Method for preparing recombinant human collagen sponge

Abstract

The invention provides a method for preparing recombinant human collagen sponge. The method comprises the step of carrying out concentration and assembly treatment on recombinant human collagen so as to obtain the recombinant human collagen sponge, and the recombinant human collagen does not have terminal peptide. According to the method disclosed by the embodiment of the invention, the recombinant human collagen sponge which has collagen with a complete triple-helix structure, excellent biological activity, low immunogenicity, high liquid absorption performance, high mechanical strength and no animal-derived virus pollution risk can be prepared by the method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and in particular relates to a method for preparing a recombinant human collagen sponge. More particularly, it relates to a recombinant human collagen sponge and uses thereof. BACKGROUND

[0002] Collagen is the main component of animal connective tissue and is also the most abundant functional protein in mammals, accounting for about 25% to 30% of the total amount of protein. Due to its unique structural characteristics, excellent biocompatibility and biodegradability, collagen has become an ideal biomedical material and is widely used in the fields of tissue engineering, wound repair, hemostatic materials, cosmetic filling, etc. Traditional collagen is mainly extracted from animal tissues (such as bovine Achilles tendon, pigskin, etc.), which has good biological activity, but has the following obvious defects: (1) potential risk of virus transmission and disease infection; (2) strong immunogenicity, which may cause allergic reactions; (3) limited source of raw materials, poor batch consistency; (4) may contain heavy metal, antibiotic residues and other impurities. The emergence of recombinant collagen technology provides a new way to solve the above problems.

[0003] As an important medical device product, the performance of collagen sponge is directly related to the clinical effect. An ideal collagen sponge should have the following characteristics: (1) no risk of animal-derived virus contamination; (2) low immunogenicity; (3) retaining the triple helix structure and complete biological activity of natural collagen; (4) good liquid absorption performance and mechanical strength; (5) appropriate degradation rate in vivo; (6) no odor, non-toxicity, non-irritation.

[0004] At present, there is a lack of collagen sponge products on the market that can meet the above requirements at the same time, so it is of great clinical significance and market value to develop a new type of recombinant human collagen sponge with high performance and a preparation method thereof. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application provides a method for preparing a recombinant human collagen sponge.

[0006] The present application is based on the following findings of the inventors: The inventors found that the existing sponge materials prepared from recombinant collagen (especially humanized collagen) often have problems of poor liquid absorption performance, insufficient mechanical strength, and easy enzymatic hydrolysis. In order to overcome these problems, the inventors provide a method for preparing a recombinant human collagen sponge. According to embodiments of the present application, the method can prepare a recombinant human collagen sponge having a complete triple helix structure, excellent biological activity, low immunogenicity, high liquid absorption performance, high mechanical strength, and no risk of animal-derived virus contamination.

[0007] In a first aspect of the present application, a method for preparing a recombinant human collagen sponge is provided. According to embodiments of the present application, the method comprises subjecting a recombinant human collagen, which does not have a telopeptide, to a concentration and assembly treatment, so as to obtain the recombinant human collagen sponge. The method according to embodiments of the present application can prepare a recombinant human collagen sponge having a complete triple helix structure, high biological activity, low immunogenicity, high liquid absorption, high mechanical strength, and no risk of animal-derived virus contamination; at the same time, the process route is simplified, the operation is convenient, the cost is reduced, and the quality of the obtained product is stable and uniform between batches.

[0008] In a second aspect of the present application, a recombinant human collagen sponge is provided. According to embodiments of the present application, the recombinant human collagen sponge is obtained by the method according to the first aspect of the present application. The recombinant human collagen sponge according to embodiments of the present application not only has a complete triple helix structure, but also has excellent biological activity, low immunogenicity, high liquid absorption, and high mechanical strength, and no risk of animal-derived virus contamination.

[0009] In a third aspect of the present application, the use of the recombinant human collagen sponge according to the second aspect of the present application in the preparation of a hemostatic material, a wound dressing, or a tissue engineering scaffold is provided.

[0010] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0011] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings. Figure 1 Electrophoretogram of the recombinant human type I collagen prepared for Example 1 of the present application.

[0012] Figure 2 Appearance morphology diagram of the recombinant human type I collagen sponge prepared for Example 1 of the present application.

[0013] Figure 3An electropherogram of the recombinant human collagen type III prepared for Example 2 of the present application.

[0014] Figure 4 A topographic image of the recombinant human collagen type III sponge prepared for Example 2 of the present application.

[0015] Figure 5 An SEM image (50x) of the recombinant human collagen type III sponge tested in Test Example 5 of the present application.

[0016] Figure 6 An SEM image (100x) of the recombinant human collagen type III sponge tested in Test Example 5 of the present application. DETAILED DESCRIPTION

[0017] Hereinafter, examples of the present application will be described in detail. The examples described below are illustrative only and are not to be taken as limiting the present application.

[0018] It should be noted that the terms "first", "second" and the like in the description do not necessarily connote an absolute order or quantity but are used to modify an entity or to distinguish entities from one another. Thus, the features defined with "first", "second" and the like can include one or more of the features. Further, in the description, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0019] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be approximations that allow for significant variation. Various ranges of values have been stated as being desired or preferred. It is contemplated that the endpoints of the ranges and any values will be one or another of the following: within one unit of the stated range or value; within 5% of the stated range or value; within 1% of the stated range or value; or within.1% of the stated range or value. Other values that are not listed are also contemplated.

[0020] In order that the application can be more fully understood, certain technical and scientific terms are specifically defined herein. Unless otherwise clearly indicated, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains.

