A low immunogenic collagen, its preparation method and application

Abstract

The application discloses low immunogenic collagen and a preparation method and application thereof, and the preparation method comprises the following steps: (1) performing enzymolysis on animal skin; (2) performing salt precipitation treatment on the enzymolysis glue liquid; (3) adding organic amino phosphoric acid or a salt thereof to replace and purify the crude collagen protein prepared through the salt precipitation treatment after redissolving the crude collagen protein, and then performing salt precipitation treatment to obtain purified collagen protein; and (4) redissolving, dialyzing and freeze-drying the purified collagen protein to obtain low immunogenic collagen. Through replacement and removal of DNA impurity molecules on collagen molecular chains by using phosphate in organic amino phosphoric acid or the salt thereof, the amino in the organic amino phosphoric acid or the salt thereof can promote the combination of the phosphate and the collagen under the action of intermolecular hydrogen bonds between the amino and residual carboxyl on the collagen, and the amino and the phosphate play a synergistic effect, so that the removal effect of the DNA impurity in the collagen can be further improved, and the immunogenicity of the collagen is reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical material preparation, and in particular to a low-immunogenic collagen, its preparation method, and its application. Background Technology

[0002] Collagen is widely found in the connective tissues (skin, bones, tendons, ligaments, and sclera, etc.) of invertebrates and vertebrates, primarily serving a structural support role. It is the most abundant functional protein in animals, accounting for approximately 30% of the body's total protein. Due to its excellent structure and function, collagen has been widely used in pharmaceuticals, biomedicine, health care, and beauty treatments. Currently, industrially produced collagen is mostly extracted from the skin and bones of terrestrial mammals such as cattle and pigs. When extracting collagen from organisms using enzymes, impurities such as fats, calcium, and non-collagenous components in the biological materials used can affect the purity of the collagen, leading to a decrease in the overall extraction rate and poor immunogenicity. Therefore, certain pretreatment of the raw materials is necessary. Among the many influencing factors, collagen purity, removal of telopeptides, and residual DNA play a crucial role in the immunogenicity of telopeptide-removed collagen.

[0003] In the preparation of animal-derived collagen raw materials, collagen extraction is typically achieved through purification processes such as pre-treatment with decellularization, enzymatic hydrolysis, salting out, and dialysis. Numerous methods for extracting collagen from animal sources have been reported, for example:

[0004] Chinese patent CN113462736A discloses a method for preparing telopeptide-free collagen from pig skin. Using three-month-old SPF pig skin as raw material, the method involves defatting with a defatting agent, extracting collagen from the pig skin using enzymatic hydrolysis to remove telopeptides, purifying the collagen using salting out and ultrafiltration, and then freeze-drying to obtain sponge-like telopeptide-free collagen. However, this method yields a low collagen yield, and the resulting freeze-dried collagen product is dense and compact, suitable only for direct use in medical and tissue engineering materials. Its application in other fields or research faces challenges such as difficulty in dissolution.

[0005] Chinese patent CN101126104B discloses a method for preparing natural active collagen using an acid-enzyme combination. This invention employs an acid-enzyme combination, which can prepare both structurally intact, acid-soluble natural collagen and pepsin-soluble collagen with better biocompatibility from the same batch of raw materials. This method uses the supernatant and precipitate from acid extraction of pigskin as raw materials for collagen extraction, yielding two types of collagen with different properties. The yield is high, but the purity of the final collagen is low.

[0006] In summary, existing technologies for preparing collagen using enzymatic hydrolysis often suffer from drawbacks such as low purity, low yield, and limited application areas. While acid-enzymatic methods for extracting collagen by removing telopeptides can eliminate immunogenicity caused by telopeptide structure, animal-derived collagen still contains substances that trigger immune responses, including DNA, cells, and other proteins that were not completely removed during extraction. Residual DNA in biological materials can induce widespread immune responses in humans. Therefore, reducing the residual DNA content in biological materials has become a crucial technical indicator for effectively controlling the immunogenicity risk of a product. Currently, there are no reports on using organoaminophosphates or their salts as treatment agents to reduce residual DNA in collagen. Summary of the Invention

[0007] This invention overcomes the shortcomings of existing technologies and provides a low-immunogenic collagen, its preparation method, and its application. It uses Replaceable Purification Technology (RPT) to extract collagen, specifically by using organic aminophosphates or their salts to remove DNA impurities from the collagen, thereby preparing a low-immunogenic, high-purity collagen.

