Injection-type human collagen soft tissue filler material and preparation method therefor

Abstract

Disclosed are an injection-type human collagen soft tissue filler material and a preparation method therefor. The present invention pertains to the technical field of biomedical materials. The injection-type human collagen soft tissue filler material comprises a pretreated recombinant human collagen and an osmotic pressure regulator and is in an isotonic state. The pretreatment of the recombinant human collagen is isoelectric precipitation and centrifugal separation. The preparation method therefor comprises sterilizing, by means of filtration, and pretreating raw materials, and homogenizing the pretreated recombinant human collagen and the osmotic pressure regulator. The injection-type human collagen soft tissue filler material of the present invention mainly comprises human collagen, has a large molecular weight and a triple-helix structure, has a uniform particle size distribution, and can achieve a filling and supporting effect. The preparation process for the material is simple and convenient, involves no special treatment process, does not introduce toxic and hazardous substances that tend to remain, and stabilizes the performance of products.

Description

Injection type human collagen soft tissue filling material and preparation method thereof TECHNICAL FIELD

[0001] The present application relates to an injection type human collagen soft tissue filling material and a preparation method thereof, and belongs to the technical field of biomedical materials. BACKGROUND

[0002] The plastic and cosmetic industry is developing rapidly with the continuous improvement of people's living standards, and breast augmentation, rhinoplasty, wrinkle removal, facial plastic surgery, etc. are popular. The current demand for facial plastic surgery includes: removing wrinkles, improving facial contours, repairing sunken scars or lips, repairing damaged tissues, etc. Therefore, the market demand for soft tissue filling materials. So far, people have successively researched materials such as white wax, fat transplantation, silicone, gel implant, etc. for soft tissue plastic surgery, and clinical application has found that: all have the problems of hardening, neoplastic mass, rapid absorption, chronic inflammation, etc. In recent years, the US FDA has successively approved several products for soft tissue filling, the main components of which are animal collagen, sodium hyaluronate, PMMA microspheres, PCL microspheres, PLA microspheres, etc., which have had good effects. For some users, there are still redness, swelling, allergy 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, is an important material for the structure of vertebrate bodies, and is also 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 total body protein. About half of the collagen in the body exists in the skin, and collagen accounts for 70% of the dry weight of the dermis and tendon, which is equivalent to 6% of the body weight. Recombinant human collagen is obtained by transforming human collagen DNA sequence into tissue engineering bacteria or cells, and then fermenting, separating, extracting and purifying it. It fundamentally solves the virus hidden danger of animal extracted collagen.

[0004] The similar products in the market at present, such as Taiwan Shuangmei and Changchun Feilan, have some problems: 1) the raw material is extracted from animals, which has the risk of animal-borne zoonosis and virus, and also has the problem of immunogen, which may cause more clinical adverse reactions of human body, including nodules, granulomas, allergies and the like; 2) the collagen protein extracted from animals must be subjected to pretreatment processes including degreasing, deproteinization, decellularization and virus inactivation, and must be treated with sensitive chemical reagents such as isopropyl alcohol and ethanol, and the chemical residues are difficult to control; 3) the control of target protein is difficult, such as the product is a mixture of type I and type III collagen; in addition, the product of Shanxi Jinbo prepared by using collagen protein also has some problems: 1) the collagen protein has small molecular weight and low collagen protein concentration, so that it mainly plays a role in supplementing collagen protein and has no filling support effect; 2) the raw material is recombinant humanized collagen protein, the sequence of which is mainly a full-length or partial sequence of human collagen protein sequence in a loop structure, and the telomere is removed, which has structural and performance differences from naturally occurring human collagen protein; the product is prepared by using a freeze-drying process, and needs to be dissolved in normal saline before use. SUMMARY

[0005] In order to solve at least one problem of the prior art, the present application provides an injection type human collagen soft tissue filling material and a preparation method thereof.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme: an injection type human collagen soft tissue filling material, comprising pretreated recombinant human collagen and an osmotic pressure adjusting agent, and the injection type human collagen soft tissue filling material is in an isotonic state.

[0007] The pretreatment of the recombinant human collagen is an isoelectric point precipitation and centrifugal separation process.

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

[0009] Preferably, more than 80% of the particle size distribution of the injection type human collagen soft tissue filling material is in the range of 25-280 μm.

