Preparation method and application of freeze-dried powder containing recombinant I-type and III-type collagen composition

Abstract

The invention is applicable to the field of anti-aging filling, and provides a preparation method and application of freeze-dried powder containing recombinant I-type and III-type collagen compositions, and the freeze-dried powder containing the recombinant I-type and III-type collagen compositions comprises the following components: an A-phase part and a B-phase part. The freeze-dried powder is prepared by freezing water in the liquid medicine in advance by adopting a vacuum freeze-drying method of a freeze-drying machine, then sublimating the frozen water in the liquid medicine in a vacuum sterile environment, and thus freeze-drying. And secondly, the freeze-dried product is loose in form after being dried, the color is basically not changed, and the freeze-dried product can be quickly dissolved after being added with water and recovers the physicochemical properties and biological activity of the original aqueous solution. The product is manufactured and filled in a ten thousand-grade environment, a microfiltration membrane is adopted for filtration and sterilization in the process, no preservative is added in the formula, and the product is safer and low in irritation.

Description

Technical Field

[0001] The present invention belongs to the field of anti-aging filling, and in particular relates to a method for preparing a freeze-dried powder containing a recombinant type I and type III collagen composition and an application thereof. Background Art

[0002] In recent years, with the continuous development of fundamental theories, technologies, and clinical research in areas such as genetic recombination and proteomics, recombinant collagen has become an increasingly important biomaterial, providing new insights and approaches for research and development in the field of medical aesthetics. Recombinant humanized collagen is a full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene, or a combination of functional human collagen fragments, produced using recombinant DNA technology.

[0003] Currently, many collagen-based products are being marketed for anti-aging and skin-filling applications, primarily animal-derived and recombinant collagen. However, despite rigorous purification and processing, animal-derived collagen, such as bovine or porcine collagen, still carries a risk of allergies. Recombinant type III collagen, while highly safe, degrades rapidly in the body, making its anti-aging effects relatively limited. Summary of the Invention

[0004] The object of the present invention is to provide a method for preparing a freeze-dried powder containing a recombinant type I and type III collagen composition, aiming to solve the technical problems existing in the prior art identified in the background technology.

[0005] The present invention is achieved by a method for preparing a lyophilized powder containing a recombinant type I and type III collagen composition, wherein the lyophilized powder containing the recombinant type I and type III collagen composition comprises the following components:

[0006] Phase A and Phase B,

[0007] The phase A comprises: 100 parts of water;

[0008] The phase B comprises: 1-10 parts of mannitol, 0.01-0.5 parts of recombinant humanized type I collagen, 0.01-0.5 parts of recombinant humanized type III collagen, and 0.1-2 parts of trehalose;

[0009] The method for preparing the lyophilized powder containing the recombinant type I and type III collagen composition comprises the following steps:

[0010] Step 1: Re-add the water in phase A to the liquid preparation system, and then add mannitol and trehalose in sequence, heating and stirring for 2-4 hours until fully dissolved;

[0011] Step 2: During the heating and stirring process, the solution is tested every hour to monitor the state and composition of the solution;

[0012] Step 3: Cool the stirred solution to 20° C. to 25° C., add the recombinant humanized type I collagen and recombinant humanized type III collagen in phase B to the liquid preparation system, and continue stirring until uniform to obtain a solvent solution;

[0013] Step 4, performing physical and chemical tests on the obtained solvent solution;

[0014] Step 5: The solvent solution is dispensed into vials through a filling and stoppering machine, and semi-stoppering is performed;

[0015] Step 6: Transfer the packaged vials to a vacuum freeze dryer for freeze-drying to obtain freeze-dried powder;

[0016] Step 7: Conduct quality inspection on the freeze-dried powder, including cleaning and sterilization container, appearance inspection, particle size and biological activity.

[0017] As a further embodiment of the present invention, the monitoring of the state and composition of the solution in step 2 specifically includes:

[0018] pH value, which monitors the acidity or alkalinity of a solution;

[0019] Transparency: check whether the solution is clear and whether there are impurities or precipitation;

[0020] Temperature: confirm whether the temperature of the solution meets the preset operating conditions;

[0021] Conductivity, to assess the concentration of ions in the solution and determine whether the composition is uniform;

[0022] viscosity, which measures the fluidity of a solution;

[0023] Component concentration: analyze whether the concentration of each component is within the target range.

