WO2023143392A1

WO2023143392A1 - Cross-linking agent residue-free collagen filler for injection and preparation method therefor

Info

Publication number: WO2023143392A1
Application number: PCT/CN2023/073215
Authority: WO
Inventors: 范代娣; 古娟; 史静静; 段志广; 徐茹; 严建亚; 刘琳
Original assignee: 陕西巨子生物技术有限公司
Priority date: 2022-01-27
Filing date: 2023-01-19
Publication date: 2023-08-03
Also published as: CN114404668B; CN114404668A

Abstract

Disclosed are a cross-linking agent residue-free collagen filler for injection and a preparation method therefor. The collagen filler for injection comprises collagen cross-linked by carbodiimide (EDC), water for injection and an excipient, and is free of cross-linking agent residues, wherein the collagen is a macromolecular I-type recombinant collagen, and is composed of multiple repetitions of a short amino acid sequence from natural human I-type collagen as a repeating unit, the number of repetitions is 75-110 times, and the short amino acid sequence is GAPGAPGSQGAPGLQ. The I-type recombinant collagen has good stability. In the method for preparing the cross-linking agent residue-free collagen filler for injection, EDC is used for cross-linking the collagen, the used cross-linking agent has a low concentration, the operation is simple and easy, the cross-linking degree can be increased by means of secondary cross-linking, and EDC can be basically completely removed only by means of a simple cleaning operation using a phosphoric acid buffer solution after the cross-linking reaction is completed.

Description

Collagen filler for injection without cross-linking agent residue and preparation method thereof

technical field

The invention belongs to the field of biotechnology and relates to a recombinant collagen filler for injection. The recombinant collagen filler for injection basically does not contain a chemical cross-linking agent and has better biological safety.

Background technique

Collagen is a biological polymer protein, the main component of animal connective tissue, and also the most abundant and widely distributed functional protein in mammals, accounting for 25-30% of the total protein. Collagen is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging, etc. It is one of the most critical raw materials in the biotechnology industry. It is used in medical materials, cosmetics , Food industry are widely used.

The source of natural human collagen is limited. The natural collagen currently used in industry is mainly extracted from animal skin or bone by acid, alkali or enzymatic methods, and its main source is animal connective tissue. However, collagen extracted from animal tissues has risks such as animal-derived diseases, and at the same time, large-scale preparations have caused huge pressure on animal feeding on the supply side.

With the widespread application of genetic engineering technology, the bottleneck problem of large-scale production of human collagen has been successfully solved by using suitable engineering strains (Escherichia coli, Pichia pastoris, etc.) to express human collagen exogenously. However, when engineering strains such as Pichia pastoris genetically engineered strains are used to express human collagen, human collagen will be degraded during fermentation, purification and storage, which increases production costs and affects the human collagen produced by this method. performance.

It is speculated that the reason for the degradation of human collagen may be that its amino acid sequence contains more sites that are prone to hydrolysis. Therefore, technicians construct recombinant collagen by selecting and repeating short amino acid sequences from natural human collagen in order to avoid sites prone to hydrolysis, thereby improving the stability of collagen while maintaining the excellent properties of natural human collagen. performance. However, the amino acid composition and distribution of the recombinant collagen constructed by repeating the short amino acid sequence from natural human collagen is relatively monotonous. It is easy to reach a stable equilibrium state, so it is easy to hydrolyze and denature in aqueous solution, and there is a tendency that the shorter the repeating unit of the short amino acid sequence and the more the number of repetitions, the more unstable the recombinant collagen molecule is in aqueous solution.

One can attempt to obtain recombinant collagens that are more resistant to degradation by mutating short amino acid sequences derived from native collagen as repeating units. However, the homology between the recombinant collagen obtained by such mutation and natural human collagen is reduced, and immunogenicity problems may arise, so it is not suitable for use in biomaterials that need to be in long-term contact with the human body.

On the other hand, tissue engineering materials such as dermal fillers are an important application direction of human collagen. As tissue engineering materials, collagen is required to have good mechanical strength and stability in aqueous solution (it can be stored in aqueous solution for a long time) . In general, the greater the molecular weight of collagen, the better its mechanical strength, and the poorer its stability in aqueous solution, especially for recombinant collagen constructed by repeating short amino acid sequences from natural human collagen. in this way.