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

[0011] The present application also provides a preparation method of the injection type human collagen soft tissue filling material, and the steps are as follows:

[0012] S1, raw material filtration and sterilization

[0013] The human collagen is filtered and sterilized through a sterilization filter;

[0014] S2, pretreatment

[0015] S2.1, isoelectric point precipitation: the isoelectric point of the recombinant human collagen filtered and sterilized above is adjusted using an isoelectric point adjusting agent, and after the isoelectric point is 5-9, it is left to stand for 10-20 hours for isoelectric point precipitation;

[0016] S2.2, centrifugal separation: the mixture after isoelectric point precipitation is centrifugally separated, the centrifugal precipitate is scraped, washed with a detergent and centrifugally washed and separated, the detergent is removed, and the washing is repeated once to obtain the pretreated recombinant human collagen;

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

[0018] The pretreated recombinant human collagen is added with an osmotic pressure adjusting agent, the amount of the osmotic pressure adjusting agent is calculated according to the isosmotic state of the pretreated recombinant human collagen as a whole, and then homogenized to obtain the injectable human collagen soft tissue filling material.

[0019] Preferably, the isoelectric point adjusting agent 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 adjusting agent is between 9 and 12.

[0020] Preferably, in step S2.2, the centrifugal force during centrifugal precipitation separation is 10,000-30,000xg, and the centrifugal time is 10-60 minutes; the centrifugal force during centrifugal washing separation is 10,000-40,000xg, and the centrifugal time is 10-60 minutes.

[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 in step S3 uses one of dispersion shear or high-pressure homogenization; preferably high-pressure homogenization.

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

[0025] Preferably, the dispersion shearing condition is: first dispersing and shearing at a rotation speed of 4000-4500 rpm for 1-3 min, then adjusting the rotation speed to 9000-9500 rpm for dispersing and shearing for 1-3 min, and then adjusting the rotation speed to 14500-15000 rpm for dispersing and shearing for 1-3 min.

[0026] Preferably, the preparation method of the injection type human collagen soft tissue filling material further comprises: step S4, filling the finished human collagen soft tissue filling material in a pre-filled syringe under sterile conditions, and completing the outer packaging.

[0027] The injection type human collagen soft tissue filling material of the present application is prepared by using recombinant human collagen, which avoids zoonosis and virus risk and reduces immunogenicity; the recombinant human collagen used in the present application has a large molecular weight and a triple helix structure, and the human collagen soft tissue filling material obtained by the preparation method of the present application still has the characteristics of a triple helix structure and a large molecular weight, and can play a filling and supporting effect; the preparation method of the present application uses physical means such as isoelectric point precipitation and centrifugal separation for pretreatment of human collagen, and uses an osmotic pressure regulator to adjust the osmotic pressure, without special treatment process and chemical solvent residue risk; the preparation method of the present application combines isoelectric point precipitation, centrifugal separation and homogenization treatment, so that the human collagen soft tissue filling material has a stable particle size distribution, more than 80% of the particle size distribution is in the range of 25-250 μm, has good particle size dispersity, good dispersion stability, and is beneficial to improving the filling effect. BRIEF DESCRIPTION OF DRAWINGS

[0028] Fig. 1 is a circular dichroism spectrum of the raw material recombinant human collagen of the present application;

[0029] Fig. 2 is a circular dichroism spectrum of the injection type human collagen soft tissue filling material of Example 1 of the present application;

[0030] Fig. 3 is a particle size distribution graph of the injection type human collagen soft tissue filling material of Example 1 of the present application;

[0031] Fig. 4 is a particle size distribution graph of the injection type human collagen soft tissue filling material of Example 2 of the present application;

[0032] Fig. 5 is a particle size distribution graph of the injection type human collagen soft tissue filling material of Example 7 of the present application;

[0033] Fig. 6 is a SDS-PAGE electrophoresis photo of the human collagen soft tissue filling material of Example 1 of the present application and the human collagen soft tissue filling material after reduction treatment. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings. The described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments, components not mentioned by the manufacturer are all conventional products that can be purchased in the market.

[0035] According to the description of the background, recombinant human collagen is used, and circular dichroism spectrum detection is carried out according to the method in YY / T1849-2022 Recombinant Collagen Medical Device Standard, as shown in FIG. 1, the recombinant human collagen shows a triple helix structure, and the recombinant human collagen is used as the raw material of the present application.

[0036] Embodiment 1

[0037] A method for preparing an injection type human collagen soft tissue filling material, the steps of which are as follows:

[0038] 1. Filtration and sterilization of raw materials

[0039] 1L of recombinant human collagen with a molecular weight of 300-400 kDa is filtered and sterilized through a sterilization filter to ensure aseptic operation;

[0040] 2. Pretreatment

[0041] 1) Isoelectric point precipitation: The isoelectric point of the above-mentioned filtered and sterilized recombinant human collagen is adjusted using an isoelectric point adjuster composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point adjuster is 10.2. After the isoelectric point is adjusted to 6, the mixture is left to stand for 10 hours for isoelectric point precipitation.

[0042] 2) Centrifugal separation: The above-mentioned isoelectric point precipitated mixture is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 10000xg for 15 minutes. The centrifugal precipitate is scraped and washed with injection water, and then placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 10000xg for 15 minutes. The washing is repeated once to remove the washing agent, and the pretreated recombinant human collagen is obtained with a yield of 90.1%.