[0024] As a further solution of the present invention, the physical and chemical tests on the solvent solution obtained in step 4 specifically include:

[0025] Properties test, pH value test and content test.

[0026] As a further solution of the present invention, step 5 further includes:

[0027] Check the cleaning and sterilization process and the final rinse water of the stoppers for visible foreign matter;

[0028] Check the capping quality every hour and dynamically monitor the environment, including the number of suspended particles, settling bacteria and surface microorganisms.

[0029] As a further solution of the present invention, the freeze-drying process described in step 6 specifically includes:

[0030] Keep it at 0℃ for 30min, then reduce to -45℃ and keep it at -45℃ for 3h;

[0031] The temperature was raised to -6°C and maintained for 8 h, and the vacuum was maintained, and the temperature was raised to 10°C and maintained for 2 h;

[0032] Keep vacuuming and raise the temperature to 30℃ for 2h;

[0033] Keep vacuuming and raise the temperature to 35°C for 4 h;

[0034] Keep vacuuming to obtain the lyophilized powder.

[0035] Another object of the present invention is to provide a freeze-dried powder containing the recombinant type I and type III collagen composition as described above for use in facial anti-aging and facial filling.

[0036] The beneficial effects of the present invention are:

[0037] Dosage form advantages: Lyophilization is a very effective method for drying heat-sensitive products and substances that need to maintain biological activity (collagen). Lyophilized powder is made by pre-freezing the water in the drug solution using the vacuum freeze-drying method of a freeze dryer, and then sublimating the frozen water in the drug solution in a vacuum and sterile environment to obtain freeze-dried powder. Secondly, the freeze-dried product has a loose shape and basically does not change its color after drying. After adding water, it can quickly dissolve and restore the physical and chemical properties and biological activity of the original aqueous solution. Third, the product is produced and filled in a 10,000-level environment. Microporous filter membranes are used for filtration and sterilization during the process. No preservatives are added to the formula, making the product safer and less irritating. Fourth, the moisture content of the product after freeze-drying is very low, which improves the stability of the product and reduces the chance of contamination. This not only facilitates transportation but also extends the shelf life of the product.

[0038] Product advantages: The recombinant type I-III collagen freeze-dried powder used in the formula of this invention is technologically advanced internationally and is the only original product in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A flow chart of a method for preparing a lyophilized powder containing a recombinant type I and type III collagen composition provided in an embodiment of the present invention;

[0040] Figure 2 The effect of using the smear function provided by the embodiment of the present invention Figure 1 ;

[0041] Figure 3 The effect of using the smear function provided by the embodiment of the present invention Figure 2 ;

[0042] Figure 4The effect of using the smear function provided by the embodiment of the present invention Figure 3 ;

[0043] Figure 5 The effect of using the smear function provided by the embodiment of the present invention Figure 4 ;

[0044] Figure 6 The effect of using the smear function provided by the embodiment of the present invention Figure 5 ;

[0045] Figure 7 The effect of using the smear function provided by the embodiment of the present invention Figure 6 ;

[0046] Figure 8 The injection filling effect provided by the embodiment of the present invention Figure 1 ;

[0047] Figure 9 The injection filling effect provided by the embodiment of the present invention Figure 2 ;

[0048] Figure 10 The injection filling effect provided by the embodiment of the present invention Figure 3 ;

[0049] Figure 11 The injection filling effect provided by the embodiment of the present invention Figure 4 ;

[0050] Figure 12 The injection filling effect provided by the embodiment of the present invention Figure 5 . DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0052] A method for preparing a lyophilized powder containing a recombinant type I and type III collagen composition, wherein the lyophilized powder containing the recombinant type I and type III collagen composition comprises the following components:

[0053] Phase A and Phase B,

[0054] The phase A comprises: 100 parts of water;

