Sterilizing and filtering process of collagen

Abstract

The invention provides a degerming and filtering process of collagen. According to the method disclosed by the invention, filtration and sterilization process parameters and conditions of the collagen are scientifically screened, and the influence of a plurality of parameters on collagen activity maintenance in the process is systematically studied. The sterilization method obtained through optimization is easy and convenient to operate, economical, environmentally friendly and good in repeatability, and damage of dry heat sterilization, moist heat sterilization, irradiation sterilization and the like to the structure and activity of the collagen is avoided. In a word, the method provided by the invention not only can ensure the sterilization effect, but also can maintain the three-dimensional structure of the collagen.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a collagen sterilization and filtration process. Background Art

[0002] Collagen is an ideal biomedical material with a unique triple helix structure and the properties of high tensile strength, low antigenic activity, low irritation and low cytotoxicity. It can promote cell growth and adhesion, and synergize with new cells and tissues to repair trauma.

[0003] In the medical field, sterile collagen can be prepared into medical dressings for hemostasis and burn trauma treatment. Collagen can pave the way for the migration and proliferation of epidermal cells, provide good nutrition, absorb tissue permeate, create a good wound microenvironment, and facilitate the proliferation and repair of epithelial cells, thereby promoting wound healing. It can be used for the treatment of burns and trauma; it can also be used as a tissue engineering scaffold material to provide a good living space and microenvironment for seed cells. Cells can grow according to the prefabricated three-dimensional scaffold and secrete new extracellular matrix, and finally produce tissues or organs with a certain shape and three-dimensional structure in vitro. In the field of medical beauty, collagen can be injected into facial tissue as a tissue filler to play a supporting and filling role, thereby achieving the purpose of correcting wrinkles.

[0004] Collagen was approved as a filler in the medical aesthetics field earlier than hyaluronic acid, but its clinical application progress is slower than hyaluronic acid. One of the important factors is that the collagen structure is easily damaged. Traditional sterilization methods such as moist heat sterilization or irradiation sterilization will cause the collagen triple helix structure to be damaged to varying degrees and lose its biological activity. The sterilization process of collagen has always been a major problem in this field, which has greatly limited its widespread application.

[0005] There is an urgent need in this field to develop a collagen sterilization process that is simple to operate, economical, environmentally friendly and reproducible. Summary of the invention

[0006] The purpose of one aspect of the present invention is to provide a sterilization process for collagen, which comprises the following steps: taking a certain amount of acid solution of a certain concentration and putting it into a beaker, weighing a certain amount of collagen, cutting it into several pieces with sterilized scissors, and then slowly adding it to the beaker, stirring it at a certain temperature until it is completely dissolved, and standing to defoam. When the bubbles disappear, filter the collagen solution into a clean beaker to obtain a filtrate. Take the above filtrate, adjust the pH value of the filtrate with an alkali solution, and then pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product.

[0007] In a preferred embodiment of the present invention, the acid solution is selected from any one of phosphoric acid, hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, fumaric acid, oxalic acid, formic acid, acetic acid, propionic acid, butyric acid, tartaric acid, citric acid, and malic acid, or a combination thereof.

[0008] In a preferred embodiment of the present invention, the concentration of the acid solution is 0.01M-0.1M, preferably 0.01M-0.05M.

[0009] In a preferred embodiment of the present invention, the concentration of collagen dissolved in the acid solution is 0.5-40 mg / mL, preferably 1-25 mg / mL, and more preferably 1-10 mg / mL.

[0010] In a preferred embodiment of the present invention, the stirring temperature is 5-45°C, preferably 10-37°C, more preferably 15-25°C.

[0011] In a preferred embodiment of the present invention, the filtration is primary filtration or secondary filtration.

[0012] In a preferred embodiment of the present invention, the pore size of the filter membrane used for filtration is 0.22 μm or 0.45 μm.

[0013] In a preferred embodiment of the present invention, the alkali solution for adjusting the pH value of the filtrate is selected from any one of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, meglumine or a combination thereof.

[0014] In a preferred embodiment of the present invention, the concentration of the alkali solution for adjusting the pH value of the filtrate is 0.01M-0.5M, preferably 0.01M-0.1M, and more preferably 0.01M-0.05M.

[0015] In a preferred embodiment of the present invention, the pH of the filtrate is adjusted to 4-8, preferably 4-7, and more preferably 4-5 with an alkali solution.