The single-chain molecular weight of natural human collagen is about 110-130kD. From a practical point of view, technicians generally believe that the molecular weight of collagen suitable for replacing natural human collagen as tissue engineering materials needs to reach more than 100kD. However, as mentioned above, the source of natural human collagen is limited, animal-derived collagen has the risk of spreading diseases, and human collagen expressed by genetic engineering is prone to degradation during fermentation, purification and storage. Recombinant collagen constructed by repeating short amino acid sequences is unstable in aqueous solution, and recombinant collagen obtained through mutational transformation has immunogenicity problems. Therefore, how to obtain collagen suitable for replacing natural human collagen as tissue engineering materials has become an important issue. Limiting issues in the technical field.

Moreover, those skilled in the art will improve the mechanical properties of the recombinant collagen and prolong its degradation time in vivo through cross-linking. However, for collagen injection products, the cross-linking agents used for cross-linking (such as glutaraldehyde, etc.) will eventually enter with the collagen and exist in the human body for a long time, and these cross-linking agents will be toxic to the human body. In this case, those skilled in the art expect to find a suitable recombinant collagen, so that the cross-linking reaction to the recombinant collagen is simple and easy, the cross-linking efficiency is high, and the cross-linking agent can be basically completely removed through simple operations.

Contents of the invention

In order to solve the above-mentioned technical problems existing in the prior art, the inventors conducted in-depth research. The inventors first conducted a technical literature survey on recombinant collagens constructed by repeating short amino acid sequences from natural human collagen, and selected some of the existing technologies from natural human type I collagen. White (the most widely used tissue engineering material) short amino acid sequences, and then use these short amino acid sequences as repeating units to construct recombinant collagens with different molecular weights, and investigate the long-term storage stability of these recombinant collagens in aqueous solution, in order to obtain A large molecular weight (above 100kD) recombinant collagen that can be stored stably in aqueous solution for a long time and can meet the mechanical strength requirements of tissue engineering materials.

As a result of the above research, the inventor unexpectedly discovered that by repeating a section of pentadecadecanoid (G A P G A P G S Q G A P G L Q) from natural human type I collagen 75 to 110 times, The obtained recombinant type I collagen has exceptionally excellent stability. It is embodied in: (1) although the length of its repeating unit is the shortest among all recombinant collagens tested by the inventor, its stability in aqueous solution is the best; The more monotonous the distribution, the greater the surface charge load of the recombinant collagen thus constructed, the less likely it is to reach a stable equilibrium state, and thus the easier it is to hydrolyze. (2) It is even more stable than the recombinant type I collagen obtained by repeating the pentapenteptide 52 times, 62 times or 72 times, and it is generally believed that the more the number of repetitions, the greater the molecular weight, and the surface of the recombinant collagen The greater the charge load, the less likely it is to reach a stable equilibrium state, and thus more susceptible to hydrolysis.

Based on the above findings, the inventors have accomplished the present invention. That is, the present invention includes:

1. A macromolecular type I recombinant collagen, which is composed of a short amino acid sequence from natural human type I collagen as a repeating unit, wherein the short amino acid sequence is as SEQ ID No.: 1( G A P G A P G S Q G A P G L Q), the number of repetitions is 75 to 110 times.

2. The macromolecular type I recombinant collagen according to item 1, wherein the number of repetitions is 80-105 times, preferably 82-102 times.

3. The macromolecular type I recombinant collagen according to item 1, which has a molecular weight of more than 100 kD.

4. The macromolecular type I recombinant collagen according to item 1, further bearing a tag for easy purification.

5. The macromolecular type I recombinant collagen according to item 4, wherein the tag is a His tag, a Flag tag or a c-Myc tag.

6. Use of the macromolecular type I recombinant collagen according to any one of items 1 to 5 as a tissue engineering material.

7. The use according to item 6, wherein the tissue engineering material is selected from the group consisting of dermal fillers, artificial bone, artificial skin, oral cavity absorbable biofilms, bone implants, vascular scaffolds, intercellular matrix scaffolds and collagen sponge.

8. The macromolecular type I recombinant collagen according to any one of items 1 to 5 is used as a subcutaneous filler, artificial bone, artificial skin, oral cavity absorbable biofilm, bone implant, vascular scaffold, and intercellular matrix scaffold and the use of collagen sponge.