[0043] 3. Injection type human collagen soft tissue filling material

[0044] The pre-processed recombinant human collagen is added with an osmotic pressure regulator, the amount of the osmotic pressure regulator is calculated according to the isotonic state of the whole pre-processed recombinant human collagen, and then homogenized, and then homogenized, and then homogenized at a pressure of 50 bar for 7 s, and then homogenized at a rate of 5 bar / s to 250 bar for 4 s, and then homogenized at a rate of 5 bar / s to 390 bar for 2 s, to obtain an injectable human collagen soft tissue filling material.

[0045] The injectable human collagen soft tissue filling material is detected: the circular dichroism spectrum is detected using the method in YY / T1849-2022 Recombinant Collagen Medical Device Row Standard, as shown in FIG. 2, the injectable human collagen soft tissue filling material still has a triple helix structure; the particle size distribution of the injectable human collagen soft tissue filling material is detected using a particle size detector, as shown in FIG. 3, more than 80% of the particle size is distributed in the range of 28-205 μm, the span is 2.036, the particle size distribution range is narrow, has good dispersibility, and the dispersion is stable; part of the injectable human collagen soft tissue filling material is reduced and treated with an 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 FIG. 6, the injectable human collagen soft tissue filling material maintains a molecular weight of 300-400 kDa under non-reducing conditions; according to the method of Chinese Pharmacopoeia (2020 Edition) Four General Rules 0632, 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 is prepared by the following method steps:

[0048] 1. Filtration and sterilization of raw materials

[0049] 1L of recombinant human collagen with a molecular weight of 250-350 kDa is filtered and sterilized through a sterilization filter to ensure sterile operation;

[0050] 2. Pretreatment

[0051] 1) Isoelectric point precipitation: the isoelectric point of the above filtered and sterilized recombinant human collagen is adjusted using an isoelectric point regulator, the isoelectric point regulator is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 11, the isoelectric point is 7.1, and the isoelectric point is precipitated after standing for 13 h;

[0052] 2) centrifugal separation: the mixture after the above isoelectric point precipitation is placed in a centrifuge with a fixed angle rotor for centrifugal precipitation separation at a centrifugal force of 13000xg for 20min, the centrifugal precipitate is scraped, washed with injection water detergent, and placed in a centrifuge with a fixed angle rotor for centrifugal washing separation at a centrifugal force of 20000xg for 20min, 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. An injectable human collagen soft tissue filling material

[0054] The above pretreated recombinant human collagen is added with an osmotic pressure regulator, the amount of the osmotic pressure regulator is calculated according to the isosmotic state of the pretreated recombinant human collagen as a whole, and then homogenized, and then homogenized at a pressure of 55bar for 8s, and then pressurized to 255bar at a rate of 5bar / s for 5s, and then pressurized to 395bar at a rate of 5bar / s for 3s to obtain an injectable human collagen soft tissue filling material. The injectable human collagen soft tissue filling material still has a triple helix structure, maintains a molecular weight of 250-350kDa under non-reducing conditions, as shown in Figure 4, more than 80% of the particle size is distributed in the range of 31-178μm, the span SPAN is 1.823, the particle size distribution range is narrow, and the dispersion is good and stable; according to the method of Chinese Pharmacopoeia (2020 edition) four general rules 0632, the osmotic pressure of the injectable human collagen soft tissue filling material product is 296mOsmol / kg.

[0055] Example 3

[0056] An injectable human collagen soft tissue filling material is prepared by the following method steps:

[0057] 1. Filtration and sterilization of raw materials

[0058] 1L of recombinant human collagen with a molecular weight of 100-150kDa is filtered and sterilized through a sterilization filter to ensure aseptic operation;

[0059] 2. Pretreatment

[0060] 1) isoelectric point precipitation: the isoelectric point of the above filtered and sterilized recombinant human collagen is adjusted using an isoelectric point regulator, the isoelectric point regulator is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 10.2, the isoelectric point is 8.0, and the isoelectric point precipitation is carried out after standing for 16h;

[0061] 2) centrifugal separation: the mixture after the above isoelectric point precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 18000xg for 25min, the centrifugation precipitate is scraped, washed with a physiological saline detergent, and placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 25000xg for 25min, 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. An injectable human collagen soft tissue filling material

[0063] The above pretreated recombinant human collagen is added with an osmotic pressure regulator, the amount of the osmotic pressure regulator is calculated according to the isosmotic state of the pretreated recombinant human collagen as a whole, and then homogenized, and then homogenized at a pressure of 60bar for 7.5s, and then homogenized at a pressure of 260bar at a rate of 5bar / s for 5s, and then homogenized at a pressure of 400bar at a rate of 5bar / s for 2.5s to obtain an injectable human collagen soft tissue filling material. The test shows that 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 25-103μm; the span is 1.692, the particle size distribution range is narrower, the dispersion is good, and the dispersion is stable; according to the method of General Chapter 0632 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 293mOsmol / kg.

