Injectable human collagen soft tissue filling material and preparation method thereof

Abstract

The present invention discloses an injectable human collagen soft tissue filling material and a preparation method thereof, belonging to the technical field of biomedical materials. The injectable human collagen soft tissue filling material comprises pretreated recombinant human collagen and an osmotic pressure regulator, and the injectable human collagen soft tissue filling material is in an isotonic state; the pretreatment of the recombinant human collagen is processed by isoelectric precipitation and centrifugal separation process; the preparation method thereof comprises filtering and sterilizing the raw materials, pretreatment, and homogenizing the pretreated recombinant human collagen and the osmotic pressure regulator. The injectable human collagen soft tissue filling material of the present invention is mainly human collagen, has a large molecular weight and a triple helix structure, and has a uniform particle size distribution, which can play a filling and supporting effect; the preparation process thereof is simple and convenient, without special processing technology, and will not introduce toxic and harmful residual substances, so that the product performance is stable.

Description

Technical Field

[0001] The invention relates to an injectable human collagen soft tissue filling material and a preparation method thereof, belonging to the technical field of biomedical materials. Background Art

[0002] The plastic surgery industry is developing rapidly with the continuous improvement of people's living standards. Breast augmentation, rhinoplasty, wrinkle removal, facial plastic surgery, etc. are all popular. The current demand for facial plastic surgery includes: removing wrinkles, improving facial contours, balancing scars or lips, repairing injured tissues, etc. Therefore, the market demand for soft tissue filling materials is high. Up to now, people have studied materials such as white wax, fat transplantation, silicone resin, and gel implants for soft tissue plastic surgery. Clinical applications have found that there are problems such as hardening, tumor-like masses, rapid absorption, and chronic inflammation. In recent years, the US FDA has successively approved several products for soft tissue filling. Its main ingredients are animal collagen, sodium hyaluronate, PMMA microspheres, PCL microspheres, PLA microspheres, etc., which have achieved good results. For some users, there are still redness, swelling, allergies, and rapid degradation at the use site.

[0003] Collagen, also known as collagen, is the most abundant and widely distributed group of hard proteins in vertebrates. It is an important material for the body structure of vertebrates and the most abundant protein in mammals. Collagen is composed of polysaccharide protein molecules and is the main protein component of connective tissue, accounting for 20-30% of the total protein in the body. About half of the collagen in the body is found in the skin, and collagen accounts for 70% of the dry weight of the dermis and tendons, equivalent to 6% of the body weight. Recombinant human collagen is obtained by transforming the human collagen DNA sequence into tissue engineering bacteria or cells, and then fermenting, separating, extracting and purifying it. It fundamentally solves the hidden dangers of virus extraction of collagen from animals.

[0004] There are similar products on the market at this stage, such as Taiwan Shuangmei and Changchun Feiman, but there are some major problems: 1) The raw materials are extracted from animals, which may cause animal-borne zoonotic diseases and viruses, as well as immunogenic problems, which may cause more clinical adverse reactions in the human body, including nodules, granulomas, allergies, etc.; 2) Collagen extracted from animal sources must use pre-treatment processes including defatting, deproteinization, decellularization, and virus inactivation, and needs to be treated with sensitive chemical reagents such as isopropanol and ethanol, and its chemical residues are difficult to control; 3) It is difficult to control the target protein, such as The product is a mixture of type I and type III collagen; in addition, Shanxi Jinbo's product uses collagen to prepare hyaluronic acid injections, which also has some problems: 1) The collagen molecular weight of the product is small and the collagen concentration is low, which causes it to mainly supplement collagen without filling and supporting effects; 2) The raw material is recombinant humanized collagen, and its sequence is mainly a full-length or partial sequence loop structure of the human collagen sequence, and telomere resection has been performed, which has structural and performance differences from naturally existing human collagen; the product adopts a freeze-drying process and needs to be dissolved in physiological saline before use. Summary of the invention

[0005] In order to address at least one problem existing in the above-mentioned prior art, the present invention provides an injectable human collagen soft tissue filling material and a preparation method thereof. The injectable human collagen soft tissue filling material is mainly based on recombinant human collagen, has a large molecular weight and a triple helix structure, and has a uniform particle size distribution, which can play a filling and supporting effect; its preparation process is simple and convenient, does not require any special processing process, and will not introduce toxic and harmful residual substances, thereby making the product performance stable.

[0006] In order to achieve the above-mentioned object, the present invention adopts the following technical scheme: an injectable human collagen soft tissue filling material, comprising pre-treated recombinant human collagen and an osmotic pressure regulator, and the injectable human collagen soft tissue filling material is in an isotonic state;

[0007] The recombinant human collagen is pretreated by isoelectric precipitation and centrifugal separation.

[0008] Preferably, the molecular weight of the recombinant human collagen is 100-400 kDa.

[0009] Preferably, more than 80% of the particle sizes of the injectable human collagen soft tissue filling material are distributed in the range of 25 to 280 μm.

[0010] Preferably, the osmotic pressure regulator is one of potassium chloride or sodium chloride.