The inventors are conducting more in-depth research on the reason why the macromolecular type I recombinant collagen of the present invention has exceptionally excellent stability. Preliminary research results show that this may be because: the stability of recombinant collagen that repeats with a certain amino acid sequence as a repeating segment is closely related to the surface charge, and the surface charge is related to the composition of amino acids and the spatial structure of the protein. After a specific number of repetitions, a certain spatial structure is just formed, so that the surface load is in a balanced or near-balanced state, so it will show an abnormally stable state. The inventor just found that the 15 amino acid repeat sequence collagen is in the range of load balance, so the macromolecular type I recombinant collagen of the present invention has exceptionally excellent stability.

Moreover, the inventors also found that the macromolecular type I recombinant collagen of the present invention is particularly suitable for cross-linking using EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide). Using EDC to cross-link the macromolecular type I recombinant collagen of the present invention, the concentration of the cross-linking agent used is low, the operation is simple and easy, and the degree of cross-linking can be improved through secondary cross-linking, and after the completion of the cross-linking reaction, only a simple The phosphate buffer washing operation can basically completely remove EDC (according to the determination of carbodiimide residues in the fourth general rule of "Pharmacopoeia of the People's Republic of China" 3206, the carbodiimide residues are not detected).

Therefore, the present invention also includes:

4-1. A collagen filler for injection, which comprises collagen cross-linked by EDC, water for injection and auxiliary materials.

4-2. The collagen filler for injection according to claim 4-1, wherein the collagen is a macromolecular type I recombinant collagen composed of a short amino acid sequence from natural human type I collagen as a repeating unit Repeated multiple times, wherein the short amino acid sequence is shown in SEQ ID No.: 1 (GA P G A P G S Q G A P GL Q), and the number of repetitions is 75 to 110 times.

4-3. The collagen filler for injection according to claim 4-2, wherein the number of repetitions is 80-105 times, preferably 82-102 times.

4-4. The collagen filler for injection according to claim 4-2, wherein the molecular weight of the macromolecular type I recombinant collagen is above 100 kD.

4-5. The collagen filler for injection according to claim 4-2, wherein the macromolecular type I recombinant collagen is also provided with a label for easy purification.

4-6. The collagen filler for injection according to claim 4-2, wherein the Oita The recombinant type I collagen is tagged with His tag, Flag tag or c-Myc tag to make it easy to purify.

4-7. The collagen filler for injection according to claim 4-1, which substantially does not contain carbodiimide (EDC). Here, basically not containing carbodiimide means that according to the measurement of carbodiimide residue in the fourth general rule of "Pharmacopoeia of the People's Republic of China" 3206, the carbodiimide residue is not detected.

4-8. The collagen filler for injection according to claim 4-1, wherein the adjuvant is selected from phosphate buffer, lidocaine (for example 0.3w/v%), vitamins and/or high Molecular microspheres (eg PVA microspheres or protein microspheres).

4-9 The preparation method of the collagen filler for injection according to any one of claims 4-1 to 4-8, comprising:

Step 1: Dissolve collagen in 0.05-0.5mM EDC solution prepared with water for injection, and perform cross-linking to obtain a primary cross-linking solution; wherein, the cross-linking temperature is -80°C to 10°C, and the cross-linking time is 10 ~48 hours;

Step 2: Freeze-dry the primary cross-linking solution to obtain a sponge 1;

Step 3: Soak the sponge 1 in a 1mM-20mM EDC solution prepared with 50%-99vol% ethanol aqueous solution, and cross-link at room temperature for 5-48 hours to obtain a secondary cross-linked product, which is a gel shape solid;

Step 4: freeze-drying the secondary cross-linked product to obtain a sponge 2;

Step 5: crushing the sponge 2 into particles with a diameter of 0.2-1 mm;

Step 6: Wash the particles in 50-1200 times the volume of phosphate buffer or water for injection, and change the solution every 1-120 minutes, accumulatively 5-15 times;

Step 7: Swell the particles washed in step 6 in phosphate buffer solution so that the collagen content is 20-150 mg/mL to obtain the collagen filler for injection.

4-10. The preparation method according to claims 4-8, wherein the osmotic concentration of the phosphate buffer in the step 7 is 270mOsmol/L-350mOsmol/L.