[0064] Example 4

[0065] An injectable human collagen soft tissue filling material is prepared by the following method steps:

[0066] 1. Filtration and sterilization of raw materials

[0067] 1L of recombinant human collagen with a molecular weight of 150-250kDa is filtered and sterilized through a sterilization filter to ensure aseptic operation;

[0068] 2. Pretreatment

[0069] 1) isoelectric point precipitation: the isoelectric point of the above filtered and sterilized recombinant human collagen is adjusted using an isoelectric point regulator, the isoelectric point regulator is composed of dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydroxide and water, and the pH of the isoelectric point regulator is 11.5, the isoelectric point is 8.8, and the isoelectric point precipitation is carried out after standing for 20h;

[0070] 2) centrifugal separation: the mixture after the above isoelectric point precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 30000xg for 30min, the centrifugation 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 40000xg for 30min, 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、injection type human collagen soft tissue filling material

[0072] The above pretreated recombinant human collagen is added with an osmotic pressure regulator, the amount of the osmotic pressure regulator is calculated according to the isosmotic state of the whole pretreated recombinant human collagen, and then homogenized, and then homogenized, homogenized at a pressure of 60bar for 7s, then pressurized to 255bar at a rate of 5bar / s for 5.5s, and then pressurized to 390bar at a rate of 5bar / s for 2s, to obtain a human collagen soft tissue filling material, which is detected to still have a triple helix structure, and has a molecular weight of 150-250kDa under non-reducing conditions, and more than 80% of the particle size is distributed in the range of 34-108μm; the span is 1.645, the particle size distribution range is narrower, has good dispersibility, and the dispersion is stable; according to the method of Chinese Pharmacopoeia (2020 edition) four general rules 0632, the osmotic pressure of the injection type human collagen soft tissue filling material product is 310mOsmol / kg.

[0073] Example 5

[0074] An injection type human collagen soft tissue filling material is prepared by the following method steps:

[0075] 1、raw material filtration and sterilization

[0076] 1L of recombinant human collagen with a molecular weight of 250-350kDa is filtered and sterilized through a sterilization filter to ensure sterile operation;

[0077] 2、pretreatment

[0078] 1) isoelectric point precipitation: the isoelectric point of the above filtered and sterilized recombinant human collagen is adjusted using an isoelectric point regulator, the isoelectric point regulator is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 11, the isoelectric point is 7.2, and the isoelectric point precipitation is carried out after standing for 13h;

[0079] 2) centrifugal separation: the mixture after the above isoelectric point precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 18000xg for 15min, 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 20000xg for 20min, the detergent is removed, and the washing is repeated once, to obtain the pretreated human collagen protein, with a yield of 92.6%;

[0080] 3. An injectable human collagen soft tissue filling material

[0081] The above pretreated recombinant human collagen is added with an osmotic pressure regulator, the amount of the osmotic pressure regulator is calculated according to the isosmotic state of the pretreated recombinant human collagen as a whole, and then homogenized, first dispersed and sheared at a speed of 4000rpm for 1min, then the speed is adjusted to 9000rpm for dispersed shearing for 1min, and then the speed is adjusted to 14500rpm for dispersed shearing for 1min, to obtain a human collagen soft tissue filling material. The test shows that the injectable human collagen soft tissue filling material still has a triple helix structure, maintains a molecular weight of 250-350kDa under non-reducing conditions, as shown in Figure 5, more than 80% of the particle size is distributed in the range of 45-280μm, the span is 2.214, the particle size distribution range is narrow, and the dispersion is good and stable; according to the method of Chinese Pharmacopoeia (2020 edition) four general rules 0632, the osmotic pressure of the injectable human collagen soft tissue filling material product is 302mOsmol / kg.

[0082] Example 6

[0083] An injectable human collagen soft tissue filling material is prepared by the following method steps:

[0084] 1. Filtration and sterilization of raw materials

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

[0086] 2. Pretreatment

[0087] 1) isoelectric point precipitation: the isoelectric point of the above filtered and sterilized recombinant human collagen is adjusted using an isoelectric point regulator, the isoelectric point regulator is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 11.5, the isoelectric point is 8.8, and the isoelectric point precipitation is carried out after standing for 20h;

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

[0089] 3. An injectable human collagen soft tissue filling material

[0090] The above pretreated recombinant human collagen is added with an osmotic pressure regulator, the amount of the osmotic pressure regulator is calculated according to the isosmotic state of the pretreated recombinant human collagen as a whole, and then homogenized, first dispersed and sheared at a speed of 4300rpm for 2min, then the speed is adjusted to 9300rpm for dispersed shearing for 2min, and then the speed is adjusted to 14800rpm for dispersed shearing for 2min to obtain the human collagen soft tissue filling material. The test shows that the injectable human collagen soft tissue filling material still has a triple helix structure, maintains a molecular weight of 150-250kDa under non-reducing conditions, and has a particle size of 80% or more distributed in the range of 42-263μm, a span of 2.187, a narrow particle size distribution range, good dispersibility, and stable dispersion. According to the method of Chinese Pharmacopoeia (2020 edition) four general rules 0632, the osmotic pressure of the injectable human collagen soft tissue filling material product is 294mOsmol / kg.

