Freeze-drying process of recombinant human type iii collagen and freeze-dried protein prepared thereby and uses thereof
By optimizing the freeze-drying process and employing low-temperature freezing and gradient drying, highly bioactive and stable recombinant human type III collagen freeze-dried protein was prepared, solving the problem of insufficient bioactivity and stability in existing technologies and achieving highly efficient wound healing and skin repair.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI UNIV
- Filing Date
- 2026-02-26
- Publication Date
- 2026-05-29
Smart Images
Figure CN122103313A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of medical aesthetics technology, and in particular to a freeze-drying process for recombinant human type III collagen and the freeze-dried protein prepared therefrom and its applications. Background Technology
[0002] As the body's first line of defense, the skin is easily irritated by external stimuli, leading to sensitivity or damage. With increasing emphasis on skin health, the need to address wound healing and skin barrier issues is becoming increasingly urgent. For sensitive or damaged skin, traditional medical dressings primarily function as physical coverings and absorb exudate, lacking the support of bioactive ingredients and thus failing to accelerate wound healing. Meanwhile, the repairing ingredients in ordinary skincare products are mostly plant extracts and vitamins, which have limited repair effects on damaged skin and may cause skin allergies due to ingredient compatibility issues.
[0003] Collagen is the main structural protein of human skin. Type III collagen is mainly found in infant skin and the early stages of wound healing, exhibiting good biocompatibility and repair-promoting activity. Early products generally used animal-derived collagen, which, while effective, suffered from high immunogenicity, low purity, and difficulty in guaranteeing activity, easily causing skin allergies and poor repair effects. With the development of biosynthetic technology, recombinant human type III collagen has been synthesized and prepared through genetic engineering. This results in higher purity, better activity, and greater homology with the human body, overcoming the drawbacks of animal-derived collagen and becoming one of the core ingredients in the field of skin repair and wound healing.
[0004] Directly synthesized protein solutions are difficult to preserve their activity, which is detrimental to subsequent use, necessitating drying and storage. Freeze-drying is a processing technology that removes moisture from materials in a low-temperature vacuum environment. Its core advantage lies in preserving the structure and function of bioactive components under low-temperature conditions, avoiding component inactivation caused by high-temperature processing. For heat-sensitive biomolecules like collagen, freeze-drying can maintain their bioactivity while forming a loose, porous product structure. This structure improves product permeability and rapidly absorbs wound exudate, creating an ideal moist environment for skin repair and wound healing. However, existing freeze-drying processes for recombinant human type III collagen still have room for improvement in terms of bioactivity and stability. Summary of the Invention
[0005] The main objective of this invention is to provide a freeze-drying process for recombinant human type III collagen, the freeze-dried protein prepared therefrom, and its applications, aiming to solve at least one of the aforementioned technical problems.
[0006] To achieve the above objectives, the present invention provides a freeze-drying process for recombinant human type III collagen, comprising the following steps: (1) Pre-freezing The protein solution of recombinant human type III collagen was frozen at low temperature; (2) Sublimation drying Under vacuum conditions, recombinant human type III collagen, frozen into a solid state, was subjected to gradient sublimation drying in four stages with progressively increasing temperatures. (3) Analysis and drying Under vacuum conditions, recombinant human type III collagen that has undergone sublimation drying is subjected to gradient desorption drying in two stages with successively increasing temperatures to obtain lyophilized recombinant human type III collagen.
[0007] Furthermore, in step (1), the specific conditions for the freezing treatment are: the temperature of the heat transfer oil reaches -40 to -45°C within 10 to 30 minutes and is maintained for 4 to 6 hours.
[0008] Furthermore, in step (2), the vacuum degree is maintained at 10 Pa in all four stages; in the first stage, the temperature of the heat transfer oil reaches -35 to -30°C within 20 minutes and is maintained for 6 to 10 hours; in the second stage, the temperature of the heat transfer oil reaches -30 to -25°C within 20 minutes and is maintained for 6 to 8 hours; in the third stage, the temperature of the heat transfer oil reaches -25 to -20°C within 20 minutes and is maintained for 6 to 8 hours; in the fourth stage, the temperature of the heat transfer oil reaches 0 to 5°C within 20 minutes and is maintained for 2 to 3 hours.
[0009] Furthermore, in step (3), the vacuum degree is maintained at 10 Pa in both stages; the temperature of the heat transfer oil in the first stage reaches 5-15°C within 20 minutes and is maintained for 2-3 hours; the temperature of the heat transfer oil in the second stage reaches 20-30°C within 20 minutes and is maintained for 2-4 hours.