4-11. The preparation method according to claims 4-8, wherein after the step 7, a moist heat sterilization step is further included, and the conditions of the moist heat sterilization are 110-130° C. for 30-60 minutes.

4-12 The preparation method according to claims 4-8, wherein the collagen is macromolecular type I recombinant collagen, which is composed of a short amino acid sequence from natural human type I collagen as a repeating unit for multiple repetitions , wherein the short amino acid sequence is shown in SEQ ID No.: 1 (G A P G A P G S Q G A P G L Q), and the number of repetitions is 75-110 times.

Description of drawings

FIG. 1 is an SDS-PAGE electrophoresis image of a portion of type I recombinant collagen prepared in Example 1. Take No.4, 6, 7, and 10 as examples for control proteins.

Fig. 2 is the HPLC picture of part of the test samples prepared in Example 2 after standing for 12 months. Take No.4, 7, 10, and 13 as examples for control proteins.

Fig. 3 is the infrared spectrogram of No.1 and No.6 type I recombinant collagen.

Fig. 4 is the Raman spectrum of No.1 and No.6 type I recombinant collagens.

Detailed ways

The present invention will be described in detail through specific examples below. It should be pointed out that these descriptions are only exemplary descriptions and do not limit the scope of the present invention.

General description: the enzymes used in the specific embodiment are all purchased from TaKaRa Company, the plasmid DNA extraction kit and the DNA gel recovery kit are purchased from Beijing Suolaibao Company, and the gene recombination kits (Reorganization Kits) are purchased from For Tiangen Biology, the specific operation was carried out in accordance with the instructions of the kit.

Example 1. Preparation of Various Type I Recombinant Collagens Using Yeast Expression System

1. Construction of yeast expression strains

Yeast expression strains expressing No. 1-18 type I recombinant collagen shown in Table 1 were constructed. The specific operation is: after optimization according to the codon preference of Pichia pastoris, synthesize the corresponding target gene by whole gene synthesis, and add SnaBI and Not I restriction sites at both ends of the gene respectively, and use SnaBI and NotI enzymes to synthesize the corresponding target gene. The target gene was double-enzymatically digested, and then ligated with pPIC9k, which was also digested with SnaB I and Not I enzymes, under the action of T4 ligase. After overnight ligation at 16°C, it was transferred into Top10 competent cells and coated with ampicillin-resistant plates. Pick the positive transformant, extract the plasmid, linearize it with SacI, transfer it into Pichia pastoris GS115 competent cells by electric shock, and screen the multi-copy transformant with G418 resistance plate, which is the expression strain of type I recombinant collagen.

Table 1 Type I recombinant collagen expressed by each yeast expression strain

2. Induced expression of target protein

(1) Pick a single colony of the yeast expression strain and add it to 5ml YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), cultivate overnight at 30°C and 200rpm for activation;

(2) inoculate 100 ml of BMGY liquid medium with 1% inoculum size, cultivate at 30°C and 200 rpm until OD600=6.0-9.0;

(3) Under the action of 1500g centrifugal force, centrifuge at 25°C for 6min to collect the bacteria, and suspend it in 200ml BMMY liquid medium, so that the initial concentration is OD600=1.0, at 30°C, Cultivate under the condition of 200rpm;

(4) Methanol was added every 24 hours, with a final concentration of 0.5-1.0%, to induce expression;

(5) After induction for 72 hours, the culture solution was taken and centrifuged at 12,000 rpm for 2 minutes, and the supernatant was taken.

3. Preparation of type I recombinant collagen

After the supernatant prepared by fermentation is concentrated by 30kD ultrafiltration membrane, it is separated by CM ion exchange column, eluted with 35% NaCl solution, and the eluate is collected, desalted and concentrated, and then freeze-dried to obtain type I Preparation of recombinant collagen. Take 0.1g of lyophilized powder and dissolve it in 100ml of normal saline, fully dissolve it and perform SDS-PAGE gel electrophoresis to confirm the molecular weight and protein electrophoresis purity.

The results showed that all the 18 expression strains constructed could successfully express the target protein, and the electrophoresis purity of the prepared protein after separation and purification was all above 99%. The electrophoresis results are shown in Figure 1 .