[0091] Example 7

[0092] An injectable human collagen soft tissue filling material is prepared by the following method steps:

[0093] 1. Filtration and sterilization of raw materials

[0094] 1L of recombinant human collagen with a molecular weight of 350-400kDa is filtered and sterilized through a sterilization filter to ensure aseptic operation;

[0095] 2. Pretreatment

[0096] 1) isoelectric point precipitation: the isoelectric point of the above filtered and sterilized recombinant human collagen is adjusted using an isoelectric point regulator, the isoelectric point regulator is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point regulator is 10.2, the isoelectric point is 5.5, and the isoelectric point precipitation is carried out after standing for 10h;

[0097] 2) Centrifugal separation: The mixture after precipitation at the isoelectric point was placed in a centrifuge with a fixed-angle rotor and centrifuged at a centrifugal force of 10000xg for 15 min. The centrifuged precipitate was scraped off, washed with water-injection detergent, and then placed in a centrifuge with a fixed-angle rotor and centrifuged at a centrifugal force of 10000xg for 15 min. The detergent was removed, and the washing was repeated once to obtain pretreated human collagen with a yield of 86.2%.

[0098] 3. Human collagen soft tissue filler

[0099] An osmotic pressure regulator was added to the pretreated recombinant human collagen. The amount of osmotic pressure regulator added was calculated based on the fact that the pretreated recombinant human collagen was in an isotonic state. Homogenization was then performed, followed by dispersion and shearing at 4500 rpm for 3 minutes, then at 9500 rpm for 3 minutes, and finally at 15000 rpm for 3 minutes to obtain human collagen soft tissue filler. Testing showed that this human collagen soft tissue filler still possessed a triple helix structure and maintained a molecular weight of 350–400 kDa under non-reducing conditions, as shown in Figure 5. More than 80% of the particle size was distributed in the range of 35–250 μm, with a span (SPAN) of 2.146. The particle size distribution range was relatively narrow, indicating good dispersibility and stable dispersion. According to the method in General Chapter 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 filler product was 299 mOsmol / kg.

[0100] The above description illustrates that the preparation method of the present invention maintains the triple helix structure and high molecular weight characteristics of the injectable human collagen soft tissue filler. The preparation method of the present invention has no significant impact on the performance of the product and can ensure the stability of the product performance. The injectable human collagen soft tissue filler of the present invention has a small particle size and a narrow distribution range, and has good dispersibility. This allows for more uniform distribution at the filling site during filling, avoiding local overfilling or underfilling, thereby improving the naturalness and aesthetics of the filling effect. At the same time, the stable particle size dispersion of the injectable human collagen soft tissue filler of the present invention can also reduce the risk of allergic reactions caused by excessively high local concentrations. In addition, it can maintain stability at the filling site for a longer period of time, thereby prolonging the duration of the filling effect.

[0101] Comparative Example 1

[0102] The specific steps for preparing an injectable human collagen soft tissue filler are as follows:

[0103] 1. Raw material filtration and sterilization

[0104] The recombinant human collagen 1L with molecular weight of 350-400 kDa is filtered by a sterilization filter to ensure sterile operation.

[0105] 2. Pretreatment

[0106] 1) Isoelectric point precipitation: The isoelectric point of the human collagen after the above sterilization filtration is adjusted using an isoelectric point adjusting agent composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point adjusting agent is 10.2. After the isoelectric point is adjusted to 4.7, the isoelectric point precipitation is performed by standing for 10 h.

[0107] During the standing process, it is found that the human collagen is gradually dissolved at the isoelectric point of 4.7, and the amount of the generated precipitate is very small. The pretreated recombinant human collagen obtained by the centrifugal separation condition of Example 1 has a yield of only 13.5%.

[0108] The subsequent preparation process is stopped.

[0109] Comparative Example 2

[0110] An injection type human collagen soft tissue filling material is prepared by the following method steps:

[0111] 1. Filtration and sterilization of raw materials

[0112] The recombinant human collagen 1L with molecular weight of 350-400 kDa is filtered by a sterilization filter to ensure sterile operation.

[0113] 2. Pretreatment

[0114] 1) Isoelectric point precipitation: The isoelectric point of the human collagen after the above sterilization filtration is adjusted using an isoelectric point adjusting agent composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point adjusting agent is 10.2. After the isoelectric point is adjusted to 4.2, the isoelectric point precipitation is performed by standing for 10 h.