[0011] The present invention also provides a method for preparing an injectable human collagen soft tissue filling material, the steps of which are as follows:

[0012] S1. Filtration and sterilization of raw materials

[0013] The human collagen is sterilized by filtering through a sterilizing filter;

[0014] S2. Preprocessing

[0015] S2.1, isoelectric point precipitation: use an isoelectric point regulator to adjust the isoelectric point of the recombinant human collagen after filtration and sterilization. When the isoelectric point is 5 to 9, let it stand for 10 to 20 hours for isoelectric point precipitation;

[0016] S2.2, centrifugal separation: centrifugal separation of the mixture after isoelectric precipitation, scraping the centrifugal precipitate, washing with a detergent and centrifugal washing separation, removing the detergent, repeating the washing once, to obtain the pretreated recombinant human collagen;

[0017] S3, injectable human collagen soft tissue filling material

[0018] An osmotic pressure regulator is added to the pretreated recombinant human collagen, and the amount of the osmotic pressure regulator added is calculated based on the fact that the pretreated recombinant human collagen is in an isotonic state as a whole, and then homogenized to obtain an injectable human collagen soft tissue filling material.

[0019] Preferably, the isoelectric point regulator in step S2.1 is composed of one or more of disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium hydroxide and potassium hydroxide and water, and the pH of the isoelectric point regulator is between 9 and 12.

[0020] Preferably, in step S2.2, during centrifugal sedimentation separation, the centrifugal force is 10000-30000 xg, and the centrifugal time is 10-60 min; during centrifugal washing separation, the centrifugal force is 10000-40000 xg, and the centrifugal time is 10-60 min.

[0021] Preferably, the detergent in step S2.2 is one of injection water, physiological saline or phosphate buffer.

[0022] Preferably, in step S2.2, the centrifugal rotor used in the above process is a fixed-angle rotor.

[0023] Preferably, the homogenization treatment in step S3 adopts one of dispersed shearing or high-pressure homogenization; preferably high-pressure homogenization.

[0024] Preferably, the high-pressure homogenization conditions are: first homogenize at a pressure of 50-60 bar for 7-8 seconds, then increase the pressure to 250-260 bar for homogenization for 4-5 seconds, then increase the pressure to 390-400 bar for homogenization for 2-3 seconds, and the pressure increase rate is 5 bar / s.

[0025] Preferably, the dispersion shearing conditions are: first, disperse and shear at a rotation speed of 4000-4500 rpm for 1-3 min, then adjust the rotation speed to 9000-9500 rpm for 1-3 min, and then adjust the rotation speed to 14500-15000 rpm for 1-3 min.

[0026] Preferably, the method for preparing the injectable human collagen soft tissue filling material further comprises: step S4, filling the finished human collagen soft tissue filling material into a prefilled syringe under aseptic conditions, and completing the outer packaging.

[0027] The beneficial effects of the present invention are as follows: 1. The injectable human collagen tissue filling material of the present invention is based on recombinant human collagen, which avoids the risks of zoonotic diseases and viruses and reduces immunogenicity; 2. The molecular weight of the recombinant human collagen used in the present invention is large and has a triple helix structure. The human collagen tissue filling material obtained by the preparation method of the present invention still has the characteristics of a triple helix structure and a large molecular weight, and can play a filling and supporting effect; 3. The preparation method of the present invention first pre-treats human collagen by physical means such as isoelectric precipitation and centrifugal separation, and then uses an osmotic pressure regulator to adjust the osmotic pressure. There is no special treatment process and no risk of chemical solvent residues; 4. The preparation method of the present invention combines isoelectric precipitation, centrifugal separation, homogenization and other processes to achieve a stable particle size distribution of the human collagen tissue filling material, with more than 80% of the particle size distribution being between 25 and 250 μm, having good particle size dispersibility, and good dispersion stability, which is beneficial to improving the filling effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the circular dichroism spectrum of the raw material recombinant human collagen of the present invention;

[0029] Figure 2 This is a circular dichroism spectrum of the injectable human collagen soft tissue filling material according to Example 1 of the present invention;

[0030] Figure 3 This is a particle size distribution diagram of the injectable human collagen soft tissue filling material of Example 1 of the present invention;

[0031] Figure 4 This is a particle size distribution diagram of the injectable human collagen soft tissue filling material according to Example 2 of the present invention;

[0032] Figure 5 This is a particle size distribution diagram of the injectable human collagen soft tissue filling material of Example 7 of the present invention;

[0033] Figure 6These are SDS-PAGE electrophoresis photographs of the human collagen soft tissue filling material of Example 1 of the present invention and the human collagen soft tissue filling material after reduction treatment. DETAILED DESCRIPTION

[0034] The technical scheme in the implementation of the present invention is clearly and completely described below in conjunction with the accompanying drawings. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers. If the manufacturers are not specified in the reagents, instruments, and components used, they are all conventional products that can be purchased commercially.

[0035] According to the description of the background technology, recombinant human collagen is used, and circular dichroism spectroscopy detection is performed according to the method in the YY / T1849-2022 recombinant collagen medical device standard, such as Figure 1 As shown, the recombinant human collagen is shown to have a triple helical structure, and the recombinant human collagen is used as the raw material of the present invention.