[0010] Furthermore, the recombinant human type III collagen protein solution is COLIII-20 protein solution. COLIII-20 protein is a recombinant human type III collagen protein COLIII-20 disclosed in Chinese patent application CN118255873A. The COLIII-20 protein solution is obtained by microbial fermentation using genetic engineering methods. For specific preparation methods, please refer to the detailed embodiments section of CN118255873A.
[0011] The present invention also provides a lyophilized protein of recombinant human type III collagen, which is prepared according to the above-described lyophilization process.
[0012] The present invention also provides the application of the above-mentioned lyophilized recombinant human type III collagen protein in the preparation of products with wound repair function.
[0013] The beneficial effects of this invention are reflected in: The freeze-drying process of this invention can obtain freeze-dried collagen raw materials with a full appearance, stable structure, and high bioactivity. Dressings prepared using highly active and biocompatible recombinant human type III collagen avoid the problems associated with traditional dressings and skincare products, alleviate skin sensitivity, maintain moisturizing and soothing effects, and promote wound healing.
[0014] This invention optimizes the freeze-drying process in terms of time and temperature to address the characteristics of collagen, resulting in a highly bioactive freeze-dried collagen product. For medical applications, it prepares dressings that combine physical protection and biological repair functions, effectively solving the problems of traditional dressings lacking active ingredients and having limited repair efficiency. This meets the precise repair needs of wound healing and post-cosmetic surgery procedures. Through highly homologous recombinant human type III collagen and targeted active ingredients, it achieves precise repair of skin structure and strengthening of barrier function. Attached Figure Description
[0015] Figure 1 These are protein state diagrams after programmed freeze-drying in Example 1 and Comparative Example 1.
[0016] Figure 2 These are electrophoresis images of the lyophilized proteins obtained in Example 1 and Comparative Examples 1, 2, and 3 (in the images, lane 1 is Example 1, lane 2 is Comparative Example 1, lane 3 is Comparative Example 2, and lane 4 is Comparative Example 3).
[0017] Figure 3 These are the coagulation effect graphs and bar charts of different concentrations of lyophilized protein in Experiment Example 2. Detailed Implementation
[0018] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.
[0019] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods; unless otherwise specified, the methods used in the embodiments of the present invention are methods mastered by those skilled in the art. COLIII-20 protein is the recombinant human type III collagen COLIII-20 disclosed in Chinese patent application CN118255873A. COLIII-20 protein solution is obtained by microbial fermentation using genetic engineering methods; the preparation method is described in the specific embodiments section of CN118255873A.
[0020] Example 1 Lyophilized COLIII-20 protein (1) Pre-freezing The COLIII-20 protein solution was frozen at low temperature, specifically: The temperature of the heat transfer oil reaches -45°C in 30 minutes, and the vacuum degree is maintained at 10 Pa for 6 hours. (2) Sublimation drying Under vacuum conditions, the frozen COLIII-20 protein was subjected to gradient sublimation drying in four stages with progressively increasing temperatures. Specifically: The temperature of the heat transfer oil in the first stage reaches -35°C within 20 minutes, and the vacuum degree is maintained at 10 Pa for 8 hours. In the second stage, the temperature of the heat transfer oil reaches -30°C within 20 minutes, and the vacuum degree is maintained at 10 Pa for 8 hours. In the third stage, the temperature of the heat transfer oil reaches -25°C within 20 minutes, and the vacuum degree is maintained at 10 Pa for 8 hours. In the fourth stage, the temperature of the heat transfer oil reaches 0°C within 20 minutes, and the vacuum degree is maintained at 10 Pa for 2 hours. (3) Analysis and drying Under vacuum conditions, recombinant human type III collagen that has undergone sublimation drying was subjected to gradient desorption drying in two stages with progressively increasing temperatures. Specifically: The temperature of the heat transfer oil in the first stage reaches 5°C within 20 minutes, and the vacuum degree is maintained at 10 Pa for 2 hours. In the second stage, the temperature of the heat transfer oil reaches 25°C within 20 minutes, and the vacuum degree is maintained at 10 Pa for 4 hours. The lyophilized protein of COLIII-20 was obtained.
[0021] Comparative Example 1 Comparison of COLIII-20 protein lyophilization The freeze-drying process of this comparative example is basically the same as that of Example 1, except that the temperature of the heat transfer oil in step (1) reaches -25°C within 20 minutes.