[0021] In the present document, the terms "comprising" or "including" or "having" are intended to be open-ended terms that do not exclude additional elements or steps.

[0022] In the present document, the terms "optionally", "optional" or "may" are generally used to mean that there are more options and that an item to be selected from the options is only an example and is not a limitation from the more options. For example, in the context of feature being optionally included, the feature can also be excluded in certain instances. In addition, while the above descriptions may contain

[0023] In this context, the term "recombinant human collagen" refers to the use of genetic engineering technology to introduce human collagen-encoding genes into host cells (such as Pichia pastoris in this invention) to produce human collagen through fermentation culture. Compared with traditional animal-derived (bovine, porcine) collagen, it is safe in origin, has no risk of animal-derived virus transmission, no immunogenicity problem, and the amino acid sequence is completely consistent with human body's own collagen.

[0024] In this context, the term "depeptization" refers to the short peptide fragments (terminal peptides) at both ends of the collagen molecule, which are the main antigenic determinants causing immune rejection. The process of removing these terminal peptides by enzymatic hydrolysis (such as pepsin) is depeptization. The purpose of this treatment in this context is to further reduce immunogenicity, making the product safer after implantation in the human body and less likely to trigger allergic or immune reactions.

[0025] In this context, the term "Pichia pastoris expression system" refers to a very commonly used eukaryotic protein expression platform. Pichia pastoris is a methanol-tolerant yeast that can efficiently express foreign proteins and has the ability to correctly fold and post-translationally modify proteins (such as glycosylation). In this context, as a "cell factory" for producing recombinant human collagen, it can produce high-quality collagen with complete triple helix structure on a large scale and at low cost.

[0026] In this context, the term "triple helix structure" refers to the specific higher-order spatial structure of collagen, formed by the intertwining of three polypeptide chains into a stable rope-like structure. This is the basis of its mechanical strength and biological function. In this context, the preparation method emphasizes the complete preservation of this structure, ensuring that the sponge product has good mechanical strength and biological activity in promoting cell adhesion and proliferation.

[0027] In this context, the term "self-assembly" refers to the process of spontaneous ordered aggregation of collagen molecules under specific physicochemical conditions (such as specific pH, ionic strength) to form higher-order collagen fibers. In this context, high-concentration collagen protein solution is self-assembled into fibers in a high-pH (12.0) buffer, which is the basis for forming the sponge network structure.

[0028] In this context, the term "vacuum freeze-drying" refers to a drying technique that first freezes the material into a solid state and then removes water by sublimation in a vacuum environment. It can maximize the preservation of the physical structure and biological activity of the material. It is the core process for preparing porous sponge materials. In this context, a special "non-desiccation drying" program is used to control the bound water content of the product, which is crucial for the subsequent resuspension speed and the liquid absorption performance of the final sponge.

[0029] In this article, the term "severe dehydration" refers to a physical cross-linking method that processes the material for a long time under high temperature and high vacuum conditions. It can form covalent bonds between collagen molecules, thereby enhancing the stability of the material. In this article, it specifically refers to significantly improving the mechanical strength and enzyme resistance of the sponge through physical means without introducing chemical cross-linking agents, so that it can maintain in the body for a longer time, and there is no risk of toxic cross-linking agent residues.

[0030] In this article, the term "pre-concentration and assembly" or "concentration assembly" refers to the process route of the present invention. First, a high-concentration (10-50 mg / mL) collagen solution is obtained by freeze-drying and re-dissolving, and then self-assembly is performed. Compared with the traditional process of "assembling a low-concentration solution and then centrifuging and concentrating", this route avoids centrifugal loss, so that the collagen yield is as high as ~100%, and the sponge structure formed is more uniform, and the performance (liquid absorption, strength) is better.

[0031] In this article, the term "assembly buffer" refers to a buffer solution used to provide a specific environment to promote the self-assembly of collagen. In this article, 40 mM phosphate buffer at pH 12.0 is used as the assembly buffer, and this high-alkaline environment is a key parameter for successfully achieving high-concentration collagen self-assembly.

[0032] In this article, the term "water absorption multiple" refers to the maximum mass multiple of liquid that can be absorbed by unit mass of sponge. It is a key indicator for measuring the performance of sponge as a dressing or hemostatic material. The water absorption multiple of the product in this article can reach 20-50 times, indicating that it has excellent liquid absorption capacity.

[0033] In this article, the term "enzyme resistance" refers to the ability of the material to resist degradation by enzymes in the body, such as collagenase. After severe dehydration treatment, the enzyme resistance of the sponge is enhanced, which means that it can support the wound for a longer time and is more conducive to the slow healing of the wound surface.

[0034] The present application provides a method for preparing a recombinant human collagen sponge, a recombinant human collagen sponge and uses thereof, which will be described in detail below.

[0035] Method for preparing a recombinant human collagen sponge In the first aspect, the present application provides a method for preparing a recombinant human collagen sponge. According to an embodiment of the present application, the method comprises subjecting a recombinant human collagen, which does not have terminal peptides, to a concentrated assembly treatment to obtain the recombinant human collagen sponge. The method of the embodiments of the present application can prepare a recombinant human collagen sponge with complete triple helix structure, high biological activity, low immunogenicity, high liquid absorption and high mechanical strength, and no risk of animal-derived virus contamination; at the same time, the process route is simplified, the operation is convenient, the cost is reduced, and the quality of the obtained product is stable and uniform between batches.