[0008] In a first aspect, the present invention provides a low-immunogenic collagen protein, wherein the low-immunogenic collagen protein is obtained by extracting collagen using an organic aminophosphate or its salt to remove DNA impurities from the collagen protein.

[0009] Further, the organic amino phosphate or its salt is selected from one or more of 2-aminoethyl phosphate, sodium 2-aminoethyl phosphate, sodium (4-amino-1-hydroxy-1-phospho-butyl)-phosphate, sodium (4-aminophenyl) phosphate, (4-aminophenyl) phosphate, or aminotrimethylene phosphate.

[0010] A second aspect of the present invention provides a method for preparing low-immunogenic collagen, the method comprising the following steps:

[0011] (1) Use an acidic solution of protease to enzymatically hydrolyze animal skin to obtain an enzymatic hydrolysate;

[0012] (2) The enzymatic hydrolysate obtained in step (1) is subjected to salt precipitation treatment to obtain crude collagen.

[0013] (3) The crude collagen obtained in step (2) is reconstituted to obtain a collagen solution. Organic amino phosphate or its salt is added to the solution, stirred, and then salted to obtain purified collagen.

[0014] (4) The purified collagen obtained in step (3) is reconstituted, dialyzed, and freeze-dried to obtain low-immunogenic collagen.

[0015] Furthermore, step (1) includes a pretreatment step of the animal skin.

[0016] In some embodiments of the present invention, step (1) is further included before step (1) by crushing the animal skin into granules.

[0017] In some embodiments of the present invention, step (1) is further included before step (1) by crushing the animal skin into particles with a particle size of 1-10 mm.

[0018] Furthermore, the raw material for the animal skin mentioned in step (1) is derived from the skin or tissue of animals containing collagen, such as cattle, pigs, fish, or rats.

[0019] In some embodiments of the present invention, the raw material for the animal skin in step (1) is pig skin.

[0020] Further, the enzymatic hydrolysis time in step (1) is 24h to 72h (24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h, 49h, 50h, 51h, 52h, 53h, 54h, 55h, 56h, 57h, 58h, 59h, 60h, 61h, 62h, 63h, 64h, 65h, 66h, 67h, 68h, 69h, 70h, 71h, 72h), preferably 24h to 48h.

[0021] Furthermore, the protease in step (1) is selected from one or more of pepsin, bromelain, papain or Aspergillus niger acid protease, preferably pepsin.

[0022] Further, the acid in the acidic solution in step (1) is selected from one or more of hydrochloric acid, acetic acid, phosphoric acid, lactic acid or citric acid, preferably one or more of hydrochloric acid, acetic acid or phosphoric acid.

[0023] Further, the concentration of the protease in the acidic solution is 0.2–2.0 mg / mL (e.g., 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL), preferably 0.3–1.5 mg / mL.

[0024] In some embodiments of the present invention, the concentration of the protease in the acidic solution is 0.3 mg / mL.

[0025] In some embodiments of the present invention, the concentration of the protease in the acidic solution is 0.5 mg / mL.

[0026] In some embodiments of the present invention, the concentration of the protease in the acidic solution is 1.0 mg / mL.

[0027] In some embodiments of the present invention, the concentration of the protease in the acidic solution is 1.5 mg / mL.

[0028] Further, the pH of the acidic solution in step (1) is 1-3.5 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5), preferably 1-3.

[0029] In some embodiments of the present invention, the pH of the acidic solution is 1.0.

[0030] In some embodiments of the present invention, the pH of the acidic solution is 1.5.

[0031] In some embodiments of the present invention, the pH of the acidic solution is 2.0.

[0032] In some embodiments of the present invention, the pH of the acidic solution is 3.0.

[0033] Furthermore, the salt precipitation treatment in steps (2)-(3) includes mixing the enzymatic hydrolysate or collagen solution with a precipitant and centrifuging.

[0034] Furthermore, the precipitant is selected from one or more of sodium chloride, ammonium sulfate, or sodium sulfate, preferably sodium chloride.

[0035] Further, the concentration of the precipitant is 0.1M-2.0M (e.g., 0.1M, 0.2M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2.0M), preferably 0.1M-1.0M, and more preferably 0.7M.

[0036] Further, the organic amino phosphate or its salt in step (3) is selected from one or more of 2-aminoethyl phosphate, sodium 2-aminoethyl phosphate, sodium (4-amino-1-hydroxy-1-phospho-butyl)-phosphate, sodium (4-aminophenyl) phosphate, (4-aminophenyl) phosphate or aminotrimethylene phosphate.

