A novel freeze-drying process for self-assembled peptide RADA16

Abstract

This invention relates to the field of pharmaceutical technology and discloses a novel freeze-drying process for the self-assembled peptide RADA16. This process addresses the problem that existing freeze-drying processes cannot achieve the ideal fluffy powder state for the self-assembled peptide RADA16. By optimizing the temperature, time, and operation methods of each step (pre-freezing, first-stage annealing, second-stage annealing, and freeze-drying), the freeze-dried self-assembled peptide RADA16 exhibits a fluffy powder state with significantly reduced internal moisture content, high long-term storage stability, and no impact on its basic properties. The novel freeze-drying process for the self-assembled peptide RADA16 provided by this invention shortens the freeze-drying time, improves product quality, and is suitable for the industrial production of the self-assembled peptide RADA16.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a novel freeze-drying process for a self-assembled polypeptide RADA16. Background Technology

[0002] The self-assembling peptide RADA16 (molecular formula Ac(RADA)4NH2) is an artificially designed and synthesized peptide that can self-assemble under certain conditions to form nanofibers with a β-sheet structure. This fiber material is characterized by stable composition, modifiability, high biocompatibility, and non-immunogenicity. It can mimic the porosity and structure of the extracellular matrix in vivo, providing a suitable microenvironment for cell growth, promoting the repair and reconstruction of damaged tissues, and also possessing good drug loading capacity. It has been proven to support the attachment and growth of hippocampal neurons, increase their proliferation rate, and promote their differentiation activity, which is beneficial for the repair of damaged nerves and the formation of interneuronal synapses. It has been widely studied and applied in fields such as three-dimensional cell culture, tissue engineering, and regenerative medicine.

[0003] Freeze-drying is a process in which a drug solution is frozen into a solid state at low temperature, and then the ice crystals are directly converted into water vapor through sublimation under vacuum conditions, thereby removing moisture and avoiding the presence of liquid water. This process reduces damage to the structure and activity of the compound and is widely used in pharmaceutical production, drug development, food processing and other fields.

[0004] However, the self-assembling peptide RADA16 is a special peptide compound. Due to its high content of basic and acidic amino acids, it easily binds to salts, resulting in good hydrophilicity. This hydrophilicity decreases when it becomes a solid, and it easily forms a gel upon the addition of water, making it suitable for bone repair gels. Currently, the commercially available self-assembling peptide RADA16 is processed using conventional freeze-drying techniques, such as the freeze-drying method shown in the patent document with authorization number CN217005117U. The traditional method involves placing the peptide stock solution in a low-temperature freezer for more than 2 hours until it is completely frozen. Simultaneously, the freeze dryer's cooling trap is pre-cooled at -60°C for at least half an hour. After the stock solution sample is completely frozen, it is transferred to the freeze dryer tray, the outer cover is placed, and the vacuum pump is turned on until the negative pressure inside the freeze dryer stabilizes below 100 Pa. The freeze dryer runs for 36-48 hours until the sample is completely dry.

[0005] However, this conventional freeze-drying process cannot achieve the ideal fluffy dry powder state for the self-assembled peptide RADA16. It is hydrophilic and easily absorbs moisture and crystals during freeze-drying, which affects the long-term storage stability and basic properties of the product, thus affecting its quality.

[0006] Therefore, it is necessary to provide a novel freeze-drying process for the self-assembled peptide RADA16 to solve the above-mentioned technical problems. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a novel freeze-drying process for self-assembled peptide RADA16. This process allows the self-assembled peptide RADA16 product to exhibit a fluffy dry powder state, reducing internal moisture content and improving the long-term storage stability of the self-assembled peptide RADA16 without affecting its basic properties.

[0008] The novel lyophilization process for the self-assembled peptide RADA16 provided by this invention includes the following steps:

[0009] S1. Pre-freezing: Pour the pure self-assembled peptide RADA16 into a flask as a liquid and place the flask in a refrigerator for pre-freezing.

[0010] S2, First-stage annealing: Take out the pre-frozen eggplant-shaped flask and place it in an insulated foam box containing liquid nitrogen. Quickly rotate the flask to make the self-assembled peptide RADA16 freeze evenly and quickly on the inner wall of the eggplant-shaped flask, forming white ice. Then place the frozen eggplant-shaped flask in a refrigerator to stand for a first-stage annealing.