[0036] According to an embodiment of the present application, the concentrated assembly treatment is achieved by subjecting the recombinant human collagen to a first freeze-drying treatment and a resuspension treatment to obtain a concentrated stock solution; subjecting the concentrated stock solution to a uniform mixing treatment with an assembly buffer to obtain a collagen assembly system; subjecting the collagen assembly system to a second freeze-drying treatment to obtain a procollagen sponge; and subjecting the procollagen sponge to a severe dehydration treatment.

[0037] According to an embodiment of the present application, the first freeze-drying treatment is performed in the presence of a first vacuum freeze-drying program.

[0038] It should be explained that in this document, "first vacuum freeze-drying program" is synonymous with "P1".

[0039] According to an embodiment of the present application, the concentration of the concentrated stock solution is 10-50 mg / mL, for example, it can be 10 mg / mL, 11 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 26 mg / mL, 28 mg / mL, 30 mg / mL, 32 mg / mL, 34 mg / mL, 36 mg / mL, 38 mg / mL, 40 mg / mL, 42 mg / mL, 44 mg / mL, 46 mg / mL, 48 mg / mL, 50 mg / mL, or a range value of 11-50 mg / mL, 12-50 mg / mL therebetween.

[0040] According to an embodiment of the present application, before the second freeze-drying treatment, further comprising subjecting the collagen assembly system to a standing treatment to obtain an assembly fiber.

[0041] According to an embodiment of the present application, the standing treatment is performed at a temperature of 28-32°C, for example, it can be 28°C, 29°C, 30°C, 31°C, 32°C.

[0042] According to an embodiment of the present application, the volume ratio of the concentrated stock solution to the assembly buffer is 3:1.

[0043] According to embodiments of the present application, the second lyophilization process is performed in the presence of a second vacuum lyophilization program.

[0044] It needs to be explained that in this text, the "second vacuum lyophilization program" is synonymous with "P2".

[0045] According to embodiments of the present application, the recombinant human collagen is selected from at least one of a recombinant type I human collagen and a recombinant type III human collagen.

[0046] According to embodiments of the present application, the recombinant human collagen is obtained by pre-treating a full-length recombinant human collagen with a telopeptide removal treatment.

[0047] According to embodiments of the present application, the telopeptide removal treatment includes enzymatic treatment with at least one of pepsin, trypsin and Lys-c enzyme.

[0048] According to embodiments of the present application, the enzyme is added in an amount of 1 / 10000 ~ 1 / 100 of the weight of the full-length recombinant human collagen, for example, it can be 1 / 10000, 1 / 9000, 1 / 8000, 1 / 7000, 1 / 6000, 1 / 5000, 1 / 4000, 1 / 3000, 1 / 2000, 1 / 1000, 1 / 900, 1 / 800, 1 / 700, 1 / 600, 1 / 500, 1 / 400, 1 / 300, 1 / 200, 1 / 100, or a range value of 1 / 9000 ~ 1 / 100, 1 / 8000 ~ 1 / 100 between them.

[0049] According to embodiments of the present application, the first vacuum lyophilization program is a non-resolution drying program, specifically including: freezing control: maintaining at -40℃ ~ -50℃ for 100 ~ 140 minutes; primary drying: maintaining at -30℃ ~ -50℃ for 1900 ~ 2000 minutes; resolution drying: maintaining at 10℃ ~ 30℃ for 0 minutes.

[0050] According to embodiments of the present application, the first vacuum lyophilization program is a non-resolution drying program, specifically including: freezing control: maintaining at -45℃ for 120 minutes; primary drying: maintaining at -40℃ for 1920 minutes; resolution drying: maintaining at 20℃ for 0 minutes.

[0051] According to embodiments of the present application, the assembly buffer is a 40mM phosphate buffer.

[0052] According to embodiments of the present application, the pH value of the phosphate buffer is 11.0 ~ 12.0.

[0053] According to embodiments of the present application, the pH value of the phosphate buffer is 12.0.

[0054] According to an embodiment of the present application, the phosphate is selected from at least one of disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate.

[0055] According to an embodiment of the present application, the second vacuum freeze-drying procedure is a non-elution drying procedure, specifically comprising: freezing control: maintaining at -45℃~-55℃ for 100~140 minutes; primary drying: maintaining at -30℃~-50℃ for 2800~3000 minutes; elution drying: maintaining at 10℃~30℃ for 0 minutes.

[0056] According to an embodiment of the present application, the second vacuum freeze-drying procedure is a non-elution drying procedure, specifically comprising: freezing control: maintaining at -50℃ for 120 minutes; primary drying: maintaining at -45℃ for 2880 minutes; elution drying: maintaining at 20℃ for 0 minutes.

[0057] According to an embodiment of the present application, the severe dehydration treatment is performed at a temperature of 100-120℃ and a pressure of -0.70 bar~-0.90 bar for 24-48 hours, for example, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours.

[0058] According to an embodiment of the present application, the severe dehydration treatment is performed at a temperature of 100-120℃ and a pressure of -0.80 bar for 48 hours.