[0115] During the standing process, it is found that the human collagen is gradually dissolved at the isoelectric point of 4.2, and the amount of the generated precipitate is very small. The pretreated recombinant human collagen obtained by the centrifugal separation condition of Example 1 has a yield of only 5.3%.

[0116] The preparation process is stopped.

[0117] Comparative Example 3

[0118] An injection type human collagen soft tissue filling material is prepared by the following method steps:

[0119] 1. Filtration and sterilization of raw materials

[0120] Take the molecular weight of 150-250 kDa of recombinant human collagen, take 1L amount, filter sterilization through sterilization filter to ensure sterile operation;

[0121] 2, pretreatment

[0122] 1) isoelectric point precipitation: the isoelectric point of the above-mentioned human collagen after sterilization filtration is adjusted using isoelectric point adjuster, the isoelectric point adjuster is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point adjuster is 11, the isoelectric point is 9.2, and the isoelectric point precipitation is carried out after standing for 10h; During the standing process, it was found that under the isoelectric point of 9.2, the precipitation appeared agglomeration phenomenon;

[0123] 2) centrifugal separation: the mixture after the above-mentioned isoelectric point precipitation is placed in a centrifuge with a fixed angle rotor and centrifuged at a centrifugal force of 30000xg for 30min, the centrifugal 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 40000xg for 30min, the detergent is removed, and the washing is repeated once, to obtain the pretreated human collagen, 93.1%;

[0124] 3, injection type human collagen soft tissue filling material

[0125] The above-mentioned pretreated recombinant human collagen is added with osmotic pressure regulator, the amount of osmotic pressure regulator is calculated according to the isosmotic state of the whole pretreated recombinant human collagen, and then homogenized, and then homogenized, homogenized at a pressure of 60bar for 7s, then pressurized to 255bar at a rate of 5bar / s for 5.5s, and then pressurized to 390bar at a rate of 5bar / s for 2s, to obtain the human collagen soft tissue filling material, which still has triple helix structure, maintains the molecular weight of 150-250kDa under non-reducing conditions, and the particle size of more than 80% is distributed in the range of 205-588μm, the span is 4.223, the particle size distribution range is wide, and the dispersity is relatively poor, and the agglomeration of the precipitate also affects the dispersion stability; According to the method of Chinese Pharmacopoeia (2020 edition) four general rules 0632, the osmotic pressure of the product is 310mOsmol / kg;

[0126] The isoelectric point used in the isoelectric point precipitation process of the recombinant human collagen of the comparative example is 9.2, and the precipitation appears agglomeration phenomenon, so that the particle size of the injection type human collagen soft tissue filling material is large, and the particle size distribution is also wide, and the filling effect is relatively poor.

[0127] Comparative example 4

[0128] An injection type human collagen soft tissue filling material, the preparation method steps are as follows:

[0129] 1. Raw material filtration and sterilization

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

[0131] 2. Pretreatment

[0132] 1) Isoelectric point precipitation: adjust the isoelectric point of the human collagen after filtration and sterilization by using an isoelectric point adjuster, the isoelectric point adjuster is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and water, and the pH of the isoelectric point adjuster is 10.2, the isoelectric point is 8.0, and the isoelectric point precipitation is carried out after standing for 8 hours;

[0133] 2) Centrifugal separation: place the mixture after isoelectric point precipitation in a centrifuge with a fixed angle rotor and centrifugal sedimentation separation at 15000xg for 25min, scrape the centrifugal sediment, wash with physiological saline detergent, and place in a centrifuge with a fixed angle rotor and centrifugal washing separation at 25000xg for 25min, remove the detergent, repeat the washing once, and obtain the pretreated recombinant human collagen with a yield of 80.5%;

[0134] The remaining steps are the same as those in Example 3, and an injection type human collagen soft tissue filling material is obtained. The injection type 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 has a particle size of 80% or more distributed in the range of 41-211μm, a span of SPAN of 1.936, good dispersity, and stable dispersion. According to the method of General Chapter 0632 of the People's Republic of China Pharmacopoeia (2020 edition), the product shows an osmotic pressure of 291 mOsmol / kg.

[0135] It is found through the present comparative example that the isoelectric point precipitation time is relatively short, resulting in less human collagen obtained, and low human collagen yield.

[0136] In addition, the isoelectric point precipitation time is too long, which increases the risk of product sterility, so the isoelectric point precipitation time should not be too long.