[0016] In a preferred embodiment of the present invention, the freeze-drying process is:

[0017] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0018] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M hydrochloric acid solution and put it into a beaker, weigh a certain amount of collagen, cut it into several pieces with sterilized scissors, and then slowly add it to the beaker, stir at 15-25°C until it is completely dissolved, let it stand to defoam, and control the collagen concentration to 1-25mg / mL. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0019] The freeze-drying process is as follows:

[0020] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0021] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M hydrochloric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 25mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0022] The freeze-drying process is as follows:

[0023] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0024] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M hydrochloric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 10mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0025] The freeze-drying process is as follows:

[0026] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0027] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M hydrochloric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 1mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0028] The freeze-drying process is as follows:

[0029]

[0030]

[0031] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M hydrochloric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 10mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 15°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0032] The freeze-drying process is as follows:

[0033] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0034] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M phosphoric acid solution and put it into a beaker, weigh a certain amount of collagen, cut it into several pieces with sterilized scissors, and then slowly add it to the beaker, stir at 15-25°C until it is completely dissolved, let it stand to defoam, and control the collagen concentration to 1-25mg / mL. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0035] The freeze-drying process is as follows:

[0036] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0037] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M phosphoric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 25mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0038] The freeze-drying process is as follows:

[0039] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0040] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M phosphoric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 10mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0041] The freeze-drying process is as follows:

[0042] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0043] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M phosphoric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 1mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0044] The freeze-drying process is as follows:

[0045] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0046] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M phosphoric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 10mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 15°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0047] The freeze-drying process is as follows:

[0048] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0049] Another object of the present invention is to provide the use of the collagen sterilization process of the present invention in the sterilization of collagen.

[0050] The collagen sterilization process of the present invention comprises the following steps: taking a certain amount of acid solution of a certain concentration and putting it into a beaker, weighing a certain amount of collagen, cutting it into several pieces with sterilized scissors, and then slowly adding it into the beaker, stirring it at a certain temperature until it is completely dissolved, and standing it for defoaming. When the bubbles disappear, the collagen solution is filtered into a clean beaker to obtain a filtrate. The filtrate is taken, the pH value of the filtrate is adjusted with an alkali solution, and then poured into a freeze-drying tray, and freeze-dried to obtain a sterile collagen dry product.

[0051] In a preferred embodiment of the present invention, the acid solution is selected from any one of phosphoric acid, hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, fumaric acid, oxalic acid, formic acid, acetic acid, propionic acid, butyric acid, tartaric acid, citric acid, and malic acid, or a combination thereof.

[0052] In a preferred embodiment of the present invention, the concentration of the acid solution is 0.01M-0.1M, preferably 0.01M-0.05M.

[0053] In a preferred embodiment of the present invention, the concentration of collagen dissolved in the acid solution is 0.5-40 mg / mL, preferably 1-25 mg / mL, and more preferably 1-10 mg / mL.

[0054] In a preferred embodiment of the present invention, the stirring temperature is 5-45°C, preferably 10-37°C, more preferably 15-25°C.

[0055] In a preferred embodiment of the present invention, the filtration is primary filtration or secondary filtration.

[0056] In a preferred embodiment of the present invention, the pore size of the filter membrane used for filtration is 0.22 μm or 0.45 μm.

[0057] In a preferred embodiment of the present invention, the alkali solution for adjusting the pH value of the filtrate is selected from any one of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, meglumine or a combination thereof.

[0058] In a preferred embodiment of the present invention, the concentration of the alkali solution for adjusting the pH value of the filtrate is 0.01M-0.5M, preferably 0.01M-0.1M, and more preferably 0.01M-0.05M.

[0059] In a preferred embodiment of the present invention, the pH of the filtrate is adjusted to 4-8, preferably 4-7, and more preferably 4-5 with an alkali solution.