[0055] The phase B comprises: 1-10 parts of mannitol, 0.01-0.5 parts of recombinant humanized type I collagen, 0.01-0.5 parts of recombinant humanized type III collagen, and 0.1-2 parts of trehalose;

[0056] like Figure 1 As shown, the method for preparing the lyophilized powder containing the recombinant type I and type III collagen composition comprises the following steps:

[0057] Step 1: Re-add the water in phase A to the liquid preparation system, and then add mannitol and trehalose in sequence, heating and stirring for 2-4 hours until fully dissolved;

[0058] Step 2: During the heating and stirring process, the solution is tested every hour to monitor the state and composition of the solution;

[0059] Step 3: Cool the stirred solution to 20° C. to 25° C., add the recombinant humanized type I collagen and recombinant humanized type III collagen in phase B to the liquid preparation system, and continue stirring until uniform to obtain a solvent solution;

[0060] Step 4, performing physical and chemical tests on the obtained solvent solution;

[0061] Step 5: The solvent solution is dispensed into vials through a filling and stoppering machine, and semi-stoppering is performed;

[0062] Step 6: Transfer the packaged vials to a vacuum freeze dryer for freeze-drying to obtain freeze-dried powder;

[0063] Step 7: Conduct quality inspection on the freeze-dried powder, including cleaning and sterilization container, appearance inspection, particle size and biological activity.

[0064] The freeze-dried powder containing recombinant type I and type III collagen prepared in this invention appears as a white, loose, porous block. Upon application, the product instantly transforms into a liquid by adding a solvent. One way to use it is by direct application to the face, which tightens the skin and improves its elasticity. Another way to use it is by subcutaneous injection for filling, effectively eliminating wrinkles and sagging.

[0065] In this embodiment, the monitoring solution state and composition described in step 2 specifically includes:

[0066] pH value, which monitors the acidity or alkalinity of a solution;

[0067] Transparency: check whether the solution is clear and whether there are impurities or precipitation;

[0068] Temperature: confirm whether the temperature of the solution meets the preset operating conditions;

[0069] Conductivity, to assess the concentration of ions in the solution and determine whether the composition is uniform;

[0070] viscosity, which measures the fluidity of a solution;

[0071] Component concentration: analyze whether the concentration of each component is within the target range.

[0072] In this embodiment, the physical and chemical tests on the solvent solution obtained in step 4 specifically include:

[0073] Properties test, pH value test and content test.

[0074] In this embodiment, step 5 further includes:

[0075] Check the cleaning and sterilization process and the final rinse water of the stoppers for visible foreign matter;

[0076] Check the capping quality every hour and dynamically monitor the environment, including the number of suspended particles, settling bacteria and surface microorganisms.

[0077] In this embodiment, the freeze-drying process described in step 6 specifically includes:

[0078] Keep it at 0℃ for 30min, then reduce to -45℃ and keep it at -45℃ for 3h;

[0079] The temperature was raised to -6°C and maintained for 8 h, and the vacuum was maintained, and the temperature was raised to 10°C and maintained for 2 h;

[0080] Keep vacuuming and raise the temperature to 30℃ for 2h;

[0081] Keep vacuuming and raise the temperature to 35°C for 4 h;

[0082] Keep vacuuming to obtain the lyophilized powder.

[0083] The present invention also includes a use of the freeze-dried powder containing the recombinant type I and type III collagen composition as described above for facial anti-aging and facial filling.

[0084] Example:

[0085] 1. Smearing function test

[0086] (1) Experimental population

[0087] Twenty female volunteers aged 20-50 were selected and randomly divided into two groups, with 10 participants in each group.

[0088] (2) Experimental methods

[0089] After cleansing, add purified water to the freeze-dried powder and apply directly to the face without washing; use once a day for 4 consecutive weeks;

[0090] (3) Evaluation method

[0091] Observe with the naked eye.

[0092] (4) Experimental results

[0093] The effects are shown in Table 1.

[0094] Table 1 Usage effect

[0095]

[0096]

[0097] According to the image data in Table 1 above, it can be seen that the use of freeze-dried powder containing recombinant type I-III collagen has a significant anti-aging effect on the skin, can fade fine lines and brighten the skin.