Embodiment 2: Stability experiment of various type I recombinant collagens in aqueous solution

AExperimental material

The materials used in the experiment were type I recombinant collagen No.1--18 prepared in Example 1.

BExperimental method

Prepare the experimental material in A with ddH2O to form a protein solution with a protein concentration of 1 mg/mL, filter it with a 0.22 μm sterile filter in a clean bench, then pack it into a sterile centrifuge tube and seal it, and place it at 25 °C ± 2 Under the condition of ℃, samples were taken at 0 month, 6 months, and 12 months respectively, and 3 tubes were sampled each time, and the protein purity was detected (protein purity was determined by high performance liquid chromatography), and the stability of the protein was determined according to the change of purity.

C Experimental results

The test results are as follows:

Table 2 Reconstituted collagen solution 12 months stability test result (purity, %)

It is generally believed that (1) for recombinant collagen with the same or similar molecular weight, the shorter the repeating unit, the more monotonous the amino acid composition and distribution, the greater the surface charge load, the less likely it is to reach a stable equilibrium state, and thus easier to hydrolyze; (2) For recombinant collagen with the same repeating unit, the more repetitions, the greater the molecular weight, the greater the surface charge load of the recombinant collagen, the less likely it is to reach a stable equilibrium state, and thus the easier it is to hydrolyze.

However, as can be seen from Table 2, the type I recombinant collagen (No.1～3) of the present invention shows exceptionally excellent stability in aqueous solution, and it is placed in aqueous solution for 12 months, and the purity can still be as high as more than 97% (see Figure 2A-G).

Example 3: Preliminary research on the reasons why type I recombinant collagen No. 1-3 has abnormal stability in aqueous solution

1) Infrared Spectroscopy Determination

After the recombinant collagens No.1 and No.6 in Table 1 were prepared into solutions, infrared spectroscopy was measured, and the spectral data was deconvoluted by Fourier with Bruker OPUS7.2, and the spectrum in the 1700-1600cm-1 band was intercepted For the data, use peakfit v4.12 to perform second-order derivative peak fitting processing, use orgin to plot the processed data to obtain the secondary structure distribution, and calculate the relative content of the secondary structure quantity. The comparison results of the secondary structures of the two proteins are shown in Table 3 and Figure 3.

The secondary structure of No.1 and No.6 recombinant collagen of table 3 infrared spectrum determination

2) Raman Spectroscopy Determination

After the recombinant collagens No.1 and No.6 in Table 1 were prepared into solutions, the Raman spectrum was measured, and the spectral data was smoothed and corrected with ThermoFisher Omnic9.2, and the spectral data in the 1700-1600cm-1 band was intercepted , use peakfit v4.12 to perform second-order derivative peak fitting processing, use orgin to plot the processed data to obtain the secondary structure distribution, and calculate the relative content of the secondary structure. The comparison results of the secondary structures of the two proteins are shown in Table 4 and Figure 4.

The secondary structure of No.1 and No.6 recombinant collagen determined by Raman spectroscopy in table 4

It can be seen from the measurement results of Example 3 that the recombinant collagens with the same repeating unit but different repeating numbers have large differences in secondary structures, which also implies that there are differences in the tertiary structures of the two. It can be speculated that the spatial structure of Type I recombinant collagen No. 1-3 makes the surface charge more balanced, thus showing good stability in aqueous solution.

Embodiment 4: Preparation 1 of collagen filler for injection

Step 1: Fully dissolve EDC in water for injection at 0.2mM, filter and sterilize through a 0.22μm filter membrane before subsequent use.

Step 2: Fully dissolve the recombinant collagen of No. 2 above at 50 mg/ml in the cross-linking agent prepared in Step 1. The protein dissolves completely and becomes a uniform and clear solution;

Step 3: place the solution prepared in step 2 at -20°C for 48 hours and then carry out vacuum freeze-drying to obtain a white spongy protein freeze-dried product; Step 4: soak the protein freeze-dried product to 90 volumes prepared with water for injection % pharmaceutical grade ethanol solution to prepare a 5mM EDC solution, carry out secondary cross-linking, and the cross-linking time is 36h to obtain a milky white gel-like solid.