[0037] In some embodiments of the present invention, the organic amino phosphate or its salt in step (3) is sodium (4-amino-1-hydroxy-1-phospho-butyl)-phosphate.

[0038] In some embodiments of the present invention, the organic amino phosphate or its salt in step (3) is 2-aminoethyl phosphate.

[0039] In some embodiments of the present invention, the organic aminophosphate or its salt in step (3) is sodium 2-aminoethyl phosphate.

[0040] In some embodiments of the present invention, the organic aminophosphate or its salt in step (3) is sodium (4-aminophenyl)phosphate.

[0041] In some embodiments of the present invention, the organic amino phosphate or its salt in step (3) is (4-aminophenyl) phosphate.

[0042] Further, the concentration of the collagen solution in step (3) is 5 mg / mL to 20 mg / mL (e.g., 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL), preferably 5 mg / mL to 15 mg / mL.

[0043] Further, the concentration of the organic aminophosphate or its salt in the collagen solution in step (3) is 0.005 mol / L-1.5 mol / L (e.g., 0.005 mol / L, 0.008 mol / L, 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.1 mol / L, 0.11 mol / L, 0.12 mol / L, 0.13 mol / L, 0.14 mol / L, 0.15 mol / L, 0.16 mol / L, 0.17 mol / L, 0.18 ... .19mol / L, 0.2mol / L, 0.21mol / L, 0.22mol / L, 0.23mol / L, 0.24mol / L, 0.25mol / L, 0.26mol / L, 0.27mol / L, 0.28mol / L, 0.29mol / L, 0.3mol / L, 0.4mol / L, 0.5mol / L, 0.6mol / L, 0.7mol / L, 0.8mol / L, 0.9mol / L, 1.0mol / L, 1.1mol / L, 1.2mol / L, 1.3mol / L, 1.4mol / L, 1.5mol / L), preferably 0.05mol / L-1.0mol / L.

[0044] In some embodiments of the present invention, the concentration of the organic aminophosphate or its salt in step (3) in the collagen solution is 0.05 mol / L.

[0045] In some embodiments of the present invention, the concentration of the organic aminophosphate or its salt in step (3) in the collagen solution is 1.0 mol / L.

[0046] In some embodiments of the present invention, the concentration of the organic aminophosphate or its salt in step (3) in the collagen solution is 0.2 mol / L.

[0047] Furthermore, in steps (3)-(4), the resolution is achieved by dissolving the collagen using an acid solution.

[0048] Furthermore, the acid in the acid solution is selected from one or more of hydrochloric acid, acetic acid, phosphoric acid or citric acid, preferably hydrochloric acid, acetic acid or phosphoric acid.

[0049] Furthermore, the pH of the acid solution is 1-3 (e.g., 1, 1.5, 2, 2.5, 3).

[0050] Further, the stirring time in step (3) is 10h-30h (e.g., 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 30h), preferably 10h-25h.

[0051] Further, the dialysis time in step (4) is 15h-40h (e.g., 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 30h, 35h, 36h, 37h, 38h, 39h, 40h), preferably 18h-36h.

[0052] Furthermore, the residual amount of DNA in the low-immunogenic collagen is less than 30 μg / g.

[0053] In a third aspect, the present invention provides the use of collagen as described in the first aspect, or collagen prepared by the preparation method described in the second aspect, in the preparation of tissue engineering materials, pharmaceuticals, and cosmetics.

[0054] Furthermore, the tissue engineering materials include soft tissue filling materials, cartilage repair materials, tissue implantation materials, and coatings for biomaterial implants;

[0055] Furthermore, the coating of the biomaterial implant includes coatings for breast fillers, catheters, cannulas, bone repairs, cartilage substitutes, micropumps and other drug delivery devices, artificial organs and blood vessels, and tissue reinforcement meshes;

[0056] Furthermore, the soft tissue filler material includes filler materials or joint lubricants for the face, neck, head, ears, breasts, joints;

[0057] Furthermore, the medicine includes wound healing materials, injection solutions, and delivery materials for therapeutic agents;

[0058] Furthermore, the therapeutic agent includes a chemotherapeutic agent or a biologically active factor;

[0059] Furthermore, the active factors include anti-inflammatory agents, antibiotics, analgesics, anesthetics, wound healing promoters, cell growth inhibitors, immunostimulants, immunosuppressants, or antiviral drugs.