[0137] Comparative Example 5

[0138] An injection type human collagen soft tissue filling material, the preparation method steps are as follows:

[0139] 1. Raw material filtration and sterilization

[0140] The recombinant human collagen 1L with a molecular weight of 100-150 kDa is filtered through a sterilization filter to ensure sterile operation;

[0141] 2. Pretreatment

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

[0143] 2) Centrifugal separation: The mixture after isoelectric point precipitation is placed in a centrifuge with a horizontal rotor and centrifuged at a centrifugal force of 8000xg for 25 min. The centrifuged precipitate is scraped and washed with a physiological saline detergent, and then placed in a centrifuge with a horizontal rotor and centrifuged at a centrifugal force of 8000xg for 25 min. The detergent is removed and the washing is repeated once to obtain the pretreated recombinant human collagen 71.3%.

[0144] The remaining steps are the same as in Example 3. The injection type human collagen soft tissue filling material is obtained. The injection type 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 has a particle size of 80% or more distributed in the range of 54-209 μm, a span of SPAN of 1.847, and good dispersity and dispersion stability. According to the method of General Chapter 0632 of the People's Republic of China Pharmacopoeia (2020 Edition), the osmotic pressure of the product is 306 mOsmol / kg.

[0145] It is found through the present comparative example that the centrifugal force of the centrifuge with a horizontal rotor is insufficient, which affects the separation and collection of human collagen, and the separation effect is poor and the yield is low.

[0146] Comparative Example 6

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

[0148] 3. Injection type human collagen soft tissue filling material

[0149] The pretreated recombinant human collagen is added with an osmotic pressure adjuster, and the amount of the osmotic pressure adjuster is calculated according to the isosmotic state of the pretreated recombinant human collagen as a whole. Then, homogenization is performed, and the human collagen soft tissue filling material is obtained by homogenization at a pressure of 400 bar for 90 s.

[0150] The remaining steps are exactly the same;

[0151] The injection type 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 has a particle size of 80% or more distributed in the range of 52-434 μm, a span of 3.156, a relatively wide particle size distribution range, and a relatively poor dispersibility. The injection type human collagen soft tissue filling material product has an osmotic pressure of 310 mOsmol / kg, as determined according to the method of General Chapter 0632 of the Pharmacopoeia of the People's Republic of China (2020 edition). The injection type human collagen soft tissue filling material of the present comparative example has a relatively large particle size and a relatively wide particle size distribution, which affects the deep skin penetration effect and makes the filling effect relatively poor.

[0152] Comparative Example 7

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

[0154] 3. An injection type human collagen soft tissue filling material

[0155] The osmotic pressure regulator is added to the pretreated recombinant human collagen, and the amount of the osmotic pressure regulator is calculated according to the isotonic state of the pretreated recombinant human collagen as a whole. Then, homogenization treatment is performed, and then homogenization treatment is performed. The dispersion shearing is performed at a rotation speed of 15000 rpm for 6 min to obtain an injection type human collagen soft tissue filling material.

[0156] The remaining steps are exactly the same.

[0157] The injection type 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 has a particle size of 80% or more distributed in the range of 52-434 μm, a span of 3.156, a relatively wide particle size distribution range, and a relatively poor dispersibility. The injection type human collagen soft tissue filling material product has an osmotic pressure of 310 mOsmol / kg, as determined according to the method of General Chapter 0632 of the Pharmacopoeia of the People's Republic of China (2020 edition). The injection type human collagen soft tissue filling material of the present comparative example has a relatively large particle size and a relatively wide particle size distribution, which affects the deep skin penetration effect and makes the filling effect relatively poor.

[0158] It is found from Comparative Examples 6 and 7 that, under the conditions of constant high-pressure homogenization and constant rotation speed dispersion shearing, although the particle size of the injection type human collagen soft tissue filling material meets the requirements of the industry standard, the particle size distribution of 80% of the particle size is relatively wide, the particle size is relatively large, the filling effect is relatively poor, the facial expression naturalness is affected, and in addition, it is not easy to penetrate into the deep skin, which affects the absorption and utilization of human collagen.

[0159] Comparative Example 8

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

[0161] 3. The injectable human collagen soft tissue filling material

[0162] The osmolality regulator is added to the pretreated recombinant human collagen, the amount of the osmolality regulator is calculated according to the osmolality of the whole pretreated recombinant human collagen, and then homogenization treatment is performed, and then homogenization treatment is performed, and then dispersed emulsification is performed at a rotation speed of 2000 rpm for 20 min to obtain the injectable human collagen soft tissue filling material;

[0163] The remaining steps are exactly the same;

[0164] It is detected that 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 has a particle size of 80% or more distributed in the range of 802-1507 μm, a span of 9.245, a wide particle size distribution range, and poor dispersibility, which is more unfavorable for dispersion stability; according to the method of Chinese Pharmacopoeia (2020 edition) four general rules 0632, it is shown that the osmolality of the injectable human collagen soft tissue filling material product is 305 mOsmol / kg.