[0060] In a preferred embodiment of the present invention, the freeze-drying process is:

[0061] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0062] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M hydrochloric acid solution and put it into a beaker, weigh a certain amount of collagen, cut it into several pieces with sterilized scissors, and then slowly add it to the beaker, stir at 15-25°C until it is completely dissolved, let it stand to defoam, and control the collagen concentration to 1-25mg / mL. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0063] The freeze-drying process is as follows:

[0064] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0065] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M hydrochloric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 25mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0066] The freeze-drying process is as follows:

[0067] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0068] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M hydrochloric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 10mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0069] The freeze-drying process is as follows:

[0070] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0071] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M hydrochloric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 1mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0072] The freeze-drying process is as follows:

[0073]

[0074]

[0075] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M hydrochloric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 10mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 15°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0076] The freeze-drying process is as follows:

[0077] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0078] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M phosphoric acid solution and put it into a beaker, weigh a certain amount of collagen, cut it into several pieces with sterilized scissors, and then slowly add it to the beaker, stir at 15-25°C until it is completely dissolved, let it stand to defoam, and control the collagen concentration to 1-25mg / mL. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0079] The freeze-drying process is as follows:

[0080] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0081] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M phosphoric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 25mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0082] The freeze-drying process is as follows:

[0083] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0084] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M phosphoric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 10mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0085] The freeze-drying process is as follows:

[0086] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0087] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M phosphoric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 1mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 25°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0088] The freeze-drying process is as follows:

[0089]

[0090]

[0091] In a preferred embodiment of the present invention, the specific sterilization process of collagen is as follows: take a certain amount of 0.01M phosphoric acid solution and put it into a beaker, weigh a certain amount of collagen so that the final concentration of collagen is 10mg / ml, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker, stir at 15°C until it is completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product,

[0092] The freeze-drying process is as follows:

[0093] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h - .

[0094] Another object of the present invention is to provide a collagen product prepared according to the sterilization process of the present invention.

[0095] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0096] Compared with the prior art, the present invention has the following beneficial effects: the present invention scientifically screens the parameters and conditions of the filtration sterilization process of collagen, and systematically studies the influence of multiple parameters on the structure and activity of collagen in the process. The sterilization method of the present invention is easy to operate, economical and environmentally friendly, has good repeatability, and avoids the damage to the structure and activity of collagen by dry heat sterilization, moist heat sterilization, and irradiation sterilization. In short, the method of the present invention can maintain the three-dimensional structure of collagen while ensuring the sterilization effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 This is a diagram showing the effects of radiation sterilization at different doses in Comparative Example 1.

[0098] Figure 2 This is the electrophoresis effect diagram of the sample in Example 1.

[0099] Figure 3 This is the electrophoresis effect diagram of the sample in Example 2.

[0100] Figure 4 This is the electrophoresis effect diagram of the sample in Example 3.

[0101] Figure 5This is the electrophoresis effect diagram of the sample in Example 4.

[0102] Figure 6 This is the electrophoresis effect diagram of the sample in Example 5.

[0103] Figure 7 This is the electrophoresis effect diagram of the sample in Example 6.

[0104] Figure 8 This is the appearance characteristics diagram of sample No. 27 in Example 6.

[0105] Fig. 9 This is the appearance characteristics diagram of sample No. 28 in Example 6.

[0106] Fig.10 This is the electrophoresis effect diagram of the sample in Example 7.

[0107] Fig.11 This is the circular dichroism spectrum of the sample in Example 8.

[0108] Fig.12 This is a diagram showing the sterilization qualification test effect of Example 9. DETAILED DESCRIPTION

[0109] To make the purpose, technical solution and advantages of the present invention more clear, the embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other arbitrarily without conflict.

[0110] The electrophoresis experiment refers to YY / T 1805.2-2021 "Collagen for Tissue Engineering Medical Device Products Part 2: Type I Collagen Molecular Weight Detection-Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Method". The specific method is as follows: (1) Sample denaturation treatment: weigh an appropriate amount of the test sample (collagen content is about 5 mg), add 8 mL of water, 5×2 mL of SDS sample buffer, mix, heat in a 100°C water bath for 5 min to 10 min to denature the protein, and centrifuge (650g, 5 min to 10 min); (2) Preparation of electrophoresis gel: prepare separation gel and concentration gel in appropriate proportions, insert a comb and set aside; (3) Sample loading: after the concentration gel solution is polymerized, carefully pull out the sample comb, fill the front and back slots of the electrophoresis tank with electrode buffer, add 5 μL of protein marker, 10 μL of type I collagen reference substance and 10 μL of the sample to be tested to the sample loading hole; (4) Electrophoresis: add electrophoresis buffer into the electrophoresis tank, install the gel plate, use constant voltage electrophoresis, the initial voltage is 80 V, and adjust to 120 V when entering the separation gel. At the end of electrophoresis, the electrophoresis front should not leave the separation gel; (5) Staining: After the electrophoresis is completed, remove the film and place it in the staining solution for 1 to 2 hours with gentle shaking, or remove it when a clear blue band can be seen; (6) Decolorization: After the staining, rinse the excess dye on the surface of the gel with water, then soak it in decolorizing solution for decolorization. Replace the decolorizing solution until the gel background is transparent.