[0060] Furthermore, the cosmetics mentioned include moisturizing products or anti-wrinkle products.

[0061] The beneficial effects of this invention are:

[0062] This invention innovatively uses organic aminophosphate or its salts as a replacement agent during collagen extraction. Since the phosphate groups in organic aminophosphate or its salts share the same properties as the glycophosphate backbone of DNA, the phosphate groups can replace and remove DNA impurities bound to / doped into collagen molecular chains, thereby reducing the probability of DNA binding to collagen and allowing DNA to remain free in the collagen solution, thus reducing the amount of DNA residue in the collagen after salting out. Furthermore, the amino groups in organic aminophosphate or its salts can form intermolecular hydrogen bonds with the residual carboxyl groups on collagen. These hydrogen bonds shorten the distance between the phosphate groups and the collagen molecular chains, thus synergistically promoting binding with collagen and significantly improving the DNA replacement effect, thereby significantly reducing the immunogenicity of collagen. After the replacement treatment, the organic aminophosphate or its salts replace the DNA bound to collagen, while the unbound organic aminophosphate or its salts can be removed during subsequent processes such as salting out, resolution, and dialysis of the collagen. Therefore, while reducing the immunogenicity of collagen, the purity of the final collagen is guaranteed, and the safety and biological properties of collagen are not affected, which is beneficial for practical clinical applications. Attached Figure Description

[0063] Figure 1 SDS-PAGE electrophoresis images of collagen samples from each embodiment and comparative example. Detailed Implementation

[0064] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0065] Example 1

[0066] (1) After slicing the pig skin, grind it using a meat grinder to obtain particles with a particle size of 1 mm. Prepare an aqueous solution of pepsin with a concentration of 0.3 mg / mL, and adjust the pH of the solution to 1 using hydrochloric acid to obtain an acidic solution of pepsin. Add 100 g of pig skin particles to the aforementioned acidic solution for collagen extraction, and process for 24 h to obtain an enzymatic hydrolysate.

[0067] (2) Add sodium chloride to the enzymatic hydrolysate from step (1) until the concentration is 0.7M. After mixing evenly, centrifuge to obtain crude collagen.

[0068] (3) The crude collagen obtained in step (2) was reconstituted with hydrochloric acid solution with pH 1 to obtain a collagen solution with a concentration of 5 mg / mL. 2-aminoethyl phosphate was added to the solution to a concentration of 0.05 mol / L, and the solution was stirred for 12 h. Sodium chloride was then added to the solution to a concentration of 0.7 M. After centrifugation, purified collagen was obtained.

[0069] (4) The purified collagen obtained in step (3) was reconstituted with hydrochloric acid solution at pH 1, placed in a dialysis bag and dialyzed in purified water for 18 hours to remove salt, with the water changed every 60 minutes. The dialyzed collagen solution was then placed in a freeze-drying tray for freeze-drying to obtain low-immunogenic collagen.

[0070] Example 2

[0071] (1) After slicing the pig skin, grind it using a meat grinder to obtain microparticles with a particle size of 5 mm. Prepare an aqueous solution of pepsin with a concentration of 1.5 mg / mL, and adjust the pH of the solution to 3 using acetic acid to obtain an acidic solution of the protease. Add 500 g of pig skin microparticles treated in step (1) to the aforementioned acidic solution for collagen extraction. The treatment time is 48 h to obtain an enzymatic hydrolysate.

[0072] (2) Add sodium chloride to the enzymatic hydrolysate from step (1) to a concentration of 1.5M, mix well, and centrifuge to obtain crude collagen.

[0073] (3) The crude collagen obtained in step (2) was reconstituted with hydrochloric acid solution with pH 3 to obtain a collagen solution with a collagen concentration of 15 mg / mL. Sodium 2-aminoethyl phosphate was added to the solution to a concentration of 1 mol / L, and the solution was stirred for 24 h. Sodium chloride was added to the solution to a concentration of 1.5 M. After centrifugation, purified collagen was obtained.

[0074] (4) The purified collagen obtained in step (3) was reconstituted with hydrochloric acid solution at pH 3, and then placed in a dialysis bag and dialyzed in purified water for 36 hours to remove salt, with the water changed every 120 minutes. The dialyzed collagen solution was then placed in a freeze-drying tray for freeze-drying to obtain low-immunogenic collagen.