Step 5: After freeze-drying the milky white gel-like solid, crush it to a diameter of 0.5mm particles, and then wash to remove the cross-linking agent: phosphate buffer (dissolve 18 mg of potassium dihydrogen phosphate, 60 mg of disodium hydrogen phosphate, and 900 mg of sodium chloride in 100 ml of water for injection), wash to remove the cross-linking agent residue, and the washing ratio is 1:1000, the stirring speed is 150rpm, the washing frequency is 13 times, and the liquid is changed every 40min.

Step 6: After washing to remove the cross-linking agent, add phosphate buffer to swell according to the ratio of collagen freeze-dried product: phosphate buffer = 1:5, and form a gel (the content of collagen in this gel is 50 ± 5 mg/mL ), filled and sterilized by moist heat (121°C, 21min).

Embodiment 5: Preparation 2 of collagen filler for injection

Step 1: Fully dissolve EDC in water for injection according to the ratio of 0.1mM, filter and sterilize through a 0.22μm filter membrane before subsequent use.

Step 2: Fully dissolve the above-mentioned No.1 recombinant collagen at 60 mg/ml in the cross-linking agent prepared in Step 1, the protein is completely dissolved, and a uniform and clear solution is formed;

Step 3: Place the solution prepared in Step 2 at -40°C for 36 hours for cross-linking, then perform vacuum freeze-drying to obtain a white spongy protein freeze-dried product;

Step 4: Soak the lyophilized protein into 8mM EDC solution prepared with 80% by volume pharmaceutical grade ethanol solution prepared with water for injection, and carry out secondary cross-linking. .

Step 5: Freeze-dry the milky white gel-like solid, pulverize it into particles with a diameter of 0.5mm, and then wash to remove the cross-linking agent: wash with phosphate buffer and water for injection to remove the cross-linking agent residue, and the washing ratio is 1: 800, the stirring speed is 150rpm, the number of washings is 16 times, the 1st-10th time is washed with water for injection, the 10th-16th time is washed with phosphate buffer solution, and the medium is changed every 40min.

Embodiment 6: Preparation 3 of collagen filler for injection

Step 1: Fully dissolve EDC in the water for injection at a ratio of 0.15mM, filter and sterilize through a 0.22μm filter membrane before subsequent use.

Step 2: Fully dissolve the recombinant collagen of No. 3 above at 45 mg/ml in the cross-linking agent prepared in Step 1. The protein dissolves completely and becomes a uniform and clear solution;

Step 3: Place the solution prepared in Step 2 at 4°C for 48 hours to cross-link, then vacuum freeze-dry to obtain a white spongy protein freeze-dried product;

Step 4: Soak the lyophilized protein into a 3mM EDC solution prepared from a 95% by volume pharmaceutical grade ethanol solution prepared with water for injection, and perform secondary cross-linking for 48 hours to obtain a cross-linked milky white gel solid.

Step 5: Freeze-dry the obtained milky white gel-like solid, pulverize it into particles with a diameter of 0.8 mm, and then wash to remove the cross-linking agent: wash with phosphate buffer and water for injection to remove the cross-linking agent residue, and the washing ratio is 1: 1100, the stirring speed is 100rpm, the number of washings is 12 times, the 1st-8th time is washing with water for injection, the 10th-12th time is washing with phosphate buffer solution, and the medium is changed every 30min.

Example 7: Detection of cross-linking agent EDC residues

The preparation of sample: get the filling of embodiment 4,5,6, the collagen protein filler sample after damp heat sterilization, prepare the pancreatin of 2mg/ml with sterile water, get 0.2ml collagen protein filler sample and add 0.2ml The prepared trypsin was placed at 37°C for 24 hours, and the protein gel in the collagen filler was digested by trypsin into water-soluble peptides. According to "Chinese Pharmacopoeia" 2020 edition four general rules 3206 carbodiimide residue determination method for detection.

Reagent preparation

Dimethyl barbituric acid test solution: Weigh 1 g of dimethyl barbituric acid in 16 ml of pyridine, add water to 20 ml, and mix well. Ready to use.

Pyridine acetate solution: Prepare by mixing equal volumes of glacial acetic acid and pyridine, and prepare it immediately before use.

100μmol/L carbodiimide reference substance stock solution: Weigh 0.0192g carbodiimide, dissolve it in water and set the volume to 100ml to obtain a 1mmol/L solution, measure 1mmol/L solution in a 10ml measuring bottle, add water to set Fill up to the mark to obtain 100 μmol/L carbodiimide reference substance stock solution. Ready to use.

