Freeze-dried powder of KGF-2 composition and preparation method thereof

Abstract

The invention belongs to the technical field of medicines and skin care products, and particularly relates to freeze-dried powder of a KGF-2 composition and a preparation method of the freeze-dried powder. The preparation method comprises the following steps: adding a 1-10% dextran solution, a 1-10% trehalose aqueous solution and a 10-30% mannitol aqueous solution into a KGF2 mother solution to obtain a KGF2 mixed solution; the KGF2 mixed solution is inversely placed in a degerming cup, and a transparent mixed solution is obtained through a suction filtration pump; and sub-packaging the mixed solution into freeze-drying containers, pre-freezing, and carrying out sublimation drying for multiple times to finally form freeze-dried powder. The prepared freeze-dried powder is stable in appearance, free of collapse and disintegration and easy to dissolve, the dissolved liquid is clear, and the biological activity effect is good.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and skin care products, and specifically relates to a freeze-dried powder of a KGF-2 composition and a preparation method thereof. Background Art

[0002] KGF-2, or keratinocyte growth factor-2, is a basic protein growth factor secreted by human subcutaneous tissue cells and a member of the fibroblast growth factor (FGF) family. It is encoded by 194 amino acids, and mature KGF has 163 amino acid residues, with a glycosylation site at its N-terminus. KGF-2 is mainly secreted by subcutaneous tissue in the human body and specifically binds to specific receptors on the surface of epithelial cells. After a complex signal transmission process, it activates the expression of genes involved in division and growth in epithelial cells, thereby stimulating the metabolism of epithelial tissue.

[0003] KGF-2 is a biological factor with significant skin care effects. It can promote the growth of epidermal keratinocytes, repair damaged cells, accelerate cell metabolism, and thus improve skin condition. Freeze-dried powder technology can effectively retain the original nutrients, activity and stability of KGF-2, allowing it to better play its skin care effects. Therefore, the research and development of KGF-2 freeze-dried powder is of great significance and can provide a safer and more effective skin care solution for the skin.

[0004] KGF-2 freeze-dried powder, as the name suggests, is KGF-2 powder obtained through vacuum freeze-drying technology. This process not only retains the biological activity and stability of KGF-2 to the maximum extent, but also gives it the advantages of easy storage, transportation and use.

[0005] With the development of the bio-gene fermentation freeze-drying industry, a variety of cell growth factors secreted during cell culture have gradually replaced synthetic growth factors, and have also become the current development trend of the cosmetics industry. The technology for producing freeze-dried powder comes from the pharmaceutical industry. The freeze-dried powder is prepared in a vial and needs to be dissolved in a solution before use. The dissolution rate is slow, the solution is viscous after dissolution, and the dissolution is not complete. Therefore, the solution is viscous and sticks to the wall, and it is difficult to completely remove it from the vial. It needs to be poured out when it is used, which will cause a lot of waste. If the mother solution is freeze-dried alone, the state after freeze-drying is very unstable, and the product will be difficult to store and degrade, and it will be easy to collapse and disintegrate. The molding is poor or even impossible to mold, which greatly affects the packaging of the finished product and is also very unsightly.

[0006] Therefore, there is an urgent need to develop a lyophilized powder of a KGF-2 composition that has a good appearance, is easy to dissolve, has more stable product activity, and is easy to store and transport. Summary of the invention

[0007] In order to solve the problems of the prior art, the present invention provides a method for preparing a fluffy freeze-dried powder of a KGF-2 composition. On the basis of ensuring the activity of the production factor in the extracted supernatant, the prepared freeze-dried powder has a stable appearance, no collapse or disintegration, is easy to dissolve, the liquid after dissolution is clear, has good biological activity effect and high stability.

[0008] Specifically, the present invention provides a method for preparing a lyophilized powder of a KGF2 composition, characterized in that the method comprises the following steps: Step 1, preparing 1-10% dextran solution, 1-10% trehalose aqueous solution and 10-30% mannitol aqueous solution; Step 2, adding 1-10% dextran solution, 1-10% trehalose aqueous solution and 10-30% mannitol aqueous solution into the KGF2 mother solution to obtain a KGF2 mixed solution; Step 3, invert the KGF2 mixed solution in a sterilization cup, and use a suction pump to obtain a transparent mixed solution; Step 4: Dispense the mixed solution into freeze-drying containers, 3 ml per bottle; Step 5: Take the filled freeze-dried container and place it on the freeze-drying machine partition for pre-freezing; Step 6: Sublimation drying is performed on the freeze-drying container for multiple times according to the prescribed time to finally form a freeze-dried powder; Step 7: Cover the freeze-drying container of the harvested freeze-dried powder with an inner stopper and press on an outer cover to obtain the freeze-dried powder.