[0011] S3. Second-stage annealing: If the sample does not reach the transparent crystalline state after the first-stage annealing, it is placed in a water bath and kept at a constant temperature for the second-stage annealing.

[0012] S4. Freeze-drying: Place the annealed eggplant-shaped flasks on a freeze dryer, start the vacuum, and freeze-dry the equipment after the vacuum level stabilizes at about 30 Pa. After the freeze-drying process is completed, the completely freeze-dried self-assembled peptide RADA16 product is obtained.

[0013] Preferably, in the pre-freezing step, the purity of the self-assembled peptide RADA16 is 98%.

[0014] Preferably, during the pre-freezing step, the amount of self-assembled peptide RADA16 liquid in the flask does not exceed one-third of the flask's maximum capacity.

[0015] Preferably, in the pre-freezing step, the pre-freezing time of the eggplant-shaped bottle containing the self-assembled polypeptide RADA16 in the refrigerator is 30 minutes, and the temperature inside the refrigerator is -5°C.

[0016] Preferably, in the first annealing step, the frozen eggplant-shaped bottle is annealed in the refrigerator for 3 hours at a temperature of 2°C.

[0017] Preferably, in the two-stage annealing step, if the sample does not reach a transparent crystalline state after the first-stage annealing, the temperature for the second-stage annealing in the constant temperature water bath is 5°C, and the standing time is 15 minutes.

[0018] Preferably, in the freeze-drying step, the time for freeze-drying the eggplant-shaped flask on the freeze dryer is 24 hours, thus obtaining a completely freeze-dried self-assembled polypeptide RADA16 product.

[0019] Compared with related technologies, the novel freeze-drying process for the self-assembled polypeptide RADA16 provided by this invention has the following beneficial effects:

[0020] 1. By optimizing the temperature, time and operation of each step through pre-freezing, first-stage annealing, second-stage annealing and freeze-drying, the freeze-dried self-assembled peptide RADA16 is in a fluffy dry powder state with greatly reduced internal moisture content, high long-term storage stability and no impact on its basic properties.

[0021] 2. Compared with the prior art, the present invention shortens the entire freeze-drying time and improves production efficiency. Attached Figure Description

[0022] Figure 1 A flowchart of a novel freeze-drying process for a self-assembled polypeptide RADA16 provided by the present invention;

[0023] Figure 2 Comparison of electron micrographs of the drugs in this invention: (A) Electron micrograph of the polypeptide drug standard, (B) Electron micrograph of the self-assembled polypeptide RADA16 after conventional lyophilization, and (C) Electron micrograph of the assembled polypeptide RADA16 after lyophilization following the annealing process of this invention. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] This invention proposes a novel freeze-drying process for the self-assembled peptide RADA16, such as... Figure 1 As shown, it includes the following steps:

[0026] S1. Pre-freezing: Obtain the pure self-assembled peptide RADA16 peptide 98 from the upstream, and fill it into a flask according to the standard that the liquid should not exceed one-third of the upper limit of the bottle. Place the entire flask in a -5℃ freezer for 30 minutes to pre-freeze.

[0027] S2. First-stage annealing: After 30 minutes, remove the flask. Because the self-assembled peptide RADA16 uses a mixture of water and acetonitrile, compounds containing a certain percentage of acetonitrile are difficult to freeze into ice, and the flask is currently still completely liquid. Pour some liquid nitrogen into an insulated foam box, place the flask containing the self-assembled peptide RADA16 into the insulated foam box containing liquid nitrogen, and quickly rotate the flask to allow the self-assembled peptide RADA16 to freeze evenly and rapidly on the inner wall of the flask. The entire process takes about 1 minute, until there is no liquid left in the flask and all the peptides are evenly frozen on the inner wall. At this point, the self-assembled peptide RADA16 is in a white ice-like state. Place the frozen flask containing the self-assembled peptide RADA16 in a 2°C freezer for a first-stage annealing process, which takes 3 hours. During this time, the freeze dryer can be turned on and the compressor can be started. The freeze dryer used in this experiment is the SCIENTZ-10ND / C ordinary multi-manifold freeze dryer from Ningbo Xinzhi Freeze Drying Equipment Co., Ltd.