[0036] Example 1

[0037] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0038] 1. Filtration and sterilization of raw materials

[0039] Take 1L of recombinant human collagen with a molecular weight of 300-400kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0040] 2. Preprocessing

[0041] 1) Isoelectric point precipitation: The isoelectric point of the recombinant human collagen after filtration and sterilization is adjusted using an isoelectric point regulator, which is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 10.2. When the isoelectric point is 6, stand for 10 hours for isoelectric point precipitation;

[0042] 2) Centrifugal separation: The mixture after the isoelectric precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 10,000 x g for 15 minutes, the centrifuged precipitate is scraped, washed with injection water detergent, and placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 10,000 x g for 15 minutes, the detergent is removed, and the washing is repeated once to obtain the pretreated recombinant human collagen with a yield of 90.1%;

[0043] 3. Injectable human collagen soft tissue filling material

[0044] An osmotic pressure regulator was added to the above-mentioned pretreated recombinant human collagen, and the amount of the osmotic pressure regulator added was calculated based on the fact that the pretreated recombinant human collagen was in an isotonic state as a whole. The mixture was homogenized and homogenized at a pressure of 50 bar for 7 seconds, and then the pressure was increased to 250 bar at a rate of 5 bar / s and homogenized for 4 seconds, and then the pressure was increased to 390 bar at a rate of 5 bar / s and homogenized for 2 seconds to obtain an injectable human collagen soft tissue filling material.

[0045] Testing of injectable human collagen soft tissue filling materials: Circular dichroism spectroscopy testing is performed using the method in accordance with the YY / T1849-2022 recombinant collagen medical device standard, such as Figure 2 As shown, the injectable human collagen soft tissue filling material still has a triple helix structure; the particle size distribution test of the injectable human collagen soft tissue filling material was tested using a particle size detector, such as Figure 3 As shown in the figure, more than 80% of the particle sizes are distributed in the range of 28 to 205 μm, the span SPAN is 2.036, the particle size distribution range is narrow, it has good dispersibility, and the dispersion is stable; a portion of the injectable human collagen soft tissue filling material is reduced and unwound with electrophoresis buffer, and then the injectable human collagen soft tissue filling material and the reduced recombinant human collagen soft tissue filling material are respectively subjected to SDS-PAGE electrophoresis detection, as shown in the figure Figure 6 As shown, the injectable human collagen soft tissue filling material maintains a molecular weight of 300-400 kDa under non-reducing conditions; measured according to the method of 0632 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition), the osmotic pressure of the injectable human collagen soft tissue filling material product is 305 mOsmol / kg.

[0046] Example 2

[0047] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0048] 1. Filtration and sterilization of raw materials

[0049] Take 1L of recombinant human collagen with a molecular weight of 250-350kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0050] 2. Preprocessing

[0051] 1) Isoelectric point precipitation: The isoelectric point of the recombinant human collagen after filtration and sterilization is adjusted using an isoelectric point regulator, which is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 11. When the isoelectric point is 7.1, stand for 13 hours for isoelectric point precipitation;

[0052] 2) Centrifugal separation: The mixture after the isoelectric precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 13000 x g for 20 min, the centrifuged precipitate is scraped, washed with injection water detergent, and placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 20000 x g for 20 min, the detergent is removed, and the washing is repeated once to obtain the pretreated recombinant human collagen with a yield of 91.9%;

[0053] 3. Injectable human collagen soft tissue filling material

[0054] An osmotic pressure regulator was added to the pretreated recombinant human collagen, and the amount of the osmotic pressure regulator added was calculated based on the fact that the pretreated recombinant human collagen was in an isotonic state as a whole. The mixture was homogenized for 8 seconds at a pressure of 55 bar, and then the pressure was increased to 255 bar at a rate of 5 bar / s for 5 seconds, and then the pressure was increased to 395 bar at a rate of 5 bar / s for 3 seconds to obtain an injectable human collagen soft tissue filling material. After testing, the injectable human collagen soft tissue filling material still had a triple helix structure and maintained a molecular weight of 250 to 350 kDa under non-reducing conditions. Figure 4 As shown, more than 80% of the particle sizes are distributed in the range of 31-178 μm, the span SPAN is 1.823, the particle size distribution range is narrow, it has good dispersibility, and the dispersion is stable; according to the method of 0632 of the fourth part of the "Pharmacopoeia of the People's Republic of China" (2020 edition), the osmotic pressure of the injectable human collagen soft tissue filling material product is 296 mOsmol / kg.