[0111] The freeze-drying process of the embodiment of the present invention is:

[0112] Freezing process Temperature(℃) Setting time (min) Holding time (min) Vacuum degree(mbar) Pre-freeze -40 30 150 - A sublimation -5 30 720 0.1 Secondary sublimation 25 30 1320 0.1 Total duration - - 40h -

[0113] The secondary filter heads in the examples are: hydrophilic polyethersulfone syringe filter (PES, 0.45 μm) and sterilization grade hydrophilic polyethersulfone syringe filter (PES, 0.22 μm). Unless otherwise specified, the samples in the examples are filtered using secondary filtration.

[0114] Comparative Example 1 Irradiation Sterilization of Collagen

[0115] Take 0.05M PBS buffer and put it into a beaker. Weigh a certain amount of collagen, cut it into several pieces with sterilized scissors, and then slowly add it to the beaker to make the final concentration of collagen 10mg / mL. Stir it at room temperature until it is evenly dispersed, and let it stand to defoam until the bubbles disappear. The collagen solution prepared above was treated with irradiation sterilization. The results showed that compared with the non-irradiated group, the collagen in the irradiated group broke its chain, its appearance changed, and its degree of denaturation increased with the increase of irradiation dose. The results of different irradiation doses are shown in Figure 2. Figure 1 shown.

[0116] Comparative Example 2 Sterilization Filtration of Collagen

[0117] Take 0.05M PBS buffer and put it into a beaker. Weigh a certain amount of collagen, cut it into several pieces with sterilized scissors, and then slowly add it to the beaker to make the final concentration of collagen 10mg / mL. Stir it at room temperature until it is evenly dispersed, and let it stand to defoam until the bubbles disappear. Use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter. The results show that the filter membrane is blocked and it is difficult to complete the filtration.

[0118] Example 1 Sterile Filtration of Collagen

[0119] Preparation of phosphoric acid solution: Add 0.5 g of phosphoric acid to a 500 mL volumetric flask, dilute to the mark with ultrapure water, and prepare 0.01 M dilute phosphoric acid for later use.

[0120] Preparation of hydrochloric acid solution: Add 41.67 g of 12 mol / L concentrated hydrochloric acid into a 500 mL volumetric flask, dilute to the mark with ultrapure water, prepare 1 mol / L dilute hydrochloric acid for later use, and dilute 1 mol / L dilute hydrochloric acid stepwise to obtain 0.1 mol / L dilute hydrochloric acid and 0.01 mol / L dilute hydrochloric acid respectively.

[0121] Preparation of NaOH solution: weigh 20g of sodium hydroxide in a beaker, add ultrapure water to dissolve, transfer to a 500mL volumetric flask after cooling, make up to volume with ultrapure water, mix well, and prepare 1mol / L NaOH solution for use. Take 5g of 1mol / L NaOH solution in a 500mL volumetric flask, add ultrapure water to the mark to obtain 0.01mol / L NaOH solution; take 10g of 1mol / L NaOH solution in a 100mL volumetric flask, add ultrapure water to the mark to obtain 0.1mol / L NaOH solution.

[0122] Preparation of sample 1: Take 300mL of 0.01M dilute phosphoric acid and put it into a beaker, weigh 9g of collagen, cut it into several pieces with sterilized scissors, and then slowly add it to the beaker. The final concentration of collagen is 30mg / mL. Stir at 37°C until completely dissolved, and let it stand to defoam. When the bubbles disappear, use a syringe equipped with a 0.45μm+0.22μm secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Take the above filtrate, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product.

[0123] The preparation of the remaining samples refers to sample 1. Among them, samples 1 and 4 are lyophilized directly after the filtrate of the collagen solution is filtered, and samples 2-3 and 5-6 are lyophilized after the filtrate of the collagen solution is filtered and the pH is adjusted. The electrophoresis results of the lyophilized products of each sample are shown in Table 1 below.