[0009] In some embodiments, the method for preparing the KGF2 mother solution comprises: Step 1): Using ultrafiltration technology to concentrate and desalt the KGF-2 mother liquor; Step 2): replacing the KGF-2 supernatant with purified water during ultrafiltration; Step 3): Use a conductivity meter to detect the conductivity of the KGF-2 mother solution; Step 4): Obtain KGF-2 mother solution by removing salt through ultrafiltration.

[0010] In some embodiments, the ultrafiltration technology uses a membrane package with a pore size of 5KD.

[0011] In some embodiments, the purified water replacement ratio is 1:1.

[0012] In some embodiments, the controllable range of the conductivity is: 12 ms / cm-30 ms / cm.

[0013] In some embodiments, the pre-freezing in step 5 specifically includes pre-freezing from room temperature to -50°C for 4 hours.

[0014] In some embodiments, the step 6 of performing multiple sublimation drying on the freeze-drying container according to the prescribed time specifically includes: evacuating to 10 Pa; sublimating from -50°C to -40°C, keeping warm for 6 hours; sublimating from -40°C to -23°C, keeping warm for 12 hours; sublimating from -23°C to -20°C, keeping warm for 24 hours; sublimating from -20°C to 20°C, keeping warm for 6 hours; and sublimating from 20°C to 30°C, keeping warm for 4 hours to complete the freeze-drying process.

[0015] Another aspect of the present invention provides a lyophilized powder of the KGF2 composition obtained by the preparation method.

[0016] The present invention also provides application of the freeze-dried powder in preparing beauty and skin care products.

[0017] Compared with the prior art, the advantages and beneficial effects of the present invention are: The KGF2 mother liquor composition of the present invention comprises KGF2 mother liquor, dextran, trehalose and mannitol. Dextran, trehalose and mannitol are used as auxiliary materials. Mannitol is used as a filler in the freeze-dried product. Dextran and trehalose are used as protective agents in the freeze-dried product. Mannitol and trehalose can significantly improve the molding effect of freeze-dried powder. The obtained freeze-dried powder has beautiful appearance and can be quickly re-dissolved. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0019] Figure 1 The preparation process of freeze-dried powder of this application.

[0020] Figure 2 Electrophoresis gel running verifies the protein stability of the lyophilized powder of KGF-2 obtained by combining the preparation method of the present application with the lyophilization method at 25°C; Figure 3 Electrophoresis gel running verifies the protein stability of the lyophilized powder of KGF-2 obtained by combining the preparation method of the present application with the lyophilization method at 50°C; Figure 4 Stability test results of KGF-2 protein solutions prepared in the present application and the prior art under different conditions. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific embodiments: To facilitate a better understanding of the present invention, but not to limit the present invention, the experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0022] Example 1: Method for desalting and concentrating KGF-2 mother liquor and preparation of freeze-dried powder The present invention configures and combines a new stock solution through different raw material component structures, and the stock solution adopts a novel desalting concentration method and a different freeze-drying method. The stock solution components are matched and have stable protein degradation, which can fully protect the formability of the product during the freeze-drying process and form a good-looking powdery appearance.

[0023] KGF-2 mother liquor desalination and concentration method: Step 1: Use ultrafiltration technology and select a 5KD pore size membrane package to concentrate and desalt the KGF-2 mother liquor; Step 2: replace the KGF-2 supernatant with purified water during ultrafiltration; the replacement ratio of desalted purified water is 1:1; Step 3: Use a conductivity meter to detect the conductivity of the KGF-2 mother solution. The conductivity can be controlled in the range of 12ms / cm-30ms / cm; Step 4: removing salt by ultrafiltration to obtain KGF-2 mother solution; Preparation method of freeze-dried powder of KGF2 mother solution composition: The method comprises the following steps: Step 1, preparing 1-10% dextran solution, 1-10% trehalose aqueous solution and 10-30% mannitol aqueous solution; Step 2, adding 1-10% dextran solution, 1-10% trehalose aqueous solution and 10-30% mannitol aqueous solution into the KGF2 mother solution to obtain a KGF2 mixed solution; Step 3, invert the KGF2 mixed solution in a sterilization cup, and use a suction pump to obtain a mixed solution that is a transparent liquid; Step 4: The obtained mixed solution of the composition is dispensed into freeze-drying containers, with 3 ml per bottle; Step 5: Place the filled container bottles on the freeze dryer partition for pre-freezing Step 6: Drying the freeze-drying container by sublimation once, twice, thrice, etc. according to the prescribed time to finally form a freeze-dried powder; Step 7: Cover the harvested freeze-dried powder container with an inner stopper and press on an outer cover to obtain the freeze-dried powder.