[0028] S3. Second-stage annealing: After 3 hours, carefully observe the state of the sample in the flask. It should change from the original white, icy state to a transparent crystalline state. If this state is not achieved, place the sample in a 5°C water bath for constant temperature and stand for second-stage annealing. The time is approximately 15 minutes.

[0029] S4. Lyophilization: After the self-assembled peptide RADA16 has reached the ideal annealing state, place the flask on a freeze dryer and start the vacuum. The freeze dryer is considered stable when the vacuum level drops and stabilizes at around 30 Pa. Because of the use of flasks and manifolds for lyophilization, the lyophilization process can be clearly observed. After 24 hours, the fully lyophilized self-assembled peptide RADA16 product can be obtained.

[0030] This invention proposes a freeze-drying process for the self-assembled peptide RADA16, which differs from conventional freeze-drying procedures. It incorporates a rapid freezing process, a first-stage annealing process, and a second-stage annealing process, shortening the overall freeze-drying time while ensuring the freeze-dried self-assembled peptide RADA16 remains stable in a fluffy dry powder state, which is visible to the naked eye. Crucially, the overall moisture content of the freeze-dried self-assembled peptide RADA16 is significantly reduced, and its density is much greater under an electron microscope (e.g., ...). Figure 2 (As shown).

[0031] The process of this invention optimizes the freeze-drying process of the self-assembled peptide RADA16, improves product quality and production efficiency, and is suitable for industrial production.

[0032] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A novel freeze-drying process for self-assembled polypeptide RADA16, characterized in that, Includes the following steps: S1. Pre-freezing: Pour the pure self-assembled peptide RADA16 into a flask as a liquid and place the flask in a refrigerator for pre-freezing. S2, First-stage annealing: Take out the pre-frozen eggplant-shaped flask and place it in an insulated foam box containing liquid nitrogen. Quickly rotate the flask to make the self-assembled peptide RADA16 freeze evenly and quickly on the inner wall of the eggplant-shaped flask, forming white ice. Then place the frozen eggplant-shaped flask in a refrigerator to stand for a first-stage annealing. S3. Second-stage annealing: If the sample does not reach the transparent crystalline state after the first-stage annealing, it is placed in a water bath and kept at a constant temperature for the second-stage annealing. S4. Freeze-drying: Place the annealed eggplant-shaped flasks on a freeze dryer, start the vacuum, and freeze-dry the equipment after the vacuum level stabilizes at about 30 Pa. After the freeze-drying process is completed, the completely freeze-dried self-assembled peptide RADA16 product is obtained.

2. The novel freeze-drying process for the self-assembled polypeptide RADA16 as described in claim 1, characterized in that, In the pre-freezing step, the purity of the self-assembled peptide RADA16 was 98%.

3. The novel freeze-drying process for the self-assembled polypeptide RADA16 as described in claim 1, characterized in that, During the pre-freezing step, the liquid of the self-assembled peptide RADA16 in the flask does not exceed one-third of the flask's maximum capacity.

4. The novel freeze-drying process for the self-assembled polypeptide RADA16 as described in claim 1, characterized in that, In the pre-freezing step, the pre-freezing time of the eggplant-shaped bottle containing the self-assembled peptide RADA16 in the refrigerator is 30 minutes, and the temperature inside the refrigerator is -5°C.

5. The novel freeze-drying process for the self-assembled polypeptide RADA16 as described in claim 1, characterized in that, In the aforementioned annealing step, the frozen eggplant-shaped bottle undergoes a 3-hour annealing process in a refrigerator at a temperature of 2°C.

6. The novel freeze-drying process for the self-assembled polypeptide RADA16 as described in claim 1, characterized in that, In the two-stage annealing step, the sample that has not reached the transparent crystalline state after the first-stage annealing is placed in a constant temperature water bath for the second-stage annealing at a temperature of 5°C for 15 minutes.

7. The novel freeze-drying process for the self-assembled polypeptide RADA16 as described in claim 1, characterized in that, In the freeze-drying step, the eggplant-shaped flask is freeze-dried in a freeze dryer for 24 hours to obtain a completely freeze-dried self-assembled peptide RADA16 product.

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