[0075] Example 3

[0076] (1) After slicing the pig skin, grind it using a meat grinder to obtain microparticles with a particle size of 10 mm. Prepare an aqueous solution of pepsin with a concentration of 0.5 mg / mL, and adjust the pH of the solution to 1.5 using hydrochloric acid to obtain an acidic solution of pepsin. Add 300 g of pig skin microparticles treated in step (1) to the aforementioned acidic solution for collagen extraction. The treatment time is 30 h to obtain an enzymatic hydrolysate.

[0077] (2) Add sodium chloride to the enzymatic hydrolysate from step (1) until the concentration is 0.7M. After mixing evenly, centrifuge to obtain crude collagen.

[0078] (3) The crude collagen obtained in step (2) was reconstituted with acetic acid solution at pH 2 to obtain a collagen solution with a collagen concentration of 10 mg / mL. Sodium (4-amino-1-hydroxy-1-phospho-butyl)-phosphate was added to the solution to a concentration of 0.2 mol / L. After stirring for 18 h, sodium chloride was added to the solution to a concentration of 0.7 M. After centrifugation, purified collagen was obtained.

[0079] (4) The purified collagen obtained in step (3) was reconstituted with acetic acid solution at pH 2, placed in a dialysis bag and placed in water for desalting dialysis for 24 hours, with the water changed every 1 hour. The dialyzed collagen solution was then placed in a freeze-drying tray for freeze-drying to obtain low immunogenic collagen.

[0080] Example 4

[0081] (1) After slicing the pig skin, grind it using a meat grinder to obtain microparticles with a particle size of 5 mm. Prepare an aqueous solution of pepsin with a concentration of 0.5 mg / mL, and adjust the pH of the solution to 2 using phosphoric acid to obtain an acidic solution of the protease. Add 200 g of pig skin microparticles treated in step (1) to the aforementioned acidic solution for collagen extraction. The treatment time is 36 h to obtain an enzymatic hydrolysate.

[0082] (2) Add sodium chloride to the enzymatic hydrolysate from step (1) until the concentration is 1M. After mixing evenly, centrifuge to obtain crude collagen.

[0083] (3) The crude collagen obtained in step (2) was reconstituted with a phosphate solution with a pH of 1 to obtain a collagen solution with a collagen concentration of 12 mg / mL. Sodium (4-aminophenyl) phosphate was added to the solution to a concentration of 0.2 mol / L. After stirring for 24 h, sodium chloride was added to the solution to a concentration of 1 M. After centrifugation, purified collagen was obtained.

[0084] (4) The purified collagen obtained in step (3) was reconstituted with a phosphate solution at pH 1, and then placed in a dialysis bag and dialyzed in water for 24 hours to remove salt, with the water being changed every hour. The dialyzed collagen solution was then placed in a freeze-drying tray for freeze-drying to obtain low-immunogenic collagen.

[0085] Example 5

[0086] (1) After slicing the pig skin, grind it using a meat grinder to obtain microparticles with a particle size of 5 mm. Prepare an aqueous solution of pepsin with a concentration of 1 mg / mL, and adjust the pH of the solution to 2 using hydrochloric acid to obtain an acidic solution of pepsin. Add 400 g of pig skin microparticles treated in step (1) to the aforementioned acidic solution for collagen extraction. The treatment time is 40 h to obtain an enzymatic hydrolysate.

[0087] (2) Add sodium chloride to the enzymatic hydrolysate from step (1) to a concentration of 1.5M, mix well, and centrifuge to obtain crude collagen.

[0088] (3) The crude collagen obtained in step (2) was reconstituted with hydrochloric acid solution with pH 1.5 to obtain a collagen solution with a collagen concentration of 8 mg / mL. (4-aminophenyl) phosphate was added to the solution to a concentration of 1 mol / L. After stirring for 24 h, sodium chloride was added to the solution to a concentration of 1.5 M. After centrifugation, purified collagen was obtained.

[0089] (4) The purified collagen obtained in step (3) was reconstituted with hydrochloric acid solution at pH 1.5, placed in a dialysis bag, and dialyzed in water for 24 hours to remove salt, with the water being changed every hour. The dialyzed collagen solution was then placed in a freeze-drying tray for freeze-drying to obtain low-immunogenic collagen.

[0090] Comparative Example 1

[0091] The difference between this method and Example 1 is only in step (3). Specifically, in this preparation method, step (3) is as follows: the crude collagen obtained in step (2) is reconstituted with hydrochloric acid solution with pH 1 to obtain a collagen solution with a concentration of 5 mg / mL. Sodium chloride is added to the solution to a concentration of 0.7 M. The solution is stirred for 12 h and then centrifuged to obtain purified collagen.