[0055] Example 3

[0056] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0057] 1. Filtration and sterilization of raw materials

[0058] Take 1L of recombinant human collagen with a molecular weight of 100-150 kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0059] 2. Preprocessing

[0060] 1) Isoelectric point precipitation: The isoelectric point of the recombinant human collagen after filtration and sterilization is adjusted using an isoelectric point regulator, which is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 10.2. When the isoelectric point is 8.0, stand for 16 hours for isoelectric point precipitation;

[0061] 2) Centrifugal separation: The mixture after the isoelectric precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 18000 x g for 25 min, the centrifuged precipitate is scraped, washed with physiological saline detergent, and placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 25000 x g for 25 min, the detergent is removed, and the washing is repeated once to obtain the pretreated recombinant human collagen with a yield of 92.7%;

[0062] 3. Injectable human collagen soft tissue filling material

[0063] An osmotic pressure regulator was added to the pretreated recombinant human collagen, and the amount of the osmotic pressure regulator added was calculated based on the pretreated recombinant human collagen being in an isotonic state as a whole. The mixture was homogenized for 7.5 seconds at a pressure of 60 bar, and then the pressure was increased to 260 bar at a rate of 5 bar / s for 5 seconds, and then the pressure was increased to 400 bar at a rate of 5 bar / s for 2.5 seconds to obtain an injectable human collagen soft tissue filling material. After testing, the injectable human collagen The white soft tissue filler material still has a triple helix structure, maintains a molecular weight of 100-150kDa under non-reducing conditions, and more than 80% of the particle sizes are distributed in the range of 25-103μm; the span SPAN is 1.692, the particle size distribution range is narrower, it has good dispersibility, and the dispersion is stable; according to the method 0632 of the fourth part of the "Pharmacopoeia of the People's Republic of China" (2020 edition), the osmotic pressure of the injectable human collagen soft tissue filler material product is 293mOsmol / kg.

[0064] Example 4

[0065] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0066] 1. Filtration and sterilization of raw materials

[0067] Take 1L of recombinant human collagen with a molecular weight of 150-250kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0068] 2. Preprocessing

[0069] 1) Isoelectric point precipitation: The isoelectric point of the recombinant human collagen after filtration and sterilization is adjusted using an isoelectric point regulator, which is composed of dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydroxide and water, and the pH of the isoelectric point regulator is 11.5. When the isoelectric point is 8.8, stand for 20 hours for isoelectric point precipitation;

[0070] 2) Centrifugal separation: The mixture after the isoelectric precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 30,000 x g for 30 min, the centrifuged precipitate is scraped, washed with saline detergent, and placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 40,000 x g for 30 min, the detergent is removed, and the washing is repeated once to obtain the pretreated recombinant human collagen with a yield of 93.8%;

[0071] 3. Injectable human collagen soft tissue filling material

[0072] An osmotic pressure regulator was added to the pretreated recombinant human collagen, and the amount of the osmotic pressure regulator added was calculated based on the pretreated recombinant human collagen being in an isotonic state as a whole. The mixture was homogenized for 7 seconds at a pressure of 60 bar, and then the pressure was increased to 255 bar at a rate of 5 bar / s for 5.5 seconds, and then the pressure was increased to 390 bar at a rate of 5 bar / s for 2 seconds to obtain a human collagen soft tissue filling material. After testing, the injectable human collagen soft tissue The woven filling material still has a triple helix structure, maintains a molecular weight of 150-250kDa under non-reducing conditions, and more than 80% of the particle sizes are distributed in the range of 34-108μm; the span SPAN is 1.645, the particle size distribution range is narrower, it has good dispersibility, and the dispersion is stable; according to the method 0632 of the fourth part of the "Pharmacopoeia of the People's Republic of China" (2020 edition), the osmotic pressure of the injectable human collagen soft tissue filling material product is 310mOsmol / kg.

[0073] Example 5

[0074] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0075] 1. Filtration and sterilization of raw materials

[0076] Take 1L of recombinant human collagen with a molecular weight of 250-350kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0077] 2. Preprocessing

[0078] 1) Isoelectric point precipitation: The isoelectric point of the recombinant human collagen after filtration and sterilization is adjusted using an isoelectric point regulator, which is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 11. When the isoelectric point is 7.2, stand for 13 hours for isoelectric point precipitation;

[0079] 2) Centrifugal separation: The mixture after the isoelectric precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 18000 x g for 15 min, the centrifuged precipitate is scraped, washed with injection water detergent, and placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 20000 x g for 20 min, the detergent is removed, and the washing is repeated once to obtain the pretreated human collagen with a yield of 92.6%;

[0080] 3. Injectable human collagen soft tissue filling material

[0081] An osmotic pressure regulator was added to the above-mentioned pretreated recombinant human collagen, and the amount of the osmotic pressure regulator added was calculated based on the fact that the pretreated recombinant human collagen was in an isotonic state as a whole, and then homogenization was performed, and then homogenization was performed again, first dispersed shearing at a speed of 4000 rpm for 1 minute, then the speed was adjusted to 9000 rpm for 1 minute, and then the speed was adjusted to 14500 rpm for 1 minute to obtain a human collagen soft tissue filling material. After testing, the injectable human collagen soft tissue filling material still has a triple helix structure and maintains a molecular weight of 250 to 350 kDa under non-reducing conditions. Figure 5 As shown, more than 80% of the particle sizes are distributed in the range of 45-280 μm, the span SPAN is 2.214, the particle size distribution range is narrow, it has good dispersibility, and the dispersion is stable; according to the method of 0632 of the fourth general chapter of the "Pharmacopoeia of the People's Republic of China" (2020 edition), the osmotic pressure of the injectable human collagen soft tissue filling material product is 302 mOsmol / kg.