Preparation of working solution of carbodiimide reference substance: take 100 μmol/L carbodiimide reference substance stock solution, dilute it with water to 10 μmol/L, 20 μmol/L, 40 μmol/L, 60 μmol/L, 80 μmol/L, namely Carbodiimide reference substance working solution.

Determination method: Take 0.2ml of collagen filler sample extract and carbodiimide reference substance working solution, add 1.8ml of dimethyl barbituric acid test solution respectively; take another 0.2ml of water as a blank control, and perform the same method do. Mix the tubes evenly, let stand in the dark at room temperature for 30 minutes, add 2.0ml of pyridine acetate solution, and measure the absorbance at a wavelength of 599nm after mixing (if there is interference in the test, centrifuge at 4000 rpm for 5 minutes before measuring the absorbance).

Calculation of results: The concentration of the working solution of the carbodiimide reference substance is used to perform a linear regression on the corresponding absorbance to obtain the linear regression equation. Substitute the absorbance of the test solution into the linear regression equation to find the content and take the average value.

Calculate according to the formula

Residual amount of carbodiimide in the test product (μmol/L) = average concentration of carbodiimide in the test solution × dilution factor

For the collagen filler samples of Examples 4, 5, and 6, the average absorbance of the residual carbodiimide was measured to be 0.431, 0.442, and 0.425, respectively, and the average absorbance of water as a blank control was 0.440. Therefore, the detection result of the residual amount of carbodiimide was not detected, that is, there was no crosslinking agent residue in the sample.

Claims

A collagen filler for injection, which comprises collagen cross-linked by carbodiimide (EDC), water for injection and auxiliary materials, wherein the collagen is macromolecular type I recombinant collagen derived from natural human type I The short amino acid sequence of collagen is repeated multiple times as a repeating unit, wherein the short amino acid sequence is shown in SEQ ID No.1, the number of repetitions is 75 to 110 times, and SEQ ID No.1 is G A P G A P G S Q G A P G L Q.
The collagen filler for injection according to claim 1, wherein the number of repetitions is 80-105 times.
The collagen filler for injection according to claim 2, wherein the number of repetitions is 82-102 times.
The collagen filler for injection according to claim 1, which substantially does not contain carbodiimide (EDC).
The collagen filler for injection according to claim 1, wherein the auxiliary material is selected from phosphate buffer, lidocaine, vitamins and/or polymer microspheres.
The collagen filler for injection according to claim 5, wherein the content of the lidocaine is 0.3w/v%.
The collagen filler for injection according to claim 5, wherein the vitamin and/or polymer microspheres are PVA microspheres or protein microspheres.
The preparation method of the collagen filler for injection according to any one of claims 1 to 7, comprising:

Step 1: Dissolve the collagen in 0.05-0.5mM EDC solution prepared with water for injection, and perform cross-linking to obtain a primary cross-linking solution; wherein, the cross-linking temperature is -80°C to 10°C, and the cross-linking time is 10 to 48 hours;

Step 2: Freeze-dry the primary cross-linking solution to obtain a sponge 1;

Step 3: Soak the sponge 1 in a 1mM-20mM EDC solution prepared with 50%-99vol% ethanol aqueous solution, and cross-link at room temperature for 5-48 hours to obtain a secondary cross-linked product, which is a gel shape solid;

Step 4: freeze-drying the secondary cross-linked product to obtain a sponge 2;

Step 5: crushing the sponge 2 into particles with a diameter of 0.2-1 mm;

Step 6: Wash the particles in 50-1200 times the volume of phosphate buffer or water for injection, and change the solution every 1-120 minutes, accumulatively 5-15 times;

Step 7: Swell the particles washed in step 6 in phosphate buffer solution so that the collagen content is 20-150 mg/mL to obtain the collagen filler for injection.
The preparation method according to claim 8, wherein the osmotic concentration of the phosphate buffer in the step 7 is 270mOsmol/L-350mOsmol/L.
The preparation method according to claim 8, wherein, after the step 7, a moist heat sterilization step is further included, and the conditions of the moist heat sterilization are 110-130° C. for 30-60 minutes.