[0024] Step 5 includes pre-freezing the filled freeze-drying container from room temperature to -50°C, and then pre-freezing for 4 hours; then performing the freeze-drying operation of step 6, starting to evacuate the container to 10 Pa; sublimating from -50°C to -40°C, and keeping warm for 6 hours; sublimating from -40°C to -23°C, and keeping warm for 12 hours; sublimating from -23°C to -20°C, and keeping warm for 24 hours; sublimating from -20°C to 20°C, and keeping warm for 6 hours; and sublimating from 20°C to 30°C, and keeping warm for 4 hours; completing the freeze-drying process to form freeze-dried powder.

[0025] The preparation process of the freeze-dried powder of the present invention is summarized as follows Figure 1 shown.

[0026] Example 2: Stability test of KGF-2 protein at 25°C and 50°C The lyophilized powder obtained by combining the preparation method of this patent with the lyophilization method was verified by electrophoresis running, and the protein stability was tested at a constant temperature of 25°C. The results are shown in Figure 2 , where 1 is the sample protein and 2 is the repeated verification protein. The results show that the protein is still stable and not degraded.

[0027] The lyophilized powder obtained by combining the preparation method of this patent with the lyophilization method was verified by electrophoresis running, and the protein stability was tested at a constant temperature of 50°C. The results are shown in Figure 3 , where 1 is the sample protein and 2 is the repeated verification protein. The results show that the protein is still stable and not degraded.

[0028] The above results show that the lyophilized powder of the KGF2 composition obtained by the preparation method of the present invention is more stable in protein activity and easier to store than the general lyophilized powder on the market.

[0029] Example 3: Comparative experiment on the stability of KGF-2 protein under different conditions 1. Experimental Materials KGF-2 protein solution prepared by the present invention KGF-2 protein solution prepared by prior art Temperature controller (used to set different temperature conditions).

[0030] KGF-2 stability testing instrument (SDS.PAGE) 2. Experimental steps 1. Preparation of KGF-2 protein solution: The target KGF-2 protein solution was prepared using the present invention and the prior art (commercially available recombinant protein KGF-2), respectively, and the KGF-2 protein concentrations of the two solutions were ensured to be the same.

[0031] 2. Grouping and condition setting The two KGF-2 protein solutions were divided into three groups, each set at a different temperature. Group 1 was tested at 25°C, group 2 was tested at 4°C, and group 3 was tested at 4°C and 25°C after adding a stabilizer.

[0032] 3. Stability testing: Use the KGF-2 protein stability testing instrument to regularly test the structural and functional stability of each group of KGF-2 proteins and record the data.

[0033] 3. Data Analysis The stability of the KGF-2 protein solution prepared by the present invention and the prior art under different temperature conditions is compared. Figure 4 As shown, where: Lane 1: The KGF-2 protein of the present invention is kept constant at 4°C for 6 days. (stabilizer added) Lane 2: The KGF-2 protein of the present invention is kept constant at 25°C for 6 days. (stabilizer added) Lane 3: The KGF-2 protein of the present invention was kept constant at 4°C for 6 days.

[0034] Lane 4: The KGF-2 protein of the present invention was kept constant at 25°C for 6 days.

[0035] Lane 5: The KGF-2 protein available on the market was kept constant at 4°C for 6 days.

[0036] Lane 6: The KGF-2 protein currently available on the market was kept constant at 25°C for 6 days.

[0037] The gel image shows that the KGF-2 protein solution of the present invention is more stable than that of the prior art.

[0038] 1. Effect of temperature: At 4°C, both KGF-2 protein solutions showed high stability. As the temperature increased to 25°C, the stability of the two KGF-2 proteins was differentiated from the gel images. The KGF-2 protein on the market (lane 6) was obviously severely degraded, and the residual protein was barely detectable, but the KGF-2 protein solution of the present invention could still maintain good stability at higher temperatures.