[0082] Example 6

[0083] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0084] 1. Filtration and sterilization of raw materials

[0085] Take 1 L of recombinant human collagen with a molecular weight of 150-250 kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0086] 2. Preprocessing

[0087] 1) Isoelectric point precipitation: The isoelectric point of the recombinant human collagen after filtration and sterilization is adjusted using an isoelectric point regulator, which is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 11.5. When the isoelectric point is 8.8, stand for 20 hours for isoelectric point precipitation;

[0088] 2) Centrifugal separation: The mixture after the isoelectric precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 25000 x g for 25 min, the centrifuged precipitate is scraped, washed with saline detergent, and placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 35000 x g for 30 min, the detergent is removed, and the washing is repeated once to obtain the pretreated human collagen with a yield of 94.2%;

[0089] 3. Injectable human collagen soft tissue filling material

[0090] An osmotic pressure regulator was added to the above-mentioned pretreated recombinant human collagen, and the amount of the osmotic pressure regulator added was calculated based on the fact that the pretreated recombinant human collagen was in an isotonic state as a whole, and then homogenization was performed, and then homogenization was performed again. First, the dispersion shearing was performed at a speed of 4300 rpm for 2 minutes, and then the speed was adjusted to 9300 rpm for dispersion shearing for 2 minutes, and then the speed was adjusted to 14800 rpm for dispersion shearing for 2 minutes to obtain a human collagen soft tissue filling material. After testing, the injectable human collagen soft tissue filling material still has a triple helix structure, maintains a molecular weight of 150 to 250 kDa under non-reducing conditions, and more than 80% of the particle size is distributed in the range of 42 to 263 μm. The span SPAN is 2.187, the particle size distribution range is narrow, it has good dispersibility, and the dispersion is stable; according to the method of 0632 of the fourth general rule of the "Pharmacopoeia of the People's Republic of China" (2020 edition), the osmotic pressure of the injectable human collagen soft tissue filling material product is 294 mOsmol / kg.

[0091] Example 7

[0092] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0093] 1. Filtration and sterilization of raw materials

[0094] Take 1L of recombinant human collagen with a molecular weight of 350-400 kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0095] 2. Preprocessing

[0096] 1) Isoelectric point precipitation: The isoelectric point of the recombinant human collagen after filtration and sterilization is adjusted using an isoelectric point regulator, which is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 10.2. When the isoelectric point is 5.5, stand for 10 hours for isoelectric point precipitation;

[0097] 2) Centrifugal separation: The mixture after the isoelectric precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 10,000 x g for 15 minutes, the centrifuged precipitate is scraped, washed with injection water detergent, and placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 10,000 x g for 15 minutes, the detergent is removed, and the washing is repeated once to obtain the pretreated human collagen with a yield of 86.2%;

[0098] 3. Human collagen soft tissue filling material

[0099] An osmotic pressure regulator was added to the pretreated recombinant human collagen, and the amount of the osmotic pressure regulator added was calculated based on the fact that the pretreated recombinant human collagen was in an isotonic state as a whole. The mixture was homogenized and then sheared at a speed of 4500 rpm for 3 minutes, and then the speed was adjusted to 9500 rpm for 3 minutes, and then the speed was adjusted to 15000 rpm for 3 minutes to obtain a human collagen soft tissue filling material. After testing, the human collagen soft tissue filling material still had a triple helix structure and maintained a molecular weight of 350 to 400 kDa under non-reducing conditions. Figure 5 As shown, more than 80% of the particle sizes are distributed in the range of 35-250 μm, the span SPAN is 2.146, the particle size distribution range is relatively narrow, with good dispersibility and stable dispersion; measured according to the method of 0632 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition), the osmotic pressure of the injectable human collagen soft tissue filling material product is 299 mOsmol / kg.

[0100] The above description shows that the preparation method of the present invention enables the injectable human collagen soft tissue filling material to maintain the characteristics of triple helix structure and large molecular weight. The preparation method of the present invention has no obvious effect on the performance of the product and can ensure the stability of the product performance. The injectable human collagen soft tissue filling material of the present invention has a small particle size and a narrow distribution range, and has good dispersibility. In this way, it can be more evenly distributed in the filling area during filling, avoiding local overfilling or underfilling, thereby improving the naturalness and aesthetics of the filling effect. At the same time, the particle size dispersion of the injectable human collagen soft tissue filling material of the present invention is stable, which can also reduce the risk of allergic reactions caused by excessive local concentration. In addition, it can maintain stability for a long time at the filling site to extend the duration of the filling effect.

[0101] Comparative Example 1

[0102] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0103] 1. Filtration and sterilization of raw materials

[0104] Take 1L of recombinant human collagen with a molecular weight of 350-400 kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0105] 2. Preprocessing

[0106] 1) Isoelectric point precipitation: The isoelectric point of the human collagen after filtration and sterilization is adjusted using an isoelectric point regulator, which is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 10.2. When the isoelectric point is 4.7, stand for 10 hours for isoelectric point precipitation;

[0107] During the standing process, at an isoelectric point of 4.7, it was found that human collagen gradually dissolved, and the amount of precipitate generated was very small. The yield of the pretreated recombinant human collagen obtained under the centrifugal separation conditions of Example 1 was only 13.5%;

[0108] The subsequent steps of preparation are stopped.