[0098] 2. Injection filling effect test

[0099] (1) Experimental population

[0100] Ten female volunteers aged 30-50 were selected.

[0101] (2) Experimental instruments

[0102] Take photos with the skin facial analyzer (Reveal).

[0103] (3) Experimental methods

[0104] The subjects received injections once a month for 2-3 consecutive months. The product was dissolved in sterile saline to a colorless, transparent solution with a total collagen concentration of 4mg / ml and injected into the desired facial area using a sterile 27G syringe. Skin images were taken using a Reveal skin analyzer (USA) at 30, 60, and 90 days after injection of the recombinant type I-III collagen freeze-dried powder to compare the effects.

[0105] (4) Evaluation method

[0106] Take photos and compare the changes in users' facial wrinkles.

[0107] (5) Experimental results

[0108] The wrinkle filling effects are shown in Table 2.

[0109] Table 2 Use effect to take pictures

[0110]

[0111] As can be seen from the table above, the product has a significant filling effect on the nasolabial folds and wrinkles around the eyes, and can reduce wrinkles. Use it 2-3 times to see noticeable results.

[0112] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0113] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a freeze-dried powder containing a recombinant type I and type III collagen composition, characterized in that: The lyophilized powder containing the recombinant type I and type III collagen composition includes the following components: Phase A and Phase B, The phase A comprises: 100 parts of water; The phase B comprises: 1-10 parts of mannitol, 0.01-0.5 parts of recombinant humanized type I collagen, 0.01-0.5 parts of recombinant humanized type III collagen, and 0.1-2 parts of trehalose; The method for preparing the lyophilized powder containing the recombinant type I and type III collagen composition comprises the following steps: Step 1: Re-add the water in phase A to the liquid preparation system, and then add mannitol and trehalose, heating and stirring for 2-4 hours until fully dissolved; Step 2: During the heating and stirring process, the solution is tested every hour to monitor the state and composition of the solution; Step 3: Cool the stirred solution to 20° C. to 25° C., add the recombinant humanized type I collagen and recombinant humanized type III collagen in phase B to the liquid preparation system, and continue stirring until uniform to obtain a solvent solution; Step 4, performing physical and chemical tests on the obtained solvent solution; Step 5: The solvent solution is dispensed into vials through a filling and stoppering machine, and semi-stoppering is performed; Step 6: Transfer the packaged vials to a vacuum freeze dryer for freeze-drying to obtain freeze-dried powder; Step 7: Conduct quality inspection on the freeze-dried powder, including cleaning and sterilization container, appearance inspection, particle size and biological activity.

2. The method according to claim 1, characterized in that The state and composition of the monitoring solution described in step 2 specifically include: pH value, which monitors the acidity or alkalinity of a solution; Transparency: check whether the solution is clear and whether there are impurities or precipitation; Temperature: confirm whether the temperature of the solution meets the preset operating conditions; Conductivity, to assess the concentration of ions in the solution and determine whether the composition is uniform; viscosity, which measures the fluidity of a solution; Component concentration: analyze whether the concentration of each component is within the target range.

3. The method according to claim 1, characterized in that The physical and chemical testing of the solvent solution obtained in step 4 specifically includes: Property test, pH value test and content test.

4. The method according to claim 1, wherein The step 5 further comprises: Check the cleaning and sterilization process and the final rinse water of the stoppers for visible foreign matter; Check the capping quality every hour and dynamically monitor the environment, including the number of suspended particles, settling bacteria and surface microorganisms.

5. The method according to claim 3, characterized in that The freeze-drying process described in step 6 specifically includes: Keep it at 0℃ for 30min, then reduce to -45℃ and keep it at -45℃ for 3h; The temperature was raised to -6°C and maintained for 8 h, and the vacuum was maintained, and the temperature was raised to 10°C and maintained for 2 h; Keep vacuuming and raise the temperature to 30℃ for 2h; Keep vacuuming and raise the temperature to 35°C for 4 h; Keep vacuuming to obtain the lyophilized powder.

6. Use of the freeze-dried powder containing the recombinant type I and type III collagen composition as claimed in claim 1 for facial anti-aging and facial filling.

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