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CN-122005764-A - Collagen gel and preparation method and application thereof

CN122005764ACN 122005764 ACN122005764 ACN 122005764ACN-122005764-A

Abstract

The invention discloses a collagen gel and a preparation method and application thereof. The preparation method comprises the following steps of respectively sterilizing, mixing and crosslinking the prepared collagen solution and glutamine transaminase solution to obtain collagen gel liquid, and then performing chromatographic enzyme removal or hollow fiber enzyme removal to obtain the collagen gel. According to the invention, glutamine transaminase is used as a cross-linking agent, and residual glutamine transaminase in the collagen gel is removed through anion membrane chromatography or hollow fiber, so that the safety problem caused by a chemical cross-linking agent and the potential safety hazard of the glutamine transaminase in the process of injecting the collagen gel are avoided. In addition, the invention realizes the regulation and control of the physical state of the gel by controlling the mixing proportion of the collagen and the glutamine transaminase, and provides rich raw materials for biomedical materials.

Inventors

  • LI JIAN
  • CHEN CHEN
  • WANG XUEMIN
  • MA KUN
  • QIAN BO
  • ZHU YONGZHEN
  • WANG ZIQIU
  • YU XIAOLING
  • JI GUANGSHUO
  • NING JINJIAO
  • QIAO JINYU

Assignees

  • 汉肽生物医药集团有限公司

Dates

Publication Date
20260512
Application Date
20260209

Claims (10)

  1. 1. A method for preparing a collagen gel, comprising the steps of: (1) Dissolving collagen to prepare collagen solution; (2) Dissolving glutamine transaminase to prepare a glutamine transaminase solution; (3) Respectively sterilizing, mixing and crosslinking the collagen solution and the glutamine transaminase solution to prepare a collagen gel liquid; (4) Performing chromatographic enzyme removal or hollow fiber enzyme removal on the collagen gel liquid to obtain the collagen gel; the content of glutamine transaminase in the collagen gel liquid is 10-500 ppm.
  2. 2. The method according to claim 1, wherein in the step (1), the collagen contains glutamine and lysine.
  3. 3. The method of preparation according to claim 2, wherein the collagen is recombinant collagen, preferably the collagen comprises recombinant I、II、III、IV、V、VI、VII、VIII、IX、X、XI、XII、XIII、XIV、XV、XVI、XVII、XVIII、XIX、XX、XXI、XXII、XXIII、XXIV、XXV、XXVI、XXVII、XXVIII or XXIX type humanized collagen.
  4. 4. The method according to claim 1, wherein in the step (1), the concentration of the collagen solution is 1 to 10% (w/v), and/or In the step (2), the concentration of the glutamine transaminase solution is 3000-5000 ppm.
  5. 5. The method according to claim 1, wherein in the step (3), the crosslinking temperature is 15 ℃ to 40 ℃, and the crosslinking time is 12 h to 48 h.
  6. 6. The preparation method of claim 1, wherein in the step (4), the chromatography is anion membrane chromatography, and the membrane pore diameter cutoff molecular weight of the hollow fiber is more than or equal to 100 kDa.
  7. 7. The method of claim 6, wherein the anionic membrane comprises Sartobind Q, mustang Q, or Purcise Q.
  8. 8. A collagen gel prepared according to any one of the preparation methods of claims 1 to 7.
  9. 9. A composition comprising, as a main ingredient, the composition comprising the collagen gel of claim 8.
  10. 10. Use of the collagen gel of claim 8 or the composition of claim 9 for the preparation of a pharmaceutical, cosmetic or cosmetical product.