[0109] Comparative Example 2

[0110] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0111] 1. Filtration and sterilization of raw materials

[0112] Take 1L of recombinant human collagen with a molecular weight of 350-400 kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0113] 2. Preprocessing

[0114] 1) Isoelectric point precipitation: The isoelectric point of the human collagen after filtration and sterilization is adjusted using an isoelectric point regulator, which is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 10.2. When the isoelectric point is 4.2, stand for 10 hours for isoelectric point precipitation;

[0115] During the standing process, at an isoelectric point of 4.2, it was found that human collagen gradually dissolved, and the amount of precipitate generated was very small. The yield of the pretreated recombinant human collagen obtained under the centrifugal separation conditions of Example 1 was only 5.3%;

[0116] The preparation process is stopped.

[0117] Comparative Example 3

[0118] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0119] 1. Filtration and sterilization of raw materials

[0120] Take 1 L of recombinant human collagen with a molecular weight of 150-250 kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0121] 2. Preprocessing

[0122] 1) Isoelectric point precipitation: The isoelectric point of the human collagen after filtration and sterilization was adjusted using an isoelectric point regulator, which consisted of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator was 11. When the isoelectric point was 9.2, the isoelectric point was allowed to stand for 10 hours for isoelectric point precipitation. During the standing process, it was found that the precipitate agglomerated at an isoelectric point of 9.2.

[0123] 2) Centrifugal separation: Place the mixture after isoelectric precipitation in a centrifuge with a fixed angle rotor at a centrifugal force of 30,000 x g for 30 minutes, scrape the centrifugal precipitate, wash it with saline detergent, and place it in a centrifuge with a fixed angle rotor at a centrifugal force of 40,000 x g for 30 minutes, remove the detergent, and repeat the washing once to obtain pretreated human collagen, 93.1%;

[0124] 3. Injectable human collagen soft tissue filling material

[0125] An osmotic pressure regulator was added to the pretreated recombinant human collagen, and the amount of the osmotic pressure regulator added was calculated based on the fact that the pretreated recombinant human collagen was in an isotonic state as a whole, and then homogenized, and then homogenized for 7 seconds at a pressure of 60 bar, and then the pressure was increased to 255 bar at a rate of 5 bar / s for 5.5 seconds, and then the pressure was increased to 390 bar at a rate of 5 bar / s for 2 seconds to obtain a human collagen soft tissue filling material. After testing, the injectable human collagen soft tissue filling material still has a triple helix structure, maintains a molecular weight of 150 to 250 kDa under non-reducing conditions, and more than 80% of the particle size is distributed in the range of 205 to 588 μm, with a span of 4.223. The particle size distribution range is wide, and the dispersibility is relatively poor. Due to the agglomeration of the precipitate, its dispersion stability is also affected; according to the method of 0632 of the fourth general rule of the Pharmacopoeia of the People's Republic of China (2020 edition), the osmotic pressure of the product is 310 mOsmol / kg;

[0126] The isoelectric point used in the isoelectric precipitation process of the recombinant human collagen in this comparative example is 9.2, and the precipitate appears to be aggregated, resulting in a larger particle size of the injectable human collagen soft tissue filling material, and a wider particle size distribution, resulting in a relatively poor filling effect.

[0127] Comparative Example 4

[0128] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0129] 1. Filtration and sterilization of raw materials

[0130] Take 1L of recombinant human collagen with a molecular weight of 100-150 kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0131] 2. Preprocessing

[0132] 1) Isoelectric point precipitation: The isoelectric point of the human collagen after filtration and sterilization is adjusted using an isoelectric point regulator, which is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 10.2. When the isoelectric point is 8.0, stand for 8 hours for isoelectric point precipitation;

[0133] 2) Centrifugal separation: The mixture after the isoelectric precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 15000 x g for 25 min, the centrifuged precipitate is scraped, washed with saline detergent, and placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 25000 x g for 25 min, the detergent is removed, and the washing is repeated once to obtain the pretreated recombinant human collagen with a yield of 80.5%;

[0134] The remaining steps are the same as those in Example 3 to obtain an injectable human collagen soft tissue filling material. After testing, the injectable human collagen soft tissue filling material still has a triple helix structure, maintains a molecular weight of 100-150 kDa under non-reducing conditions, and more than 80% of the particle size is distributed in the range of 41-211 μm. The span SPAN is 1.936, with good dispersibility and stable dispersion; according to the method of 0632 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition), the osmotic pressure of the product is 291 mOsmol / kg.

[0135] Through this comparative example, it was found that the isoelectric precipitation time was relatively short, resulting in less human collagen obtained, resulting in a lower human collagen yield.

[0136] In addition, a long isoelectric precipitation time will increase the risk of product sterility, so the isoelectric precipitation time should not be too long.