[0059] According to an embodiment of the present application, the method comprises the following steps: S1: expressing full-length recombinant human collagen by using a Pichia pastoris expression system, and subjecting the full-length recombinant human collagen to endopeptidase treatment to obtain a recombinant human collagen stock solution; S2: freeze-drying the recombinant human collagen stock solution by using a first vacuum freeze-drying procedure (P1) to obtain a stock solution freeze-dried sample; S3: resolubilizing the freeze-dried stock solution to obtain a concentrated stock solution with a concentration of 10-50 mg / mL; S4: adding an assembly buffer to the concentrated stock solution at a volume ratio of 3:1, mixing uniformly to obtain a collagen assembly system, and standing at 30℃ to allow self-assembly of the collagen assembly system to form an assembly fiber; S5: freeze-drying the collagen assembly system by using a second vacuum freeze-drying procedure (P2) to obtain a procollagen sponge; and S6: subjecting the procollagen sponge to severe dehydration treatment to obtain the recombinant human collagen sponge. According to the method of the present application, a recombinant human collagen sponge with complete triple helix structure, high biological activity, low immunogenicity, high liquid absorption, high mechanical strength, and no risk of animal-derived virus contamination can be prepared; at the same time, the process route is simplified, the operation is convenient, the cost is reduced, and the quality of the obtained product is stable and batch uniform.

[0060] Recombinant human collagen sponge In a second aspect, the present application provides a recombinant human collagen sponge. According to embodiments of the present application, the recombinant human collagen sponge is prepared by the method of the first aspect of the present application. The recombinant human collagen sponge according to embodiments of the present application not only retains the complete triple helix structure, but also has excellent biological activity, low immunogenicity, high liquid absorption and high mechanical strength, and no risk of animal-derived virus contamination.

[0061] According to embodiments of the present application, the collagen yield of the recombinant human collagen sponge is ≥ 99%.

[0062] According to embodiments of the present application, the water absorption multiple of the recombinant human collagen sponge is 20-50 times of its own weight, for example, it can be 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or a range value of 22-50, 20-48 times between them.

[0063] According to embodiments of the present application, the water absorption speed of the recombinant human collagen sponge is 20-35 seconds, for example, it can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 seconds or a range value of 21-35 seconds, 22-35 seconds between them.

[0064] According to embodiments of the present application, the recombinant human collagen sponge is suitable for resisting a pulling force of 0.5N to 2N.

[0065] According to embodiments of the present application, the collagen in the recombinant human collagen sponge has a complete triple helix structure.

[0066] According to embodiments of the present application, the recombinant human collagen sponge has no risk of animal-derived virus.

[0067] Use In a third aspect, the present application provides use of the recombinant human collagen sponge of the second aspect of the present application in the preparation of a hemostatic material, a wound dressing or a tissue engineering scaffold.

[0068] The solutions of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0069] Example 1: Preparation of recombinant type I human collagen sponge (1) Preparation of recombinant human collagen type I: The recombinant human collagen type I was obtained by using the preparation method of recombinant human collagen type I described in CN119954936A, and the recombinant human collagen type I does not have a terminal peptide. The amount of pepsin added in the supernatant of the culture product broken by pepsin was 100 U / L (100 U / L is equivalent to 1 / 10000 of the weight of the full-length recombinant human collagen protein), the enzyme treatment temperature was 4°C, and the enzyme treatment time was 24 hours. The electrophoresis result of the recombinant human collagen type I is shown in FIG. 1, the sequence of the recombinant human collagen type I is shown in SEQ ID NO: 1 (COL1A1) and SEQ ID NO: 2 (COL1A2). Figure 1

[0070] ​ (2) Freeze-drying of the recombinant human collagen type I: The prepared recombinant human collagen type I stock solution (batch number JHM0820250201) was slowly poured into a freeze-drying tray, avoiding the introduction of air bubbles, and a freeze-drying program was preset, and the freeze-drying process was started. The first vacuum freeze-drying program is shown in Table 1: Table 1: Parameters of the first vacuum freeze-drying program (P1) used in the present application

[0071] (3) Resuspension and self-assembly of the recombinant human collagen type I: The moisture content of the recombinant human collagen type I stock solution freeze-dried sample was detected, and the amount of purified water needed to be added to set the collagen concentration was calculated. A certain amount of recombinant human collagen type I stock solution freeze-dried sample was taken, purified water was added to the set collagen concentration, and it was placed at 2-8°C for 48 hours until the freeze-dried sample was completely dissolved. An appropriate amount was taken and added to 10 mM hydrochloric acid solution, mixed well, and the resuspended collagen (concentrated stock solution) concentration was determined. The resuspended collagen concentration value was 4 / 3 times the target collagen sponge concentration. The assembly buffer was added to the concentrated stock solution at a volume ratio of concentrated stock solution: assembly buffer = 3:1, mixed quickly, and the collagen assembly system was prepared; the assembly temperature was 30°C, and it was placed for 16 hours to obtain the recombinant human collagen type I assembly fiber. The composition of the assembly buffer: 40 mM disodium hydrogen phosphate, pH 12.0.

[0072] (4) Preparation of the recombinant human collagen type I pre-sponge: The recombinant human collagen type I assembly fiber was slowly injected into a vacuum bottle, vacuumized to -0.5 bar, and maintained for 30 minutes. Then the vacuum-treated assembly fiber was slowly poured into a freeze-drying tray, avoiding the introduction of air bubbles, a second vacuum freeze-drying program (P2) as shown in Table 2 was preset, the freeze-drying process was started, and the recombinant human collagen type I pre-sponge was obtained.

[0073] Table 2: Parameters of the second vacuum freeze-drying program (P2) used in the present application

[0074] (5) Severe dehydration of the recombinant human collagen type I sponge: The temperature of the vacuum drying oven was set to 110°C, the pre-collagen sponge obtained in step (4) was placed in the drying oven, vacuumized to -0.80 bar, and maintained for 48 hours to complete the severe dehydration treatment, and the recombinant human collagen type I sponge was obtained. After the sponge was taken out, it was quickly placed in a plastic sealed bag for storage, and subsequent performance tests such as moisture content, tensile strength, liquid absorption rate, liquid absorption multiple, and enzyme digestion experiment were carried out. The appearance and morphology of the recombinant human collagen type I sponge prepared in the present application are shown in Figure 2 .