[0022] Comparative Example 2 Comparison of COLIII-20 protein lyophilization The freeze-drying process of this comparative example is basically the same as that of Example 1, except that the temperature of the heat transfer oil in the first three stages of step (2) is increased by 10°C, that is, the temperature of the heat transfer oil in the first stage reaches -25°C within 20 minutes; the temperature of the heat transfer oil in the second stage reaches -20°C within 20 minutes; and the temperature of the heat transfer oil in the third stage reaches -15°C within 20 minutes.
[0023] Comparative Example 3 Comparison of COLIII-20 protein lyophilization The freeze-drying process of this comparative example is basically the same as that of Example 1, except that the temperature of the heat transfer oil in the second stage of step (3) reaches 40°C within 20 minutes.
[0024] Experimental Example 1 Performance testing of lyophilized proteins The lyophilized protein of COLIII-20 obtained in Example 1 is shown in the image. Figure 1 As shown in Figure B, it can be seen that after the freeze-drying process is optimized, the collagen has a full appearance, a tight internal structure, and no cavities, bubbles, collapses, or shrinkage on the surface.
[0025] The lyophilized protein of COLIII-20 protein prepared in Comparative Example 1 is shown in the image. Figure 1 As shown in Figure A, the collagen in this sample has a generally poor appearance, a relatively loose internal structure, air bubbles on the surface, and some signs of collapse and shrinkage. Other freeze-dried protein solutions prepared in the comparative examples also exhibited similar phenomena to varying degrees, which will not be illustrated further.
[0026] I. Activity Detection The MTT assay was used to detect the proliferative capacity of the lyophilized proteins prepared in Example 1 and the comparative examples on human immortalized epidermal cells (HaCaT).
[0027] Experimental materials: Experimental group: freeze-dried COLIII-20 protein obtained in Example 1 and each comparative example.
[0028] Control groups: DMEM medium containing 10% fetal bovine serum (negative control, NC), DMEM medium containing 3% DMSO (positive control, used to verify the cytotoxic boundary), and DMEM medium without cells (blank control).
[0029] Experimental steps: Immortalized human epidermal cells (HaCaT) were taken, digested, centrifuged, resuspended, and diluted to 2 × 10⁻⁶. 4 Cells / mL, 100 μL per well were seeded into a 96-well cell culture plate; cell-free culture medium was also seeded as a blank control. The plates were incubated at 37°C, 5% CO2, and 95% humidity for 24 h.
[0030] Discard the culture medium, add culture medium to the blank control, add culture medium containing 3% DMSO to the positive control, and add culture medium containing different concentrations of recombinant human type III collagen liquid dressing to the experimental group. Incubate at 37℃, 5% CO2, and 95% humidity for 24 hours. Add 20 μL of MTT to each well and incubate for 4 h at 37°C, 5% CO2, and 95% humidity. Add 150 μL of DMSO and incubate on a shaker for 10 min to fully dissolve the crystals. Measure the absorbance at 490 nm using a microplate reader. Zero the cell using the OD value of the blank control well, and record the average value. Calculate the relative cell proliferation rate based on the average absorbance of each group. Cell viability = (Experimental group Abs - Blank control group Abs) / (Negative control group Abs - Blank control group Abs) * 100%.
[0031] Experimental results are shown in Table 1.
[0032] Table 1 Cell proliferation rate
[0033] As shown in Table 1 above, the cell proliferation rate in Example 1 is higher than that in Comparative Examples 1, 2, and 3, indicating that the collagen obtained by this freeze-drying process has better bioactivity, a more aesthetically pleasing overall appearance, and a shorter freeze-drying time.
[0034] II. Stability Testing The stability of the lyophilized COLIII-20 protein prepared in Example 1 and each comparative example was detected by SDS-PAGE electrophoresis when stored in a sealed and dry environment at 37°C for 1 month.
[0035] The results are as follows Figure 2 As shown in the electrophoresis results, compared with Comparative Examples 1, 2, and 3, the target protein band content in Example 1 is higher and the degradation degree is lower, indicating that the collagen obtained by this freeze-drying process has better stability.
[0036] Experiment Example 2 In vitro coagulation effect experiment of lyophilized protein This experiment uses the lyophilized COLIII-20 protein obtained in Example 1 as the experimental material to verify its in vitro coagulation efficacy. The core indicator is to determine the ability of the biomaterial to induce blood clot formation on the surface of the material after contact with calcified whole blood. Specifically, the procoagulant activity of different samples is evaluated by the Blood Clotting Index (BCI) test.