[0137] Comparative Example 5

[0138] An injectable human collagen soft tissue filling material, the preparation method and steps thereof are as follows:

[0139] 1. Filtration and sterilization of raw materials

[0140] Take 1L of recombinant human collagen with a molecular weight of 100-150 kDa and filter it through a sterilizing filter to ensure aseptic operation;

[0141] 2. Preprocessing

[0142] 1) Isoelectric point precipitation: The isoelectric point of the human collagen after filtration and sterilization is adjusted using an isoelectric point regulator, which is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 10.2. When the isoelectric point is 8.0, stand for 10 hours for isoelectric point precipitation;

[0143] 2) Centrifugal separation: Place the mixture after the isoelectric precipitation in a centrifuge with a horizontal rotor and centrifuge for 25 minutes at a centrifugal force of 8000 x g, scrape the centrifugal precipitate, wash it with saline detergent, and place it in a centrifuge with a horizontal rotor and centrifuge for 25 minutes at a centrifugal force of 8000 x g, remove the detergent, and repeat the washing once to obtain the pretreated recombinant human collagen, 71.3%;

[0144] The remaining steps are the same as Example 3; an injectable human collagen soft tissue filling material is obtained. After testing, the injectable human collagen soft tissue filling material still has a triple helix structure, maintains a molecular weight of 100-150 kDa under non-reducing conditions, and more than 80% of the particle size is distributed in the range of 54-209 μm. The span SPAN is 1.847, with good dispersibility and stable dispersion; according to the method of 0632 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition), the osmotic pressure of the product is 306 mOsmol / kg.

[0145] Through this comparative example, it was found that the centrifugal force of the horizontal rotor centrifuge was insufficient, which affected the separation and collection of human collagen, resulting in poor separation effect and low yield.

[0146] Comparative Example 6

[0147] An injectable human collagen soft tissue filling material, which is different from Example 3 in that:

[0148] 3. Injectable human collagen soft tissue filling material

[0149] Adding an osmotic pressure regulator to the pretreated recombinant human collagen, calculating the amount of the osmotic pressure regulator added based on the pretreated recombinant human collagen being in an isotonic state as a whole, and then homogenizing, and then homogenizing at a pressure of 400 bar for 90 seconds to obtain a human collagen soft tissue filling material;

[0150] The rest of the steps are exactly the same;

[0151] After testing, the injectable human collagen soft tissue filling material still has a triple helix structure, maintains a molecular weight of 100-150kDa under non-reducing conditions, and more than 80% of the particle sizes are distributed in the range of 52-434μm. The span SPAN is 3.156, the particle size distribution range is wide, the dispersibility is relatively poor, and the dispersion is relatively stable; according to the method of 0632 of the fourth general rule of the "Pharmacopoeia of the People's Republic of China" (2020 edition), the osmotic pressure of the human collagen soft tissue filling material product is 310mOsmol / kg; the injectable human collagen soft tissue filling material in this comparative example has a larger particle size and a wider particle size distribution, which will affect the deep skin penetration effect and make the filling effect relatively poor.

[0152] Comparative Example 7

[0153] An injectable human collagen soft tissue filling material, which is different from Example 3 in that:

[0154] 3. Injectable human collagen soft tissue filling material

[0155] Add an osmotic pressure regulator to the pretreated recombinant human collagen, calculate the amount of the osmotic pressure regulator added based on the pretreated recombinant human collagen being in an isotonic state as a whole, perform homogenization, perform homogenization again, disperse and shear at a speed of 15000 rpm for 6 minutes, and obtain an injectable human collagen soft tissue filling material;

[0156] The rest of the steps are exactly the same;

[0157] After testing, the injectable human collagen soft tissue filling material still has a triple helix structure, maintains a molecular weight of 100-150kDa under non-reducing conditions, and more than 80% of the particle size is distributed in the range of 100-850μm. The span SPAN is 6.135, the particle size distribution range is wider, and the dispersion is worse, which is not conducive to dispersion stability; according to the method of 0632 of the fourth part of the "Pharmacopoeia of the People's Republic of China" (2020 edition), the osmotic pressure of the injectable human collagen soft tissue filling material product is 301mOsmol / kg.

[0158] Through Comparative Examples 6 and 7, it was found that under constant high-pressure homogenization and constant speed dispersed shearing, although the particle size of the injectable human collagen soft tissue filling material meets the requirements of industry standards, the particle size distribution of 80% of the particles is relatively wide, the particle size is relatively large, and the filling effect is poor, which affects the naturalness of facial expressions. In addition, it is not easy to penetrate deep into the skin, affecting the absorption and utilization of human collagen.

[0159] Comparative Example 8

[0160] An injectable human collagen soft tissue filling material, which is different from Example 3 in that:

[0161] 3. Injectable human collagen soft tissue filling material

[0162] Add an osmotic pressure regulator to the pretreated recombinant human collagen, calculate the amount of the osmotic pressure regulator added based on the pretreated recombinant human collagen being in an isotonic state as a whole, perform homogenization, perform homogenization again, disperse and emulsify at a speed of 2000 rpm for 20 minutes, and obtain an injectable human collagen soft tissue filling material;

[0163] The rest of the steps are exactly the same;

[0164] After testing, the injectable human collagen soft tissue filling material still has a triple helix structure, maintains a molecular weight of 100-150kDa under non-reducing conditions, and more than 80% of the particle size is distributed in the range of 802-1507μm. The span SPAN is 9.245. The particle size distribution range is wide, the dispersion is poor, and it is not conducive to dispersion stability; according to the method 0632 of the fourth part of the "Pharmacopoeia of the People's Republic of China" (2020 edition), the osmotic pressure of the injectable human collagen soft tissue filling material product is 305mOsmol / kg.