[0124] Table 1

[0125]

[0126] The electrophoresis results are shown in Figure 2 The results showed that: ① there was little difference between the results of using phosphoric acid and hydrochloric acid as solvents; ② the activity of collagen could be partially restored after the filtrate was neutralized with alkali solution; ③ the concentration of alkali solution used to adjust the pH of the filtrate had an effect on the activity of collagen.

[0127] Example 2 Sterile Filtration of Collagen

[0128] The preparation of the samples refers to the above sample 1. The samples are all lyophilized after the filtrate of the collagen solution is filtered and the pH is adjusted. The electrophoresis results of the lyophilized products of each sample are shown in Table 2 below.

[0129] Table 2

[0130]

[0131] The electrophoresis results are shown in Figure 3 The results showed that: ① high concentration of hydrochloric acid as a solvent will destroy the activity of collagen, and ② lowering the concentration of collagen is beneficial to maintaining its activity.

[0132] Example 3 Sterile Filtration of Collagen

[0133] The preparation of each sample refers to the above sample 1. Among them, sample 10 is freeze-dried from the unfiltered acid solution, samples 11-13 are freeze-dried from the filtrate after the collagen solution is filtered, and samples 14-15 are freeze-dried from the filtrate after the collagen solution is filtered and the pH is adjusted. The electrophoresis results of the freeze-dried products of each sample are shown in Table 3 below.

[0134] Table 3

[0135]

[0136] The electrophoresis results are shown in Figure 4 The results showed that: ① further verification that adjusting the pH of the filtrate with alkaline solution can partially restore the activity of collagen, and ② the filtration process has little effect on collagen. In addition, it was observed that adjusting the pH to 4-5 relative to adjusting the pH to neutral can make the freeze-dried product more tough and better in appearance.

[0137] Example 4 Sterile Filtration of Collagen

[0138] The sample preparation refers to the above sample 1. Among them, samples 16, 18, 20, and 22 are lyophilized directly from the filtrate after the collagen solution is filtered, and samples 17, 19, 21, and 23 are lyophilized after the filtrate after the collagen solution is filtered and the pH is adjusted. The electrophoresis results of the lyophilized products of each sample are shown in Table 4 below.

[0139] Table 4

[0140]

[0141]

[0142] The electrophoresis results are shown in Figure 5 The results showed that: ① lowering the collagen concentration was beneficial to maintaining activity, and ② lowering the collagen dissolution temperature was beneficial to maintaining activity.

[0143] Example 5 Sterile Filtration of Collagen

[0144] The sample preparation refers to the above sample 1. Among them, sample 24 is freeze-dried from the unfiltered acid solution, sample 25 is freeze-dried from the filtrate after the collagen solution is filtered, and sample 26 is freeze-dried from the filtrate after the collagen solution is filtered and the pH is adjusted. The electrophoresis results of the freeze-dried products of each sample are shown in Table 5 below.

[0145] Table 5

[0146]

[0147] The electrophoresis results are shown in Figure 6 The results showed that: ① the activity of high-concentration collagen was lost significantly after dissolution, ② it was further verified that the filtration process had little effect on collagen, and ③ it was further verified that adjusting the pH of the filtrate with alkaline solution could partially restore the activity of collagen.

[0148] Example 6 Sterile Filtration of Collagen

[0149] The preparation of each sample refers to the above sample 1. All samples are lyophilized after the filtrate of the collagen solution is filtered and the pH is adjusted. The electrophoresis results of the lyophilized products of each sample are shown in Table 6 below.

[0150] Table 6

[0151]

[0152]

[0153] The electrophoresis results are shown in Figure 7 The results showed that there was little difference in the activity of collagen when using 0.01M phosphoric acid or 0.01M hydrochloric acid as solvent. In addition, the appearance of samples No. 27 and No. 28 were Figure 8 and Fig. 9 .

[0154] Example 7 Sterile Filtration of Collagen

[0155] Preparation of 0.01M acetic acid solution: Add 2.86mL of glacial acetic acid to a 500mL volumetric flask, dilute to the mark with ultrapure water, and prepare 0.1mol / L acetic acid solution for later use. Take 50mL of 0.1mol / L acetic acid solution in a 500mL volumetric flask, dilute to the mark with ultrapure water, and obtain 0.01mol / L acetic acid solution.

[0156] Preparation of 0.01M citric acid solution: weigh 1.05g citric acid into a beaker, add ultrapure water to dissolve, transfer to a 500mL volumetric flask after cooling, make up to volume with ultrapure water, mix well, and prepare 0.01mol / L citric acid solution for use.