[0092] Comparative Example 2

[0093] The only difference between this method and Example 1 is step (3). Specifically, in this preparation method, step (3) is as follows:

[0094] The crude collagen obtained in step (2) was reconstituted with hydrochloric acid solution at pH 1 to obtain a collagen solution with a collagen concentration of 5 mg / mL. Sodium dihydrogen phosphate was added to the solution to a concentration of 0.05 mol / L. After stirring for 12 h, sodium chloride was added to the solution to a concentration of 0.7 M. After centrifugation, purified collagen was obtained.

[0095] Comparative Example 3

[0096] The only difference between this method and Example 1 is step (3). Specifically, in this preparation method, step (3) is as follows:

[0097] The crude collagen obtained in step (2) was reconstituted with hydrochloric acid solution at pH 1 to obtain a collagen solution with a collagen concentration of 5 mg / mL. Ethylenediamine was added to the solution to a concentration of 0.05 mol / L, and the solution was stirred for 12 h. Then, sodium chloride was added to the solution to a concentration of 0.7 M. After centrifugation, purified collagen was obtained.

[0098] Comparative Example 4

[0099] The only difference between this method and Example 1 is step (3). Specifically, in this preparation method, step (3) is as follows:

[0100] After reconstituted the crude collagen obtained in step (2) with hydrochloric acid solution at pH 1, a certain amount of ethylenediamine phosphate buffer solution was added to make the final solution have a collagen concentration of 5 mg / mL, an ethylenediamine concentration of 0.05 mol / L, and a phosphate concentration of 0.02 mol / L. After stirring for 12 h, sodium chloride was added to the solution to a concentration of 0.7 M. After centrifugation, purified collagen was obtained.

[0101] Performance example

[0102] Performance Example 1: Detection of DNA Residue in Low-Immunogenic Collagen

[0103] The residual DNA content of the low immunogenic collagen samples prepared in Examples 1-5 and Comparative Examples 1-4 was detected using a Quant-IT PicoGreen dsDNA Reagent and Kits kit. DNA standard solutions with concentrations of 80, 40, 20, 10, 5, and 0 μg / L were prepared for use. The lyophilized collagen samples prepared in each example and comparative example were digested with digestion buffer to obtain test solutions. Lysis buffer and magnetic swabs were added to the test solutions to allow the DNA in the test solutions to adsorb and bind to the magnetic swabs. The DNA on the magnetic swabs was then further eluted and purified to obtain purified DNA solutions for each sample. 100 μL of the DNA standard solutions and the purified DNA solutions from each sample were added to each well of a 96-well black ELISA plate. Add 100 μl of PicoGreen (fluorescent reaction solution) to the wells containing the standard and the purified sample, respectively. After reacting for 5 min in the dark, measure the fluorescence value of each sample using a fluorescence microplate reader and substitute it into the standard curve to calculate the DNA concentration in the sample. The detection results are shown in Table 1.

[0104] Table 1. DNA residue in collagen samples from different embodiments and comparative examples.

[0105] Sample Name DNA residue, μg / g Example 1 27 Example 2 30 Example 3 25 Example 4 27 Example 5 30 Comparative Example 1 154 Comparative Example 2 40 Comparative Example 3 130 Comparative Example 4 42

[0106] As shown in the table above, the residual DNA levels in Examples 1-5 were all no higher than 30 μg / g, while the residual DNA levels in Comparative Examples 1-4 were all higher than 40 μg / g. This is mainly because Examples 1-5 used organic aminophosphate or its salts as a replacement agent during collagen extraction. The phosphate groups in organic aminophosphate or its salts have the same properties as the sugar-phosphate backbone of DNA. The phosphate groups can replace and remove DNA impurity molecules bound / doped onto collagen molecular chains, thereby reducing the probability of DNA binding to collagen and allowing DNA to remain free in the collagen solution, thus reducing the residual DNA level in collagen after salting out. Furthermore, the amino groups in organic aminophosphate or its salts can form a molecule with the residual carboxyl groups on collagen. Intermolecular hydrogen bonds shorten the distance between phosphate groups and collagen molecular chains, thereby promoting the binding of phosphate groups to collagen and improving the DNA replacement effect. After replacement treatment, organic aminophosphate or its salts replace DNA and bind to collagen. This process does not affect the safety and biological properties of collagen. At the same time, unbound organic aminophosphate or its salts can be removed by subsequent processes such as salt precipitation purification, resolution, and dialysis of collagen. While ensuring the purity of collagen, the immunogenicity of collagen is significantly reduced, which is beneficial for clinical applications.