[0075] Example 2: (1) Preparation of recombinant human collagen type III; the recombinant human collagen type III obtained by the preparation method of recombinant human collagen type III described in CN119874880A does not have a terminal peptide. The amount of pepsin added in the culture product broken supernatant is 100 U / L (100 U / L is equivalent to 1 / 10000 of the weight of the full-length recombinant human collagen protein), the temperature of enzyme cutting treatment is 4°C, and the enzyme cutting treatment time is 24 hours. The electrophoresis result of the recombinant human collagen type III is shown in Figure 3 , and the sequence of the recombinant human collagen type III is shown in SEQ ID NO: 3 (COL3A1).

[0076] (2) Freeze-drying of recombinant human collagen type III: The recombinant human collagen type III stock solution (batch number JHM0920250301) was slowly poured into the freeze-drying tray, avoiding the introduction of air bubbles, and a freeze-drying program was preset, and the freeze-drying process was started. The freeze-drying program is shown in Table 1.

[0077] (3) Resuspension and self-assembly of recombinant human collagen type III: The moisture content of the recombinant human collagen type III stock solution freeze-dried sample was detected, and the amount of purified water to be added to set the collagen concentration was calculated. A certain amount of recombinant human collagen type III stock solution freeze-dried sample was taken, purified water was added to set the collagen concentration, and it was placed at 2-8°C for 48 hours until the freeze-dried sample was completely dissolved. An appropriate amount was taken and added to 10 mM hydrochloric acid solution, mixed well, and the resuspended collagen (concentrated stock solution) concentration was determined. The resuspended collagen concentration value was 4 / 3 times the target collagen sponge concentration, which refers to the concentration of collagen in the final sponge. The assembly buffer was added to the concentrated stock solution at a volume ratio of concentrated stock solution: assembly buffer = 3:1, mixed quickly, and the collagen assembly system was obtained; the assembly temperature was 30°C, and it was placed for 16 hours to obtain recombinant human collagen type III assembly fibers. The composition of the assembly buffer: 40 mM disodium hydrogen phosphate, pH 12.0.

[0078] (4) Preparation of recombinant human collagen type III pre-sponge: The recombinant human collagen type III assembly fibers were slowly injected into a vacuum bottle, vacuumized to -0.5 bar, and maintained for 30 minutes. Then the vacuum-treated assembly fibers were slowly poured into the freeze-drying tray, avoiding the introduction of air bubbles, a second vacuum freeze-drying program (P2) shown in Table 2 was preset, the freeze-drying process was started, and the recombinant human collagen type III pre-sponge was obtained.

[0079] (5) Severe dehydration of recombinant human collagen type III sponge: The temperature of the vacuum drying oven was set to 110°C, the pre-collagen sponge obtained in step (4) was placed in the drying oven, vacuumized to a drying oven pressure of -0.80 bar, and maintained for 48 hours to complete the severe dehydration treatment, and the recombinant human collagen type III sponge was obtained. The sponge was taken out and quickly placed in a plastic sealed bag, and the moisture, tensile strength, liquid absorption rate and liquid absorption multiple, enzyme digestion experiment, etc. were detected. The recombinant human collagen type III sponge prepared by the present application is shown in Figure 4 .

[0080] Comparative Example 1: Preparation of recombinant human collagen type I sponge by assembling first and then concentrating Preparation method: DS1: Take the appropriate amount of recombinant human collagen type I (the recombinant human collagen type I does not have a terminal peptide) stock solution in Example 1, add it to a beaker, number ① (type I), and start stirring. According to the volume ratio of 3:1 of the recombinant human collagen type I stock solution to the self-assembly buffer (40 mM Na2HPO4, pH 12.0), add the self-assembly buffer to the recombinant human collagen type I stock solution No. ①, continue stirring for 10 min, and verify the pH value of 7.0-7.2 to obtain a self-assembly system; DS2: Place the self-assembly system in a 30°C constant temperature box and stand for 16 h to obtain self-assembled fibers; DS3: Centrifuge the self-assembled fibers at 9000 rpm (8900*G, Thermo ST 16R table centrifuge) for 30 min, and take the precipitate to obtain high-concentration self-assembled fibers; DS4: According to the concentration and volume of the supernatant after centrifugation in DS3, preliminarily calculate the concentration of the self-assembled fibers after centrifugation; DS5: According to the target collagen sponge concentration, add water to prepare, stir well, and verify the collagen concentration; DS6: According to the sponge specification requirements, the above mixed system is loaded into different molds; DS7: Vacuum freeze-drying; DS8: Freeze-dried product is placed in a vacuum drying box for DHT (-0.80 bar, 110°C, 48 h) to obtain a recombinant human collagen type I sponge.