[0165] Through this comparative example 8, it was found that the particle size of the human collagen soft tissue filling material obtained by dispersion and emulsification did not meet the industry standard requirements, and the particle size distribution of 80% of the particles was wide, the particle size was large, and the filling effect was worse, which could easily cause excessive local filling and even cause allergies.

[0166] In summary, the human collagen tissue filling material of the present invention is mainly composed of human collagen, has a large molecular weight and a triple helix structure, and has a uniform particle size distribution, which can play a filling and supporting effect; its preparation process is simple and convenient, without any special treatment process, and will not introduce toxic and harmful residual substances, so that the product performance is stable; the human collagen tissue filling material prepared by the present invention has good stability, excellent biocompatibility, no virus hidden dangers, and good soft tissue filling and repair effects.

[0167] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit and essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0168] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An injectable human collagen soft tissue filling material, characterized in that: The injectable human collagen soft tissue filling material is composed of pre-treated recombinant human collagen and an osmotic pressure regulator, and the injectable human collagen soft tissue filling material is in an isotonic state; The steps of the method for preparing the injectable human collagen soft tissue filling material are as follows: S1. Filtration and sterilization of raw materials The recombinant human collagen is filtered and sterilized through a sterilizing filter; S2. Preprocessing S2.1, isoelectric point precipitation: use an isoelectric point regulator to adjust the isoelectric point of the recombinant human collagen after filtration and sterilization. When the isoelectric point is 5-9, let it stand for 10-20 hours for isoelectric point precipitation; S2.2, centrifugal separation: centrifugal separation of the mixture after isoelectric precipitation, scraping the centrifugal precipitate, washing with a detergent and centrifugal washing separation, removing the detergent, repeating the washing once, to obtain the pretreated recombinant human collagen; S3, injectable human collagen soft tissue filling material Adding an osmotic pressure regulator to the pretreated recombinant human collagen, calculating the amount of the osmotic pressure regulator to be added based on the pretreated recombinant human collagen being in an isotonic state as a whole, and then performing a homogenization process to obtain an injectable human collagen soft tissue filling material; The homogenization process adopts one of dispersed shearing or high pressure homogenization; The high-pressure homogenization conditions are as follows: first homogenize at a pressure of 50-60 bar for 7-8 seconds, then increase the pressure to 250-260 bar for 4-5 seconds, then increase the pressure to 390-400 bar for 2-3 seconds, and the pressure increase rate is 5 bar / s; The dispersion shearing conditions are as follows: first, the dispersion shearing is performed at a speed of 4000-4500 rpm for 1-3 min, then the dispersion shearing is performed at a speed of 9000-9500 rpm for 1-3 min, and then the dispersion shearing is performed at a speed of 14500-15000 rpm for 1-3 min; More than 80% of the injectable human collagen soft tissue filling material has a particle size distribution of 25-280 μm; The molecular weight of the recombinant human collagen is 100-400 kDa.

2. The injectable human collagen soft tissue filling material according to claim 1, characterized in that: The osmotic pressure regulator is one of potassium chloride or sodium chloride.

3. A method for preparing the injectable human collagen soft tissue filling material according to claim 1 or 2, characterized in that: The steps of the preparation method are as follows: S1. Filtration and sterilization of raw materials The recombinant human collagen is sterilized by filtering through a sterilizing filter; S2. Preprocessing S2.1, isoelectric point precipitation: use an isoelectric point regulator to adjust the isoelectric point of the recombinant human collagen after filtration and sterilization. When the isoelectric point is 5-9, let it stand for 10-20 hours for isoelectric point precipitation; S2.2, centrifugal separation: centrifugal separation of the mixture after isoelectric precipitation, scraping the centrifugal precipitate, washing with a detergent and centrifugal washing separation, removing the detergent, repeating the washing once, to obtain the pretreated recombinant human collagen; S3, injectable human collagen soft tissue filling material An osmotic pressure regulator is added to the pretreated recombinant human collagen, and the amount of the osmotic pressure regulator added is calculated based on the fact that the pretreated recombinant human collagen is in an isotonic state as a whole, and then homogenized to obtain an injectable human collagen soft tissue filling material.

4. The method for preparing an injectable human collagen soft tissue filling material according to claim 3, characterized in that: The isoelectric point regulator in step S2.1 is composed of one or more of disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium hydroxide and potassium hydroxide and water, and the pH of the isoelectric point regulator is between 9 and 12.

5. The method for preparing an injectable human collagen soft tissue filling material according to claim 3, characterized in that: In the step S2.2, during centrifugal sedimentation separation, the centrifugal force is 10000~30000xg, and the centrifugal time is 10~60min; during centrifugal washing separation, the centrifugal force is 10000~40000xg, and the centrifugal time is 10~60min; the centrifugal rotor used in the above process is a fixed-angle rotor; the detergent in the step S2.2 is one of injection water, physiological saline or phosphate buffer.

Images