[0165] It is found by the present comparative example 8 that the particle size of the human collagen soft tissue filling material obtained by dispersed emulsification does not meet the requirements of the industry standard, and the particle size distribution of 80% of the particle size is wide, the particle size is large, the filling effect is poorer, and local filling is more likely to be excessive, and even cause allergy.

[0166] In summary, the human collagen tissue filling material of the present application mainly uses human collagen, has a large molecular weight and a triple helix structure, has uniform particle size distribution, and can play a filling and supporting effect; the preparation process is simple and convenient, does not require special treatment process, and does not introduce toxic and harmful residual substances, so that the product performance is stable; the human collagen tissue filling material prepared by the present application has good stability, excellent biocompatibility, no virus hidden danger, and good soft tissue filling and repair effect.

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

[0168] Furthermore, it should be understood that although the specification is described in terms of embodiments, not every embodiment includes every feature described. The specification can include implicit combinations of explicitly mentioned features and / or explicit combinations of implicitely mentioned features. Each embodiment depends on the explicit combinations of features and / or the implicit combinations of features made specifically within that embodiment, and each such embodiment can be combined with every other such embodiment to create further embodiments.

Claims

1. An injectable human collagen soft tissue filler material, characterized in that, The injection type human collagen soft tissue filling material comprises pre-processed recombinant human collagen and an osmotic pressure regulator, and is in an isotonic state. The pre-processing of the recombinant human collagen is isoelectric point precipitation and centrifugal separation.

2. The injectable human collagen soft tissue filler material according to claim 1, wherein, More than 80% of the injection type human collagen soft tissue filling material has a particle size distribution of 25-280 μm.

3. The injectable human collagen soft tissue filler material of claim 1, wherein, The molecular weight of the recombinant human collagen is 100-400 kDa.

4. The injectable human collagen soft tissue filler material of claim 1, wherein, The osmotic pressure regulator is one of potassium chloride and sodium chloride.

5. A method for preparing the injectable human collagen soft tissue filler material according to any one of claims 1 to 4, characterized in that, The preparation method comprises the following steps: S1, raw material filtration and sterilization The recombinant human collagen is filtered and sterilized by a sterilization filter; S2, pre-processing S2.1, isoelectric point precipitation: the isoelectric point of the filtered and sterilized recombinant human collagen is adjusted by using an isoelectric point regulator, and after the isoelectric point reaches 5-9, the mixture is left to stand for 10-20 hours for isoelectric point precipitation; S2.2, centrifugal separation: the mixture after isoelectric point precipitation is centrifugally separated, the centrifugal precipitate is scraped, a washing agent is used for washing and centrifugal washing separation, the washing agent is removed, and the washing is repeated once to obtain pre-processed recombinant human collagen; S3, injection type human collagen soft tissue filling material The pre-processed recombinant human collagen is added with an osmotic pressure regulator, the amount of the osmotic pressure regulator is calculated according to the isotonic state of the whole pre-processed recombinant human collagen, and then homogenization treatment is performed to obtain the injection type human collagen soft tissue filling material.

6. The method of claim 5, wherein the collagen is human collagen. The isoelectric point regulator in step S2.1 is composed of one or more of sodium phosphate dibasic, potassium phosphate dibasic, potassium phosphate monobasic, sodium phosphate monobasic, sodium hydroxide and potassium hydroxide and water, and the pH of the isoelectric point regulator is between 9 and 12.

7. The method of claim 5, wherein the collagen is human collagen. In step S2.2, the centrifugal force is 10000-30000xg and the centrifugal time is 10-60 minutes during centrifugal precipitation separation, and the centrifugal force is 10000-40000xg and the centrifugal time is 10-60 minutes during centrifugal washing separation; the centrifugal rotor used in the above process is a fixed-angle rotor; and the washing agent in step S2.2 is one of injection water, physiological saline or phosphate buffer.

8. The method for preparing an injectable human collagen soft tissue filler according to claim 5, characterized in that, The homogenization treatment in step S3 adopts one of dispersion shearing or high-pressure homogenization.

9. A method for preparing an injectable human collagen soft tissue filler according to claim 5 or 8, characterized in that, The homogenization treatment in step S3 adopts high-pressure homogenization; the high-pressure homogenization conditions are: first homogenization at a pressure of 50-60 bar for 7-8 seconds, then homogenization at a pressure of 250-260 bar for 4-5 seconds, and then homogenization at a pressure of 390-400 bar for 2-3 seconds, and the pressure increasing rate is 5 bar / s.

10. A method for preparing an injectable human collagen soft tissue filler according to claim 5 or 8, characterized in that, The homogenization treatment in step S3 adopts dispersion shearing, and the dispersion shearing conditions are: first dispersion shearing at a rotation speed of 4000-4500 rpm for 1-3 minutes, then dispersion shearing at a rotation speed of 9000-9500 rpm for 1-3 minutes, and then dispersion shearing at a rotation speed of 14500-15000 rpm for 1-3 minutes.

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