[0081] Preparation of recombinant human collagen type III sponge by assembling first and then concentrating Preparation method: DS1: Take the appropriate amount of recombinant human collagen type III (the recombinant human collagen type III does not have a terminal peptide) stock solution in Example 2, add it to a beaker, number ② (type III), and start stirring. According to the volume ratio of 3:1 of the recombinant human collagen type III stock solution to the self-assembly buffer (40 mM Na2HPO4, pH 12.0), add the self-assembly buffer to the recombinant human collagen type III stock solution No. ②, continue stirring for 10 min, and verify the pH value of 7.0-7.2 to obtain a self-assembly system; DS2: Place the self-assembly system in a 30°C constant temperature box and stand for 16 h to obtain self-assembled fibers; DS3: Centrifuge the self-assembled fibers at 9000 rpm (8900*G, Thermo ST 16R table centrifuge) for 30 min, and take the precipitate to obtain high-concentration self-assembled fibers; DS4: According to the concentration and volume of the supernatant after centrifugation in DS3, preliminarily calculate the concentration of the self-assembled fibers after centrifugation; DS5: According to the target collagen sponge concentration, add water to prepare, stir well, and verify the collagen concentration; DS6: Put the mixed system into different molds according to sponge specifications; DS7: Vacuum freeze-drying; DS8: Put the freeze-dried product into a vacuum drying oven for DHT (-80 bar, 110°C, 48h) to obtain the recombinant type III human collagen sponge.

[0082] Comparative Example 3: The experimental process was basically the same as that of Example 1, except that the general freeze-drying procedures P1-1 and P2-1 shown in Tables 3 and 4 were used to prepare the recombinant type I human collagen sponge. The reconstitution speed of the freeze-dried stock sample, the water absorption multiple of the collagen sponge, and the water absorption speed were shown in Table 5.

[0083] Table 3: General vacuum freeze-drying procedure (P1-1) parameters used in the comparative example

[0084] Table 4: General vacuum freeze-drying procedure (P2-1) parameters used in the comparative example

[0085] Table 5: Effect of different freeze-drying procedures on the performance of recombinant type I human collagen sponge

[0086] As can be seen from Table 5, under the same collagen concentration, the recombinant type I human collagen sponge prepared by using the freeze-drying procedures P1 and P2 has a faster reconstitution speed, a higher water absorption multiple, and a faster water absorption speed than that prepared by using the general freeze-drying procedures P1-1 and P2-1.

[0087] Comparative Example 4: The experimental process was basically the same as that of Example 2, except that the general freeze-drying procedures P1-1 and P2-1 shown in Tables 3 and 4 were used to prepare the recombinant type III human collagen sponge. The reconstitution speed of the freeze-dried stock sample, the water absorption multiple of the collagen sponge, and the water absorption speed were shown in Table 6.

[0088] Table 6: Recombinant type III human collagen sponge prepared by using 20 mg / mL recombinant type III human collagen concentration according to freeze-drying procedures P1, general freeze-drying procedures P1-1, freeze-drying procedures P2, and general freeze-drying procedures P2-1.

[0089] Table 6: Effect of different freeze-drying procedures on the performance of recombinant type III human collagen sponge

[0090] As can be seen from Table 6, under the same collagen concentration, the recombinant human collagen type III sponge prepared by using the freeze-drying procedures P1 and P2 has a faster reconstitution speed, a higher water absorption multiple, and a faster water absorption speed than the general freeze-drying procedures P1-1 and P2-1.

[0091] Test Example 1: Liquid absorption experiment of recombinant human collagen sponge Liquid absorption experiment: Take about 20 mg of the recombinant human collagen type I sponge prepared in Example 1, the recombinant human collagen type III sponge prepared in Example 2, the recombinant human collagen type I sponge prepared in Comparative Example 1, and the recombinant human collagen type III sponge prepared in Comparative Example 2, respectively, and accurately weigh them, and record them as m1. Immersed in a beaker containing 20℃±1℃ water, gently immerse in water until completely wet, and all air is removed, and attention is paid not to be damaged. After the water is absorbed, a corner is gently clamped with a small clamp, taken out of the water, and the clamp is held lightly on the water surface to drain for 1 min, and then weighed again, recorded as m2, and calculated according to the following formula. A total of 5 samples were randomly selected, and the average value was reported as the water absorption multiple: A=(m2-m1) / m1 In the formula, A: water absorption multiple of the sample; m1: mass before wetting, unit: gram (g); m2: mass after wetting, unit: gram (g). The liquid absorption experiment results (water absorption multiple, water absorption speed) of the recombinant human collagen type I sponge prepared in Example 1 and the recombinant human collagen type III sponge prepared in Example 2 are shown in Table 7: Table 7: Liquid absorption performance of the recombinant human collagen sponge prepared in Example

[0092] As can be seen from Table 7, under the same collagen concentration and the same preparation conditions, the recombinant human collagen type III sponge prepared in Example 2 has a higher water absorption multiple and a relatively faster water absorption speed than the recombinant human collagen type I sponge prepared in Example 1.

[0093] The liquid absorption experiment results (water absorption multiple, water absorption speed) of the recombinant human collagen type I sponge prepared in Comparative Example 1 and the recombinant human collagen type III sponge prepared in Comparative Example 2 are shown in Table 8: Table 8: Liquid absorption performance of the recombinant human collagen sponge prepared in Comparative Example

[0094] *Water absorption speed (s): refers to the time required for liquid absorption experiment, from the time when the sponge is put into 20℃±1℃ water, gently immerse in water until completely wet, and absorb water.

[0095] Comparing Table 7 with Table 8, the water absorption multiple and water absorption speed of the recombinant type I / III human collagen sponge prepared in Example 1 are higher and faster, respectively, and the water absorption multiple and water absorption speed of the recombinant type I human collagen sponge prepared in Comparative Example 1 are lower and slower, respectively; the water absorption multiple and water absorption speed of the recombinant type III human collagen sponge prepared in Example 2 are higher and faster, respectively, and the water absorption multiple and water absorption speed of the recombinant type III human collagen sponge prepared in Comparative Example 2 are lower and slower, respectively. This indicates that the recombinant human collagen sponge prepared by the concentration-assembly process has a higher water absorption multiple and a faster water absorption speed compared with the assembly-concentration process.

