0076] Second pure: The first pure solution was concentrated under reduced pressure and loaded onto a Daiso SP-100-8-ODS-P filler preparative column;
[0077] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 4.01)
[0078] Mobile phase B: acetonitrile
[0079] Gradient elution was performed according to the second pure method to obtain a second pure solution with an HPLC purity of 99.201%.
[0080] Salt transfer: The pure solution is concentrated under reduced pressure and then loaded onto the sample. 30-300 filler preparative column purification and separation, using sodium hydroxide
[0081] -Acetonitrile-water system was used for gradient elution to obtain a refined RGD cyclic peptide solution, which was concentrated under reduced pressure and then freeze-dried to obtain a powdered RGD cyclic peptide refined product with a weight of 1014 mg, a yield of 68.1%, and a HPLC purity of 99.811%.
[0082] Example 4
[0083] Dissolution of crude peptide: 6178 mg of crude RGD cyclic peptide was dissolved in 40 ml of purified water by stirring.
[0084] Filtration: Install a 0.45 μm microporous polyethersulfone filter membrane into the G4 sand core, fully wet it, add the crude peptide solution, connect the vacuum pump, open the control valve, and filter. After the filtration is completed, add 10 ml of purified water to the filter and filter it. The filtrate is combined with the crude peptide solution.
[0085] First purification: The crude peptide solution to be purified was loaded, and the column efficiency test of Daiso SP-100-8-ODS-P preparative column showed that the theoretical plate number was 36658 (EP);
[0086] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 7.00)
[0087] Mobile phase B: acetonitrile
[0088] Gradient elution was performed according to the first purification method to obtain a pure solution with an HPLC purity of 96.332%.
[0089] Second pure: The first pure solution was concentrated under reduced pressure and loaded onto a Daiso SP-100-8-ODS-P filler preparative column;
[0090] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 3.99)
[0091] Mobile phase B: acetonitrile
[0092] Gradient elution was performed according to the second pure method to obtain a second pure solution with an HPLC purity of 99.005%.
[0093] Salt transfer: The pure solution is concentrated under reduced pressure and then loaded onto the sample. The product was purified and separated by using a 30-300 filler preparative column and eluted with a sodium hydroxide-acetonitrile-water system according to a gradient to obtain a refined RGD cyclic peptide solution, which was concentrated under reduced pressure and then freeze-dried to obtain a powdered RGD cyclic peptide refined product with a weight of 4175 mg, a yield of 67.6%, and a HPLC purity of 99.687%.
[0094] Example 5
[0095] Dissolution of crude peptide: Dissolve 7110 mg of crude RGD cyclic peptide in 40 ml of purified water by stirring.
[0096] Filtration: Install a 0.45 μm microporous polyethersulfone filter membrane into the G4 sand core, fully wet it, add the crude peptide solution, connect the vacuum pump, open the control valve, and filter. After the filtration is completed, add 10 ml of purified water to the filter and filter it. The filtrate is combined with the crude peptide solution.
[0097] First purification: The crude peptide solution to be purified was loaded, and the column efficiency test of Daiso SP-100-8-ODS-P preparative column showed that the theoretical plate number was 38094 (EP);
[0098] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 7.01)
[0099] Mobile phase B: acetonitrile
[0100] Gradient elution was performed according to the first purification method to obtain a pure solution with an HPLC purity of 94.828%.
[0101] Second pure: The first pure solution was concentrated under reduced pressure and loaded onto a Daiso SP-100-8-ODS-P filler preparative column;
[0102] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 4.00)
[0103] Mobile phase B: acetonitrile
[0104] Gradient elution was performed according to the second pure method to obtain a second pure solution with an HPLC purity of 98.759%.
[0105] Salt transfer: The pure solution is concentrated under reduced pressure and then loaded onto the sample. The product was purified and separated by using a 30-300 filler preparative column and eluted with a sodium hydroxide-acetonitrile-water system according to a gradient to obtain a refined RGD cyclic peptide solution, which was concentrated under reduced pressure and then freeze-dried to obtain a powdered RGD cyclic peptide refined product with a weight of 4740 mg, a yield of 66.7%, and a HPLC purity of 99.784%.
[0106] Example 6
[0107] Dissolution of crude peptide: 8496 mg of crude RGD cyclic peptide was dissolved in 40 ml of purified water by stirring.
[0108] Filtration: Install a 0.45 μm microporous polyethersulfone filter membrane into the G4 sand core, fully wet it, add the crude peptide solution, connect the vacuum pump, open the control valve, and filter. After the filtration is completed, add 10 ml of purified water to the filter and filter it. The filtrate is combined with the crude peptide solution.
[0109] First purification: The crude peptide solution to be purified was loaded, and the column efficiency test of Daiso SP-100-8-ODS-P preparative column showed that the theoretical plate number was 37986 (EP);
[0110] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 7.00)
[0111] Mobile phase B: acetonitrile
[0112] Gradient elution was performed according to the first purification method to obtain a pure solution with an HPLC purity of 95.371%.