[0039] 2. Effect of stabilizer: After adding stabilizers such as trehalose and dextran, the stability of the KGF-2 protein solution of the present invention is improved.

[0040] Conclusion: The stability of the KGF-2 protein solution of the present invention may be further improved after adding a stabilizer.

[0041] Through comparative experiments, we can draw the following conclusions: Under different temperature conditions, the KGF-2 protein solution of the present invention exhibits higher stability than that of the prior art.

[0042] After adding the stabilizer, the stability of the KGF-2 protein solution of the present invention is improved more significantly.

[0043] Example 4: Freeze-dried powder morphology and dissolution Freeze-dried powder is made by freezing the stock solution into a solid state in a sterile environment and then sublimating the water to dry it in a vacuum. The vacuum freeze drying method of the freeze dryer is used to freeze the water in the stock solution in advance, and then the frozen water in the liquid is sublimated in a vacuum sterile environment to obtain freeze-dried powder. In short, the water in the stock solution is extracted in a low temperature environment, retaining its original stock solution function and forming a good-looking appearance.

[0044] The freeze-dried powder is cylindrical in shape and in the form of white powder blocks. It has a light and delicate texture. It appears very fluffy and easy to dissolve after being spread out. When the freeze-dried powder is mixed with water and the bottle is shaken, it dissolves quickly without foaming and does not feel sticky or greasy.

[0045] When drying, the frozen surface first dehydrates to form a dense dry shell, and the sublimated water vapor below escapes from the gaps between the molecules of the dried surface layer.

[0046] The appearance is uniform color, dense pores, and basically unchanged shape, forming a block or sponge-like mass structure.

[0047] The lyophilized powder was dissolved in 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 1 mL, 2 mL, 5 mL and 10 mL of purified water respectively. The obtained solution was easily soluble, had no quality and was clear and transparent.

[0048] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

[0049] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will also be appreciated by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A method for preparing a freeze-dried powder of a KGF-2 composition, characterized in that: The method comprises the following steps: Step 1: prepare 1-10% dextran solution, 1-10% trehalose aqueous solution and 10-30% mannitol aqueous solution; Step 2: adding 1-10% dextran solution, 1-10% trehalose aqueous solution and 10-30% mannitol aqueous solution into the KGF-2 mother solution to obtain a KGF-2 mixed solution; Step 3: Invert the KGF-2 mixed solution into a sterilization cup and use a suction pump to obtain a transparent mixed solution; Step 4: Dispensing the mixed solution into freeze-drying containers; Step 5: Take the filled freeze-dried container and place it on the freeze-drying machine partition for pre-freezing; Step 6: Sublimation drying is performed on the freeze-drying container for multiple times according to the prescribed time to finally form freeze-dried powder.

2. The preparation method according to claim 1, characterized in that The preparation method of the KGF-2 mother solution comprises: Step 1): Using ultrafiltration technology to concentrate and desalt the KGF-2 mother liquor; Step 2): replacing the KGF-2 supernatant with purified water during ultrafiltration; Step 3): Use a conductivity meter to detect the conductivity of the KGF-2 mother solution; Step 4): Obtain KGF-2 mother solution by removing salt through ultrafiltration.

3. The preparation method according to claim 2, characterized in that: The ultrafiltration technology uses a membrane package with a pore size of 5KD.

4. The preparation method according to claim 2, characterized in that: The purified water replacement ratio is 1:

1.

5. The preparation method according to claim 2, characterized in that: The controllable range of the conductivity is: 12ms / cm-30ms / cm.

6. The preparation method according to claim 1, characterized in that: The pre-freezing in step 5 specifically includes pre-freezing from room temperature to -50°C for 4 hours.

7. The preparation method according to claim 1, characterized in that: In step 6, performing multiple sublimation drying on the freeze-drying container according to the prescribed time specifically includes: vacuuming to 10 Pa; sublimation from -50°C to -40°C, keeping warm for 6 hours; sublimation from -40°C to -23°C, keeping warm for 12 hours; sublimation from -23°C to -20°C, keeping warm for 24 hours; from -20°C to 20°C, keeping warm for 6 hours; from 20°C to 30°C, keeping warm for 4 hours; completing the freeze-drying process.

8. The lyophilized powder of the KGF2 composition obtained according to the preparation method according to any one of claims 1 to 7.

9. Use of the freeze-dried powder according to claim 8 in preparing beauty and skin care products.

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