[0157] The sample preparation process refers to sample 1, and each sample is a filtrate of the collagen solution filtered and then freeze-dried after adjusting the pH. The electrophoresis results of the freeze-dried products of each sample are shown in Table 7 below.

[0158] Table 7

[0159]

[0160] The electrophoresis results are shown in Fig.10 The results showed that acetic acid and citric acid were not suitable as solvents for sterilization filtration of collagen.

[0161] Example 8 Circular dichroism spectrum of collagen

[0162] Principle of circular dichroism spectroscopy to characterize the three-dimensional structure of collagen: The special right-handed superhelical structure of type I collagen gives it a special circular dichroism spectrum. Usually, the negative absorption peak around 194nm and the positive absorption peak around 220nm are the unique circular dichroism spectra of collagen. Studies have shown that when collagen is denatured or the triple helix structure is destroyed, its circular dichroism spectrum will show a red shift of the negative peak and the disappearance of the positive peak. The height of the positive absorption peak can also characterize the amount of triple helix structure.

[0163] Instrument reagents: glacial acetic acid (analytical grade), deionized water, Chirascan circular dichroism spectrometer

[0164] Steps: 1. Preparation of collagen solution: Use 0.5 mol / L acetic acid solution as solvent to prepare 10 mL of collagen sample solution with a concentration of 0.1 mg / mL, fully dissolve, balance for 12 hours, and centrifuge to obtain the supernatant.

[0165] 2. Instrument settings: Set the wavelength range to 190nm-260nm, the cuvette pathlength to 0.5mm, and the scanning speed to 0.5nm / s; the detection environment is 25℃, nitrogen. Use 0.5mol / L acetic acid solution as the blank control.

[0166] 3. Repeat the sample scan 3 times and the blank scan 2 times. Measure the blank control once each time you measure the sample.

[0167] 27, 28, 29 and collagen raw materials were selected for circular dichroism analysis. The results are shown in Fig.11 The results showed that sample No. 28 was the best, followed by sample No. 27, and sample No. 29 did not meet the requirements.

[0168] Example 9 Sterilization Compliance Test of Collagen

[0169] Collagen sponge sterility test

[0170] 1. Instruments and equipment: clean workbench, biological safety cabinet, pressure steam sterilizer, biochemical incubator

[0171] 2. Reagents and solutions: Thioglycollate fluid medium, tryptic soy bean broth (TSB), pH 7.0 sterile sodium chloride-peptone buffer

[0172] 3. Preparation of bacterial suspension: Inoculate fresh culture of Staphylococcus aureus into tryptic soytone liquid medium, culture at 30-35°C for 18-24 hours, and then dilute to a suitable concentration of bacterial suspension with sterile sodium chloride-peptone buffer at pH 7.0.

[0173] 4. Preparation before the test

[0174] 5. Clean workbench: Wipe the surface of the workbench with 75% alcohol cotton before the experiment. Turn on the UV light for half an hour, then turn off the UV light and turn on the fan for half an hour before entering the laboratory.

[0175] 6. Delivery of test items and equipment: Before the test, the required test items and equipment should be transferred to the transfer window and disinfected under ultraviolet light for more than 30 minutes.

[0176] 7. Test steps:

[0177] (1) Test solution: In a clean workbench, take 100 mL of thioglycollate fluid culture medium and tryptic soy liquid culture medium and inoculate 0.3 g of the test sample (prepared according to the sterilization process of sample No. 28) into each of the collagen sponge by direct inoculation method.

[0178] (2) Negative control: Take one bottle of 100 mL of thioglycollate fluid culture medium and one bottle of trypticase soy liquid culture medium as negative controls.

[0179] (3) Positive control: Take a bottle of 100 mL of thioglycollate fluid medium and add 0.3 g of the test sample. Add no more than 100 cfu of bacterial liquid in a biological safety cabinet.

[0180] 8. Cultivation and observation

[0181] The inoculated thioglycollate fluid culture medium tubes were cultured at 30-35°C for 14 days, the inoculated trypticase soytone liquid culture medium tubes were cultured at 20-25°C for 14 days, the negative control thioglycollate fluid culture medium group was cultured at 30-35°C for 14 days, the negative control trypticase soytone liquid culture medium group was cultured at 20-25°C for 14 days, and the positive control tubes were cultured at 30-35°C for no more than 5 days. During the culture period, observe and record whether there is bacterial growth every day, and fill in the inspection record sheet.