[0113] Second pure: The first pure solution was concentrated under reduced pressure and loaded onto a Daiso SP-100-8-ODS-P filler preparative column;
[0114] Mobile phase A: 50 mM sodium dihydrogen phosphate buffer (pH 4.00)
[0115] Mobile phase B: acetonitrile
[0116] Gradient elution was performed according to the second pure method to obtain a second pure solution with an HPLC purity of 99.160%.
[0117] Salt transfer: The pure solution is concentrated under reduced pressure and then loaded onto the sample. The product was purified and separated by using a 30-300 filler preparative column and eluted with a sodium hydroxide-acetonitrile-water system according to a gradient to obtain a refined RGD cyclic peptide solution, which was concentrated under reduced pressure and then freeze-dried to obtain a powdered RGD cyclic peptide refined product with a weight of 5573 mg, a yield of 65.6%, and a HPLC purity of 99.880%.
[0118] Taking Example 6 as an example, the detection spectrum of each step is as follows: Figure 1 This is the HPLC chart of crude RGD cyclic peptide. Figure 2 This is the HPLC diagram of a pure solution of RGD cyclic peptide. Figure 3 This is the HPLC diagram of the pure solution of RGD cyclic peptide. Figure 4 This is the HPLC chart of the refined RGD cyclic peptide.
[0119] The method of the invention uses a preparation column to purify the crude RGD cyclic peptide step by step, transfer salt, and freeze-dry, thereby achieving effective separation and purification of the RGD cyclic peptide and impurities, and finally obtaining a high-purity RGD cyclic peptide with a purity of more than 99.5%, and a purification yield of 60-70%.
[0120] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.
Claims
1. A method for purifying high-purity RGD cyclic peptide, characterized in that: The crude RGD cyclic peptide was purified and salted by preparative columns to obtain high-purity RGD cyclic peptide. Wherein, step-by-step purification is performed by selecting the preparation column filler of each step and adjusting the mobile phase buffer salt of each step.
2. The method for purifying high-purity RGD cyclic peptide according to claim 1, characterized in that: The following steps are involved: (1) The crude RGD cyclic peptide was purified by a preparative column to obtain a pure sample of RGD cyclic peptide with a purity of 93-96%; (2) performing a second purification operation on the RGD cyclic peptide pure sample through a preparation column to obtain a second pure sample of RGD cyclic peptide with a purity of 98% or more; (3) The RGD cyclic peptide dipure sample is purified, separated and converted to salt by a preparative column, and then freeze-dried to obtain a high-purity RGD cyclic peptide sample with a purity of more than 99.5%.
3. The method for purifying high-purity RGD cyclic peptide according to claim 2, characterized in that: In step (1) and step (2), the preparative columns are all C18 filler preparative columns, the particle size of the filler is 5-20 μm, and the theoretical plate number of the column efficiency test of the preparative columns is 25000-50000.
4. The method for purifying high-purity RGD cyclic peptide according to claim 3, characterized in that: In step (1), the mobile phase is a phosphate buffer-acetonitrile system with a pH value of 4.5-7.
5.
5. The method for purifying high-purity RGD cyclic peptide according to claim 4, characterized in that: In step (1), the crude RGD cyclic peptide is dissolved in purified water, filtered with a filter membrane, the filtrate is collected, purified and separated by a C18 filler preparative column, and eluted with a phosphate buffer-acetonitrile system to obtain a pure sample of RGD cyclic peptide.
6. The method for purifying high-purity RGD cyclic peptide according to claim 2, characterized in that: In step (2), the mobile phase is a phosphate buffer-acetonitrile system with a pH value of 2.0-5.
0.
7. The method for purifying high-purity RGD cyclic peptide according to claim 6, characterized in that: In step (2), the first pure sample of RGD cyclic peptide is purified and separated by a C18 filler preparative column and eluted with a phosphate buffer-acetonitrile system to obtain a second pure sample of RGD cyclic peptide.
8. The method for purifying high-purity RGD cyclic peptide according to claim 2, characterized in that: In step (3), the preparation column is selected to be a polymer reverse phase filler with a filler particle size of 10-50 μm.
9. The method for purifying high-purity RGD cyclic peptide according to claim 2, characterized in that: In step (3), the mobile phase is an alkali solution-acetonitrile water / system; the alkali solution is a mixture of sodium carbonate and sodium hydroxide in a volume ratio of 1:2-4.
10. The method for purifying high-purity RGD cyclic peptide according to claim 9, characterized in that: In step (3), the pure sample of RGD cyclic peptide is purified and separated by a polymer reverse phase filler preparation column, eluted with an alkali solution-acetonitrile water / system, and freeze-dried to obtain a refined RGD cyclic peptide.