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CN-121987763-A - Freeze-drying method for improving bioactivity of recombinant mussel mucin

CN121987763ACN 121987763 ACN121987763 ACN 121987763ACN-121987763-A

Abstract

The invention is suitable for the technical field of biological pharmacy, and provides a freeze-drying method for improving the bioactivity of recombinant mussel mucin, which comprises the steps of pretreatment, pre-freezing, primary drying and secondary drying, wherein the pretreatment mixes a recombinant mussel mucin solution with pH of 3.5-5.5 with a composite protective agent composed of trehalose, L-arginine and poloxamer according to a specific proportion, the pre-freezing adopts a slow-fast-slow sectional cooling mode, and the drying adopts a vacuum drying mode combining gradient heating and gradient vacuum degree.

Inventors

  • ZHU HONG
  • WANG CHENGZHI
  • ZOU WENTAO
  • LIU JUNYANG
  • TIAN ZHENGYI
  • Yue Muchen

Assignees

  • 西安磐泽生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260325

Claims (10)

  1. 1. The freeze drying method for improving the bioactivity of the recombinant mussel mucin comprises the steps of pretreatment, prefreezing, primary drying and secondary drying, and is characterized in that the pretreatment is to mix a recombinant mussel mucin solution with the pH value of 3.5-5.5 with a composite protective agent to obtain a mixed solution, wherein the composite protective agent consists of trehalose, L-arginine and poloxamer, and the weight ratio of the trehalose to the L-arginine to the poloxamer is (4-6) to (0.8-1.2) to (0.4-0.6); The pre-freezing is to treat the mixed solution by adopting a sectional cooling strategy comprising a first slow cooling stage, a fast cooling stage and a second slow cooling stage; the primary drying and the secondary drying are vacuum drying by adopting a mode of combining gradient heating and gradient vacuum degree.
  2. 2. The method for enhancing the biological activity of recombinant mussel mucin according to claim 1, wherein the weight ratio of the trehalose, L-arginine and poloxamer in the composite protectant is 5:1:0.5.
  3. 3. The method for enhancing biological activity of recombinant mussel mucin according to claim 1, wherein the final concentration of the recombinant mussel mucin solution after mixing is 5-20mg/mL.
  4. 4. The method for enhancing the biological activity of recombinant mussel mucin according to claim 1, wherein the cooling rate in the first slow cooling stage is 0.5-1.0 ℃ per minute, the cooling rate in the fast cooling stage is 2.0-3.0 ℃ per minute, and the cooling rate in the second slow cooling stage is 0.3-0.5 ℃ per minute.
  5. 5. A freeze-drying process for enhancing the bioactivity of recombinant mussel mucin according to claim 1, wherein the sectional cooling strategy is performed by cooling from room temperature to-10 ℃ at a rate of 0.5-1.0 ℃ per minute for at least 30 minutes, cooling from-10 ℃ to-40 ℃ at a rate of 2.0-3.0 ℃ per minute for at least 60 minutes, and cooling from-40 ℃ to-50 ℃ at a rate of 0.3-0.5 ℃ per minute for at least 120 minutes.
  6. 6. A freeze-drying method for improving the bioactivity of recombinant mussel mucin according to claim 1, wherein the primary drying is carried out under a vacuum of 10-50Pa, and the shelf temperature is gradually increased from-40 ℃ to-10 ℃ for 24-36 hours.
  7. 7. A freeze-drying method for improving the bioactivity of recombinant mussel mucin according to claim 1, wherein the secondary drying is carried out under a vacuum of 5-20Pa, and the shelf temperature is gradually increased from-10 ℃ to 25 ℃ for 6-12 hours.
  8. 8. The method for lyophilizing recombinant mussel mucin according to claim 1, wherein the poloxamer is poloxamer 188.
  9. 9. The method for freeze-drying recombinant mussel mucin according to claim 1, wherein the 3, 4-Dihydroxyphenylalanine (DOPA) content retention rate of the prepared recombinant mussel mucin freeze-dried product is more than 90%, the residual moisture content is less than 1.5%, and the re-dissolution time is less than 30 seconds.
  10. 10. The method for improving the bioactivity of recombinant mussel mucin according to claim 1, wherein the prepared recombinant mussel mucin freeze-dried product has a porous network structure with uniform pore size and mutual communication under a scanning electron microscope, and the average pore size is 50-80 microns.