[0182] 9. Results

[0183] See Fig.12 The positive control group was turbid and the anaerobic layer disappeared, the test sample TSB group was clear, the test sample thioglycollate group was clear and the anaerobic layer was retained, the TSB negative control group was clear, the thioglycollate negative control group was clear and the anaerobic layer was retained, and the test samples met the requirements.

[0184] The sterilization qualification test results of the test samples prepared by referring to the sterilization method of samples No. 19, No. 21, No. 23 and No. 27 are the same as No. 28 above, and all meet the requirements.

[0185] Although the embodiments disclosed in the present invention are as above, the contents described are only embodiments adopted to facilitate understanding of the present invention and are not intended to limit the present invention. Any technician in the field to which the present invention belongs can make any modifications and changes in the form and details of the implementation without departing from the spirit and scope disclosed in the present invention, but the patent protection scope of the present invention shall still be subject to the scope defined in the attached claims.

Claims

1. A sterilization process for collagen, the sterilization process comprising the following steps: Take a certain amount of acid solution of a certain concentration and put it into a beaker, weigh a certain amount of collagen, cut it into several pieces with sterilized scissors, then slowly add it to the beaker, stir it at a certain temperature until it is completely dissolved, and let it stand to defoam. When the bubbles disappear, filter the collagen solution into a clean beaker to obtain a filtrate. Take the above filtrate, adjust the pH value of the filtrate with alkali solution, then pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product.

2. The sterilization process according to claim 1, characterized in that: The acid solution is selected from any one of phosphoric acid, hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, fumaric acid, oxalic acid, formic acid, acetic acid, propionic acid, butyric acid, tartaric acid, citric acid, and malic acid, or a combination thereof; the concentration of the acid solution is 0.01M-0.1M, preferably 0.01M-0.05M; the concentration of the collagen dissolved in the acid solution is 0.5-40mg / mL, preferably 1-25mg / mL, more preferably 1-10mg / mL; the dissolution temperature is 5-45°C, preferably 10-37°C, more preferably 15-25°C.

3. The sterilization process according to any one of claims 1 to 2, characterized in that: The filtration is primary filtration or secondary filtration, and the pore size of the filter membrane used in the filtration is 0.22 μm or 0.45 μm respectively.

4. The sterilization process according to any one of claims 1 to 3, characterized in that: The alkali solution for adjusting the pH value of the filtrate is selected from any one of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, meglumine or a combination thereof, and the concentration of the alkali solution for adjusting the pH value of the filtrate is 0.01M-0.5M, preferably 0.01M-0.1M, and more preferably 0.01M-0.05M.

5. The sterilization process according to any one of claims 1 to 4, characterized in that: The pH of the filtrate is adjusted to 4-8, preferably 4-7, more preferably 4-5 with alkaline solution.

6. The sterilization process according to any one of claims 1 to 5, characterized in that: The freeze-drying process is: 。 7. The sterilization process according to any one of claims 1 to 6, characterized in that: The specific process is as follows: take a certain amount of 0.01M hydrochloric acid solution and put it into a beaker, weigh a certain amount of collagen, cut it into several pieces with sterilized scissors, and then slowly add it to the beaker, stir at 15-25°C until it is completely dissolved, let it stand to defoam, and control the collagen concentration to 1-25mg / mL. After the bubbles disappear, use a syringe equipped with a 0.45um+0.22um secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product, The freeze-drying process is as follows: 。 8. The sterilization process according to any one of claims 1 to 6, characterized in that: The specific process is as follows: take a certain amount of 0.01M phosphoric acid solution and put it into a beaker, weigh a certain amount of collagen, cut it into several pieces with sterilized scissors, and then slowly add it to the beaker, stir at 15-25°C until it is completely dissolved, let it stand to defoam, and control the collagen concentration to 1-25mg / mL. After the bubbles disappear, use a syringe equipped with a 0.45um+0.22um secondary filter head to filter the collagen solution into a clean beaker to obtain a filtrate. Use 0.01M sodium hydroxide solution to adjust the pH of the obtained filtrate to 4-5. Take the above solution, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product, The freeze-drying process is as follows: 。 9. Use of the sterilization process according to any one of claims 1 to 8 for sterilizing collagen.

10. A collagen product, characterized in that: The product is prepared by the sterilization process described in any one of claims 1 to 8.

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