[0096] Test Example 2: Tensile test of recombinant human collagen sponge Tensile test: The recombinant type I human collagen sponge prepared in Example 1, the recombinant type III human collagen sponge prepared in Example 2, the recombinant type I human collagen sponge prepared in Comparative Example 1 and the recombinant type III human collagen sponge prepared in Comparative Example 2 were cut into 1 cm wide strips, one end was fixed, and the other end was subjected to a tensile force of 0.5 N, maintained for 1 min, and whether the sample was broken was observed and reported. The tensile force was continuously increased to 1 N and 2 N, and whether the sample was broken was observed. The results of the recombinant type I human collagen sponge prepared in Example 1 and the recombinant type III human collagen sponge prepared in Example 2 are shown in Table 9 (the data in the table is the maximum tensile force that the recombinant human collagen sponge can withstand): Table 9: Mechanical properties (tensile force resistance) of recombinant human collagen sponge prepared in Example

[0097] As can be seen from the data in Table 9, under the same collagen concentration and the same preparation conditions, the tensile resistance of the recombinant type I human collagen sponge is equivalent to that of the recombinant type III human collagen sponge.

[0098] (2) The tensile test process of the recombinant type I human collagen sponge prepared in Comparative Example 1 and the recombinant type III human collagen sponge prepared in Comparative Example 2 is described in Test Example 2, and the test results are shown in Table 10.

[0099] Table 10: Mechanical properties (tensile force resistance) of recombinant human collagen sponge prepared in Comparative Example

[0100] Comparing the tensile strength of the recombinant I and recombinant III collagen sponges in Table 9 and Table 10, the recombinant I collagen sponge prepared in Example 1 has a higher tensile strength, and the recombinant I collagen sponge prepared in Comparative Example 1 has a lower tensile strength; the recombinant III collagen sponge prepared in Example 2 has a higher tensile strength, and the recombinant III collagen sponge prepared in Comparative Example 2 has a lower tensile strength. This shows that the recombinant human collagen sponge prepared by the process of concentrating first and assembling later is tougher than the recombinant human collagen sponge prepared by the process of assembling first and concentrating later, and has a relatively higher tensile strength.

[0101] Test Example 3: Digestibility (resistance to enzymatic digestion) experiment of recombinant human collagen sponge Digestibility experiment: Take one piece of the recombinant I collagen sponge prepared in Example 1 and the recombinant III collagen sponge prepared in Example 2, the recombinant I collagen sponge prepared in Comparative Example 1 and the recombinant III collagen sponge prepared in Comparative Example 2, respectively, and immerse them in a beaker containing water, gently to completely wet them with fingers, and make sure that all air is removed, and do not break them. Take them out, remove the excess water with filter paper, and put the wet samples into 150 mL stoppered flasks, which have been filled with 100 mL of preheated to 37℃±1℃ 1% pepsin (activity about 3000 U / mg) hydrochloric acid solution [c(HCl)=0.1 mol / L]. Shake them gently at 37℃±1℃ and about 150 r / min until completely digested. Repeat the operation twice. Report the average value of the three complete digestion times.

[0102] The sponge samples prepared in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 are all subjected to severe dehydration treatment (DHT) at 110℃ and -0.80 bar for 48 hours. Then, the resistance to enzymatic digestion experiment is carried out: digestion in a pepsin solution at 37℃, and observation at different time points. The results show that for the I collagen sponge, the product prepared in Example 1 needs 3 hours and 10 minutes for complete digestion, while the product prepared in Comparative Example 1 only needs 2 hours and 40 minutes; for the III collagen sponge, the product prepared in Example 2 needs 3 hours and 10 minutes for complete digestion, while the product prepared in Comparative Example 2 only needs 2 hours and 40 minutes. This shows that under the same DHT conditions, the sponge prepared by the process of concentrating first and assembling later has a longer resistance to enzymatic digestion time.

[0103] Test Example 4: Total collagen yield The total collagen yield of the recombinant type I human collagen sponge prepared in Comparative Example 1 was compared with that of the recombinant type I human collagen sponge prepared in Example 1, and the total collagen yield of the recombinant type III human collagen sponge prepared in Comparative Example 2 was compared with that of the recombinant type III human collagen sponge prepared in Example 2. The total collagen yield calculation process of Comparative Example 1 and Comparative Example 2: Take an appropriate amount of recombinant human collagen stock solution (hereinafter referred to as stock solution), stir at 200 rpm according to stock solution: assembly buffer (v / v ratio) = 3:1, slowly add assembly buffer (40 mM Na2HPO4, pH 12.0) to the recombinant human collagen stock solution, continue to stir for 10 min, then place the sample in a 30°C constant temperature box, stand for 16 h, and obtain the self-assembly system. Centrifuge the self-assembly system: 2-8°C, 9000 rpm, 30 min (centrifuge Thermo, ST16R), take the supernatant and precipitate respectively, record the supernatant volume, detect the collagen concentration in the supernatant, and calculate the self-assembly process yield according to the formula [1-(v1*c1) / (v0*c0)]*% (wherein v0: stock solution volume, ml; c0: stock solution concentration, mg / ml; v1: supernatant volume, ml; c1: collagen concentration in the supernatant, mg / ml;). The total collagen yield comparison and explanation are shown in Table 11.