Description

Freeze-drying method for improving bioactivity of recombinant mussel mucin Technical Field The invention belongs to the technical field of biological pharmacy processes, and particularly relates to a freeze-drying method for improving bioactivity of recombinant mussel mucin. Background Mussel Mucin (MAP) is a natural biological macromolecule secreted by the foot gland of marine mussel, the molecular structure of the mussel is rich in a large number of DOPA residues, and the unique structure endows the mussel with excellent adhesion performance in a humid environment. Along with the rapid development of modern genetic engineering technology, the recombinant mussel mucin (rMAP) has successfully realized large-scale production, and rMAP is not only an excellent biological adhesive, but also has obvious anti-inflammatory, antibacterial, tissue repair and regeneration promoting and other various biological activities, and has great application prospects in the biological medicine fields of high-end medical appliances, wound repair dressings, tissue engineering scaffolds, targeted drug delivery and the like. In order to ensure long-term stability of rMAP in the process of storage and transportation and facilitate subsequent application, the rMAP is usually prepared into a freeze-dried powder form, and freeze-drying is a well-known gold standard technology for maintaining the activity of biological macromolecules such as proteins, but the freeze-drying process aiming at rMAP in the prior art has a plurality of technical problems, specifically as follows: The freezing stress and the drying stress in the freeze-drying process easily cause rMAP conformational changes, aggregation denaturation, the core functional group DOPA is extremely sensitive to oxidation and pH change, and the freeze-drying process is easily damaged, so that the key biological activities such as adhesion, anti-inflammatory and the like are greatly reduced; The existing mussel mucin related freeze-drying process only takes freeze-drying as a conventional step for obtaining solid powder, is not deeply optimized for maximally maintaining rMAP biological activity, and lacks systematic design of process parameters; Although the field of protein freeze-drying protective agents has application of single components or binary combinations of trehalose, arginine, poloxamer and the like, the combined use of three protective agents is not explored aiming at rMAP, and the synergistic protective effect is not found; most of the cooling strategies of the existing freeze-drying technology are universal rapid or slow cooling, and the special physicochemical properties of rMAP and the special composite protective agent are not combined to carry out collaborative design, so that the ice crystal morphology can not be accurately controlled to reduce protein damage; The prior art does not determine an adaptive stable pH range aiming at rMAP, and does not develop a special freeze-drying scheme for coupling a process and a protective agent formula, so that the physical and chemical indexes of rMAP freeze-dried products such as long-term stability, re-solubility and the like are poor; There is therefore a need for a freeze-drying method that increases the bioactivity of recombinant mussel mucins to solve the above problems. Disclosure of Invention The embodiment of the invention aims to provide a freeze-drying method for improving bioactivity of recombinant mussel mucin, which aims to effectively solve the problem of activity loss caused by complicated freezing and drying stress in the freeze-drying process of rMAP by creatively combining an optimized compound low-temperature protective agent formula with a precisely controlled sectional freeze-drying process curve, and particularly to protect the stability of a core functional group DOPA of the recombinant mussel mucin. In order to achieve the above purpose, the present invention provides the following technical solutions: The freeze drying method for improving the bioactivity of the recombinant mussel mucin comprises the steps of pretreatment, prefreezing, primary drying and secondary drying, wherein the pretreatment is to mix a recombinant mussel mucin solution with the pH value of 3.5-5.5 with a composite protective agent to obtain a mixed solution, and the composite protective agent consists of trehalose, L-arginine and poloxamer, wherein the weight ratio of the trehalose to the L-arginine to the poloxamer is (4-6) to (0.8-1.2) to (0.4-0.6); The pre-freezing is to treat the mixed solution by adopting a sectional cooling strategy comprising a first slow cooling stage, a fast cooling stage and a second slow cooling stage; the primary drying and the secondary drying are vacuum drying by adopting a mode of combining gradient heating and gradient vacuum degree. According to a further technical scheme, the weight ratio of the trehalose, the L-arginine and the poloxamer in the composite protective agent is 5:1:0.5. Accord