[0037] I. Experimental Materials Experimental group: Lyophilized COLIII-20 protein obtained in Example 1; Control group: saline (negative control, NC), bovine type I collagen (COLI (Bovine)), and recombinant human type III collagen (COLIII).
[0038] II. Experimental Procedure Take four clean 10mL centrifuge tubes and add 1mL of solution each to the sample group, negative control group, bovine type I collagen group, and recombinant human type III collagen group. Incubate in a 37℃ water bath for 5 minutes to ensure temperature stability. Quickly add 200μL of sodium citrate anticoagulated whole blood preheated to 37℃ to each centrifuge tube, and immediately add 20μL of preheated 0.2MCaCl2 solution (to trigger the coagulation reaction). Gently invert the centrifuge tube three times to mix, and continue incubation at 37℃ for 2 minutes. Slowly add 10mL of preheated deionized water at 37℃ along the wall of the centrifuge tube, and incubate at 37℃ for 2 minutes. Centrifuge at 2000rpm for 5 minutes, and transfer 100μL of supernatant to a 96-well plate (3 replicates per group). Measure the absorbance (Abs) at 540nm using a microplate reader. Calculate the coagulation index (BCI) using the following formula: BCI = (Abs in sample group / Abs in negative control group) × 100%. The lower the BCI value, the stronger the procoagulant activity.
[0039] III. Experimental Results The results are as follows Figure 3 The results showed that the BCI values of the lyophilized COLIII-20 protein at concentrations of 0.4 mg / mL and 0.6 mg / mL were significantly lower than those of the negative control group (NC), and also showed significant differences from commercially available animal collagen, demonstrating its good in vitro coagulation properties.
[0040] In summary, the freeze-drying process of this invention can produce freeze-dried protein raw materials with a full and beautiful appearance, good stability, high activity, and hemostatic effect. Based on this, recombinant human type III collagen dressings with wound repair function can be formulated and can be widely used in hemostasis, wound repair materials, medical aesthetics, cosmetics and other fields.
[0041] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A freeze-drying process for recombinant human type III collagen, characterized in that, Includes the following steps: (1) Pre-freezing The protein solution of recombinant human type III collagen was frozen at low temperature; (2) Sublimation drying Under vacuum conditions, recombinant human type III collagen, frozen into a solid state, was subjected to gradient sublimation drying in four stages with progressively increasing temperatures. (3) Analysis and drying Under vacuum conditions, recombinant human type III collagen that has undergone sublimation drying is subjected to gradient desorption drying in two stages with successively increasing temperatures to obtain lyophilized recombinant human type III collagen.
2. The freeze-drying process for recombinant human type III collagen as described in claim 1, characterized in that, In step (1), the specific conditions for the freezing treatment are: the temperature of the heat transfer oil reaches -40 to -45°C within 10 to 30 minutes and is maintained for 4 to 6 hours.
3. The freeze-drying process for recombinant human type III collagen as described in claim 1 or 2, characterized in that, In step (2), the vacuum level is maintained at 10 Pa in all four stages; in the first stage, the temperature of the heat transfer oil reaches -35 to -30°C within 20 minutes and is maintained for 6 to 10 hours; in the second stage, the temperature of the heat transfer oil reaches -30 to -25°C within 20 minutes and is maintained for 6 to 8 hours; in the third stage, the temperature of the heat transfer oil reaches -25 to -20°C within 20 minutes and is maintained for 6 to 8 hours; in the fourth stage, the temperature of the heat transfer oil reaches 0 to 5°C within 20 minutes and is maintained for 2 to 3 hours.
4. The freeze-drying process for recombinant human type III collagen as described in claim 1 or 2, characterized in that, In step (3), the vacuum level is maintained at 10 Pa in both stages; the temperature of the heat transfer oil in the first stage reaches 5-15°C within 20 minutes and is maintained for 2-3 hours; the temperature of the heat transfer oil in the second stage reaches 20-30°C within 20 minutes and is maintained for 2-4 hours.
5. The freeze-drying process for recombinant human type III collagen as described in any one of claims 1 to 4, characterized in that, The protein solution of the recombinant human type III collagen is COLIII-20 protein solution.
6. A lyophilized protein of recombinant human type III collagen, characterized in that, Prepared by the freeze-drying process according to any one of claims 1 to 5.
7. The application of the lyophilized recombinant human type III collagen as described in claim 6 in the preparation of products with wound repair effects.