[0104] Table 11: Effect of different preparation processes on the total yield of recombinant human collagen sponge

[0105] As can be seen from Table 11, the total collagen yield of the recombinant type I human collagen sponge prepared in Example 1 is higher, and the total collagen yield of the recombinant type I human collagen sponge prepared in Comparative Example 1 is lower; the total collagen yield of the recombinant type III human collagen sponge prepared in Example 2 is higher, and the total collagen yield of the recombinant type III human collagen sponge prepared in Comparative Example 2 is lower. This shows that the recombinant human collagen sponge prepared by first concentrating and then assembling has a higher total collagen yield than the recombinant human collagen sponge prepared by first assembling and then concentrating.

[0106] Test Example 5: SEM observation of recombinant human collagen sponge Take an appropriate amount of recombinant type I human collagen sponge prepared in Example 1 and recombinant type III human collagen sponge prepared in Example 2, recombinant type I human collagen sponge prepared in Comparative Example 1 and recombinant type III human collagen sponge prepared in Comparative Example 2, and spray gold treatment, and observe in a Hitachi SU-8010 type scanning electron microscope (Hitachi SU-8010, Hitachi, model SU-8010).

[0107] This test example exemplarily shows the scanning electron microscope results of the recombinant type III human collagen sponge prepared in Example 2, which is specifically shown inFigure 5 (50x) and Figure 6 (100x). The sponge was prepared from Figure 5 and Figure 6 It can be seen that the recombinant human collagen type III sponge prepared in Example 2 has a typical fibrous / pore structure.

[0108] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples, without contradiction.

[0109] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A method of preparing a recombinant human collagen sponge, characterized by, The application relates to a method for preparing a recombinant human collagen sponge, comprising the following steps: a concentration and assembly treatment is performed on a recombinant human collagen to obtain the recombinant human collagen sponge, wherein the recombinant human collagen does not have terminal peptides. The concentration and assembly treatment is achieved by performing a first freeze-drying treatment and a re-dissolving treatment on the recombinant human collagen to obtain a concentrated stock solution; 2. The method of claim 1, wherein, the concentrated stock solution is uniformly mixed with an assembly buffer to obtain a collagen assembly system; the collagen assembly system is subjected to a second freeze-drying treatment to obtain a procollagen sponge; the procollagen sponge is subjected to a severe dehydration treatment. The first freeze-drying treatment is performed in the presence of a first vacuum freeze-drying program; 3. The method of claim 2, wherein, Optionally, the concentration of the concentrated stock solution is 10-50 mg / mL; Optionally, before the second freeze-drying treatment, the collagen assembly system is further subjected to a standing treatment to obtain an assembly fiber; Optionally, the standing treatment is performed at 28-32 DEG C; Optionally, the volume ratio of the concentrated stock solution to the assembly buffer is 3:1; Optionally, the second freeze-drying treatment is performed in the presence of a second vacuum freeze-drying program; Optionally, the recombinant human collagen is at least one selected from a recombinant type I human collagen and a recombinant type III human collagen. The recombinant human collagen is obtained by performing a terminal peptide removal treatment on a full-length recombinant human collagen in advance; 4. The method of claim 1, wherein, Optionally, the terminal peptide removal treatment comprises an enzymatic treatment by using at least one selected from pepsin, trypsin and Lys-c enzyme; Optionally, the enzyme is added in an amount of 1 / 10000-1 / 100 of the weight of the full-length recombinant human collagen. The first vacuum freeze-drying program is a non-resolution drying program, and specifically comprises:

5. The method of claim 3, wherein, frozen control: maintaining at -40 DEG C to -50 DEG C for 100-140 minutes; primary drying: maintaining at -30 DEG C to -50 DEG C for 1900-2000 minutes; resolution drying: maintaining at 10 DEG C to 30 DEG C for 0 minute; preferably, frozen control: maintaining at -45 DEG C for 120 minutes; primary drying: maintaining at -40 DEG C for 1920 minutes; resolution drying: maintaining at 20 DEG C for 0 minute. The assembly buffer is a 40 mM phosphate buffer; 6. The method of claim 2, wherein, Optionally, the pH value of the phosphate buffer is 11.0-12.0; Optionally, the phosphate is at least one selected from disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate and potassium dihydrogen phosphate. The second vacuum freeze-drying program is a non-resolution drying program, and specifically comprises:

7. The method of claim 3, wherein, frozen control: maintaining at -45 DEG C to -55 DEG C for 100-140 minutes; primary drying: maintaining at -30 DEG C to -50 DEG C for 2800-3000 minutes; resolution drying: maintaining at 10 DEG C to 30 DEG C for 0 minute; preferably, frozen control: maintaining at -50 DEG C for 120 minutes; primary drying: maintaining at -45 DEG C for 2880 minutes; resolution drying: maintaining at 20 DEG C for 0 minute. The severe dehydration treatment is performed at a temperature of 100-120 DEG C and a pressure of -0.70 bar to -0.90 bar for 24-48 hours.

8. The method of claim 2, wherein, ​ 9. A recombinant human collagen sponge, characterized in that, obtained by the method of any one of claims 1-8.

10. The recombinant human collagen sponge according to claim 9, wherein, The collagen yield of the recombinant human collagen sponge is ≥ 99%; Optionally, the water absorption multiple of the recombinant human collagen sponge is 20-50 times of its own weight. Optionally, the water absorption speed of the recombinant human collagen sponge is 20-35 seconds. Optionally, the recombinant human collagen sponge is suitable for resisting a tensile force of 0.5N to 2N.

11. Use of the recombinant human collagen sponge of claim 9 or 10 in the preparation of a hemostatic material, a wound dressing or a tissue engineering scaffold.

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