[0107] Furthermore, a comparison of the data from Example 1 and Comparative Examples 1-4 shows that the residual amount in Example 1 (27 μg / g) was reduced by approximately 82% compared to Comparative Example 1 (154 μg / g) which did not use any replacement agent, by approximately 32% compared to Comparative Example 2 (40 μg / g) which used inorganic phosphate as the replacement agent, by approximately 68% compared to Comparative Example 3 (130 μg / g) which used organic amine, and by approximately 36% compared to Comparative Example 4 (42 μg / g) which used both phosphoric acid and organic amine. The above data indicate that in Comparative Example 1, which did not undergo DNA replacement treatment, the amount of residual DNA was the highest, posing a high risk of use. In Comparative Example 2, the amount of residual DNA was reduced compared to Comparative Example 1 by treating collagen with inorganic phosphate; however, due to the lack of hydrogen bonds between amino groups and collagen, it failed to synergistically promote the binding of phosphate groups to DNA on collagen, thus the DNA removal effect was inferior to that of Example 1. In Comparative Example 3, although the amino groups in ethylenediamine can form hydrogen bonds with collagen, its effect on the binding of collagen to DNA is limited. The sample was relatively small, and the residual amount of DNA in the sample was still very high, indicating that organic amines had no significant effect on DNA removal. In Comparative Example 4, ethylenediamine phosphate buffer solution was used to replace DNA on collagen. In this case, the phosphate group played a major role in DNA removal. Although the amino groups in ethylenediamine formed intermolecular hydrogen bonds with the groups on collagen, they were also unable to synergistically enhance the replacement of phosphate groups. Therefore, ethylenediamine phosphate buffer solution had a certain effect on the overall removal of DNA, but it was significantly less effective than the treatment of collagen with organic amino phosphate in Example 1.

[0108] Performance Example 2: SDS-PAGE electrophoresis detection of low immunogenic collagen

[0109] The low immunogenic collagen samples prepared in Examples 1-5 and Comparative Examples 1-4 were subjected to SDS-PAGE electrophoresis detection. The specific detection process was as follows: 4% stacking gel and 7% separating gel were prepared according to the SDS-PAGE polyacrylamide gel electrophoresis method in the electrophoresis method of Part IV, 0541 of the Pharmacopoeia of the People's Republic of China (2020 edition).

[0110] (1) Sample preparation: Take the lyophilized collagen samples from different examples and comparative examples, dissolve the collagen in 0.01 mol / L hydrochloric acid solution to a collagen solution with a concentration of 1 mg / mL, mix it with 4× electrophoresis loading buffer (take 2.5 mL of 0.5 mol / L Tris-HCl buffer, 2.0 mL of glycerol, 4.0 mL of 10% SDS, 0.5 mL of 0.1% bromophenol blue, and ultrapure water to make up to 10 mL and mix well) at a 1:1 ratio to obtain the test solution, heat it in a 100℃ water bath for 2 min, take it out and cool it, and wait for sample loading and analysis.

[0111] (2) Sample loading and analysis: 10 μl of pre-stained protein molecular weight standard (marker) and 20 μl of cooled sample solutions from different examples and comparative proportions were loaded into different lanes. The initial voltage was 80 V. After the sample entered the separating gel from the stacking gel, the voltage was adjusted to 110 V. Electrophoresis was stopped when bromophenol blue migrated to the bottom of the gel. The electrophoresis gel was removed and placed in Coomassie Brilliant Blue staining solution for 2 hours. After removal, destaining was performed until the gel background was transparent. After completion, the electrophoresis gel was photographed and analyzed using a gel analysis system. The electrophoresis results are shown below. Figure 1 .

[0112] from Figure 1 As can be clearly seen, the electrophoresis patterns of collagen from different examples and comparative examples all include bands composed of α, β, and γ peptide chains. From top to bottom, these are: a γ chain (trimer of the α chain) with a molecular weight of approximately 300 kDa, a β chain (dimer of the α chain) with a molecular weight of approximately 270 kDa, and α1 and α2 chains with a molecular weight of approximately 130 kDa. Furthermore, no other bands are present on the patterns, indicating that the collagen extracted in the examples and comparative examples possesses the characteristic structure of collagen—a triple helix structure. Since SDS-PAGE is a method for determining the structure and purity of proteins, it cannot respond to residual DNA, which is mainly composed of deoxyribonucleotides. Therefore, although the residual DNA content in the collagen samples from the examples and comparative examples differs, their electrophoresis patterns are essentially consistent. Additionally, image analysis showed that the residual amount of impurities in the different collagen samples was less than 1%, indicating that the collagen extracted after the purification process of the examples does not affect the characteristic structure of collagen.