Description

Collagen gel and preparation method and application thereof Technical Field The invention relates to the field of biomedical materials, in particular to collagen gel and a preparation method and application thereof. Background Collagen is the most abundant protein in mammals and is widely distributed in various tissues and organs. Collagen has good biocompatibility and biodegradability, and is widely used as a biological material in the fields of wound healing, hemostasis, bone regeneration, skin aging resistance and the like. The traditional animal extracted collagen has risks of virus transmission, immune rejection and the like, and limits the application of the collagen in the fields of biomedicine and the like. Compared with the animal tissue extraction method, the recombinant humanized collagen is produced by the genetic engineering technology, and has the advantages of no animal source risk, clear structure, good water solubility, extremely low immunogenicity and the like. However, recombinant collagen generally lacks the complex higher structure and self-crosslinking ability of natural collagen, and its mechanical strength is often poor, and degradation in vivo is too fast, which limits its application. Collagen crosslinking is an effective means for solving the problems of poor collagen strength, rapid in vivo degradation and the like. At present, a physical method (such as an ultraviolet irradiation method and a heavy dehydration method) is mostly adopted for the preparation of collagen gel (Wang Xinhui and the like, a collagen crosslinking method and research progress [ J ]. Printing and dyeing, 2024 (3): 92-97) or a chemical crosslinking method (such as CN 102924731A-a triple-crosslinked collagen, and aldehydes, imides and epoxide crosslinking agents used in the preparation method and the application thereof). The gel prepared by the physical method has weak mechanical property and poor stability, the chemical crosslinking method usually uses a cytotoxic crosslinking agent, and residual chemical substances possibly cause inflammatory reaction and have poor biological safety (Zhao Jinghua and the like, the research on the chemical modification method of the collagen and the application thereof is progressed [ J ]. Fishery research, 2017,39 (2): 147-156). Therefore, the development of the recombinant humanized collagen gel which is free of chemical cross-linking agents and stable in structure has important significance. Microbial glutamine transaminase (mTGase) is a natural acyltransferase that catalyzes a cross-linking reaction between the γ -amide group of a glutamine residue and the epsilon-amino group of a lysine residue in a protein to form a covalent bond. The enzyme has mild reaction condition and is a high-efficiency biological cross-linking agent. Although the enzyme itself can be regarded as a "natural" additive with high safety, the enzyme is not known to be safe when applied for injection. Therefore, enzyme residue is one of the key problems to be solved urgently when mTGase is utilized to catalyze and recombine the cross-linking of the humanized collagen. Disclosure of Invention In order to improve the stability and safety of the collagen gel and avoid the influence of the residue of a biological cross-linking agent (such as glutamine transaminase) on the quality of the collagen gel, the invention provides the following technical scheme. In a first aspect, the present invention provides a method for preparing a collagen gel, the method comprising the steps of: (1) Dissolving collagen to prepare collagen solution; (2) Dissolving glutamine transaminase to prepare a glutamine transaminase solution; (3) Respectively sterilizing the collagen solution and the glutamine transaminase solution, mixing and crosslinking according to a preset proportion to prepare a collagen gel liquid; (4) And performing chromatographic enzyme removal or hollow fiber enzyme removal on the collagen gel liquid to obtain the collagen gel. Preferably, in step (1), the collagen contains glutamine and lysine. Further, the collagen is recombinant collagen, More preferably, the collagen includes, but is not limited to, recombinant I、II、III、IV、V、VI、VII、VIII、IX、X、XI、XII、XIII、XIV、XV、XVI、XVII、XVIII、XIX、XX、XXI、XXII、XXIII、XXIV、XXV、XXVI、XXVII、XXVIII or XXIX-type humanized collagen. Preferably, in the step (1), the concentration of the collagen solution is 1 to 10% (w/v), for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. Preferably, in step (1), the solvent of the collagen solution includes, but is not limited to, physiological saline for injection, water for injection, phosphate buffer, tris buffer, histidine buffer or acetate buffer. Further, the pH value of the buffer solution is 5.0-8.0, for example, 5.0, 5.3, 5.6, 5.9, 6.0, 6.2, 6.8, 7.0, 7.2, 7.4, 7.7 and 8.0. Preferably, in the step (2), the concentration of the glutamine transaminase solution is 3000-5000 ppm, for example, 3000 ppm, 3500 ppm, 4000 ppm, 4500-ppm, 5000