[0113] In summary, the collagen extracted using the displacement purification technique of this invention, which uses organic aminophosphate or its salt as a displacement agent, ensures that the triple helix structure of collagen is not destroyed while reducing the amount of residual DNA. This results in low-immunogenic collagen with good biological activity, which meets the needs of clinical applications for the safety and biological characteristics of collagen.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0115] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0116] The fact that the steps of the method are listed in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. A low-immunogenic collagen, characterized in that, The low-immunogenic collagen is obtained by extracting collagen using organic aminophosphate or its salt to remove DNA impurities from the collagen.

2. The collagen as described in claim 1, characterized in that, The organic amino phosphate or its salt is selected from one or more of 2-aminoethyl phosphate, sodium 2-aminoethyl phosphate, sodium (4-amino-1-hydroxy-1-phospho-butyl)-phosphate, sodium (4-aminophenyl) phosphate, (4-aminophenyl) phosphate, or aminotrimethylene phosphate.

3. A method for preparing low-immunogenic collagen as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Use an acidic solution of protease to enzymatically hydrolyze animal skin to obtain an enzymatic hydrolysate; (2) The enzymatic hydrolysate obtained in step (1) is subjected to salt precipitation treatment to obtain crude collagen. (3) The crude collagen obtained in step (2) is reconstituted to obtain a collagen solution. Organic amino phosphate or its salt is added to the solution, stirred, and then salted to obtain purified collagen. (4) The purified collagen obtained in step (3) is reconstituted, dialyzed, and freeze-dried to obtain low-immunogenic collagen.

4. The preparation method according to claim 3, characterized in that, The protease in step (1) is selected from one or more of pepsin, bromelain, papain or Aspergillus niger acid protease; preferably, the concentration of the protease in the acidic solution is 0.2 to 2.0 mg / mL, more preferably 0.3 to 1.5 mg / mL.

5. The preparation method according to claim 3, characterized in that, The acid in the acidic solution in step (1) is selected from one or more of hydrochloric acid, acetic acid, phosphoric acid, lactic acid or citric acid; preferably, the pH of the acidic solution in step (1) is 1-3.5, more preferably 1-3.

6. The preparation method according to claim 3, characterized in that, The salt precipitation process in steps (2)-(3) includes mixing the enzymatic hydrolysate or collagen solution with a precipitant and centrifuging; preferably, the precipitant is selected from one or more of sodium chloride, ammonium sulfate or sodium sulfate, preferably sodium chloride; more preferably, the concentration of the precipitant is 0.1M-2.0M.

7. The preparation method according to claim 3, characterized in that, The organic amino phosphate or its salt in step (3) is selected from one or more of 2-aminoethyl phosphate, sodium 2-aminoethyl phosphate, sodium (4-amino-1-hydroxy-1-phospho-butyl)-phosphate, sodium (4-aminophenyl) phosphate, (4-aminophenyl) phosphate, or aminotrimethylene phosphate; preferably, the concentration of the organic amino phosphate or its salt in the collagen solution is 0.005 mol / L-1.5 mol / L, more preferably 0.05 mol / L-1.0 mol / L.

8. The preparation method according to any one of claims 3-7, characterized in that, The concentration of the collagen solution is 5 mg / mL to 20 mg / mL, preferably 5 mg / mL to 15 mg / mL.

9. The preparation method according to claim 8, characterized in that, In steps (3)-(4), resolution is performed by dissolving the collagen with an acid solution; preferably, the acid in the acid solution is selected from one or more of hydrochloric acid, acetic acid, phosphoric acid or citric acid; preferably, the pH of the acid solution is 1-3.

10. The use of collagen as described in claim 1 or 2, or collagen prepared by any one of claims 3-9, in the preparation of tissue engineering materials, pharmaceuticals, and cosmetics; Preferably, the tissue engineering materials include soft tissue filling materials, cartilage repair materials, tissue implantation materials, and coatings for biomaterial implants; Preferably, the drug includes wound healing materials, injection solutions, and delivery materials for therapeutic agents; Preferably, the cosmetic product includes a moisturizing product or an anti-wrinkle product.

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