Composite protective agent for protein irradiation sterilization as well as preparation method and application of composite protective agent

Abstract

The invention provides a composite protective agent for protein irradiation sterilization as well as a preparation method and application thereof, and the composite protective agent comprises the following components in percentage by weight volume (w / v): 1-10% of trehalose, 1-8% of mannitol, 0.5-5% of a polysaccharide compound system, 0.01-0.5% of a cross-linking agent, 0.1-2% of a free radical scavenger and the balance of a solvent, the polysaccharide compounding system is composed of chitosan and yeast beta-glucan or yeast mannan according to the mass ratio of 1: 5-5: 1, the invention further provides a preparation method of the protective agent, and the preparation method comprises the steps of dissolving, mixing, pH adjusting, filtering and the like. According to the method, free radicals can be effectively removed in the irradiation sterilization process of a protein preparation, the protein structure is stabilized, the activity retention rate of protein after being irradiated at the dosage of 25 kGy is increased to 85% or above from less than 30%, meanwhile, the long-term stability of a product is remarkably improved, and the technical problem that the activity loss of the protein biological preparation is serious in the irradiation sterilization process is solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a composite protective agent for protein irradiation sterilization, its preparation method, and its application. Background Technology

[0002] Protein-based biological agents, especially recombinant mussel adhesive protein and collagen, have shown great application potential in medical devices and biopharmaceuticals, such as high-end dressings, tissue engineering, and drug delivery systems, due to their excellent biocompatibility, biodegradability, and specific biological functions. However, the aseptic production of these products has always been a key technological bottleneck restricting their clinical translation. To ensure the biosafety of these products, effective terminal sterilization methods must be used to completely inactivate microorganisms.

[0003] Currently, the mainstream international sterilization technologies for medical devices mainly include irradiation sterilization, ethylene oxide sterilization, and moist heat sterilization. Among them, irradiation sterilization (such as gamma rays and electron beams) is considered an ideal terminal sterilization method due to its advantages such as high sterilization efficiency, strong penetration, no chemical residue, and the ability to be carried out at room temperature. However, unfortunately, while high-energy rays kill microorganisms, they also cause severe damage to the primary and higher-order structures of proteins by directly acting on protein molecules or indirectly through reactive oxygen species (such as ·OH) generated by the irradiation of water, leading to protein denaturation, aggregation, and loss of biological activity. This damage is particularly significant for structurally complex and conformationally sensitive recombinant mussel adhesive proteins and collagen. Studies have shown that after treatment with conventional irradiation doses of 15-25 kGy, the activity loss rate of these proteins generally exceeds 70%, seriously affecting the final efficacy of the product. This greatly limits the application of irradiation sterilization in protein-based biological agents.

[0004] Ethylene oxide sterilization is another commonly used method, but its residual toxicity is a major concern. Ethylene oxide dissolves in water to form toxic ethylene glycol, posing a clear safety risk to liquid protein formulations and therefore is unsuitable. While moist heat sterilization causes relatively less damage to some heat-stable proteins (such as mussel avidin, which has a certain degree of heat resistance), with protein and active group loss controlled to approximately 20%, its sterilization process conditions (high temperature and high pressure) are quite complex and demanding. This places higher requirements on the sealing and pressure resistance of the product's primary packaging (such as vials and rubber stoppers), increasing production costs and process complexity.

[0005] To mitigate irradiation damage to proteins, several protective strategies have been employed in existing technologies. The most common is the addition of small-molecule protective agents, such as trehalose and mannitol. Trehalose, as an osmotic protectant, can stabilize the native conformation of protein molecules by forming a glassy structure around them; mannitol possesses some free radical scavenging ability. However, this simple protective system has limited effectiveness and cannot effectively address the large number of highly reactive free radicals generated during irradiation, particularly failing to resolve the problem of erroneous cross-linking and aggregation between protein molecules caused by free radical attacks. Polysaccharides such as chitosan have also been studied as stabilizers, primarily providing a physical barrier through film formation; however, their free radical scavenging ability is weak when used alone, resulting in unsatisfactory protective effects on protein activity.

[0006] Therefore, developing a novel composite protective agent that can efficiently scavenge free radicals and stabilize protein spatial structures through synergistic effects of multiple mechanisms, while also being well-compatible with irradiation processes, is of vital importance for promoting the industrialization of protein-based biopharmaceuticals. This invention aims to provide a highly efficient, safe, and suitable composite protective agent solution for protein irradiation sterilization against this backdrop. Summary of the Invention

[0007] Purpose of the invention: The purpose of this invention is to provide a composite protective agent for protein irradiation sterilization. Through the synergistic effect of each component, it can effectively scavenge free radicals and stabilize protein structure during the irradiation sterilization process of protein preparations, thereby increasing the activity retention rate of proteins after irradiation at a dose of 25 kGy from less than 30% to more than 85%. At the same time, it significantly improves the long-term stability of the product and solves the technical problem of severe activity loss of protein biological preparations during irradiation sterilization.

[0008] The present invention discloses a composite protective agent for protein irradiation sterilization, comprising the following components by weight / volume percentage (w / v): 1-10% trehalose, 1-8% mannitol, 0.5-5% polysaccharide complex system, 0.01-0.5% crosslinking agent, 0.1-2% free radical scavenger, and the remainder being solvent.

[0009] Furthermore, the polysaccharide complex system is composed of chitosan and yeast β-glucan or yeast mannan, and the mass ratio of chitosan to yeast β-glucan and yeast mannan in the polysaccharide complex system is (1:1:1) to (5:1:1).

[0010] Furthermore, the yeast β-glucan is a glucan prepared by alkaline extraction or irradiation degradation, which has a β-1,3-glycosidic bond as the main chain and contains β-1,6-glycosidic bonds as the branch chain.

[0011] Furthermore, the yeast β-glucan is prepared by treating yeast cell wall raw material with 1-2 mol / L alkaline solution at 70-90℃ for 1-3 hours, collecting the insoluble matter, washing and drying to obtain yeast β-glucan.

[0012] Furthermore, the yeast mannan is a mannan prepared by autolysis or hot water extraction, which has an α-1,6-mannoglycosidic bond main chain and contains α-1,2 and α-1,3-mannoglycosidic bond side chains.

[0013] Furthermore, the hot water extraction method includes: extracting yeast cell wall raw material with water at 110-125℃ for 0.5-2 hours, collecting the supernatant, concentrating it, and then precipitating it with alcohol to obtain yeast mannan.

[0014] Furthermore, the crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; the free radical scavenger is vitamin C.

[0015] This invention provides a method for preparing a composite protective agent for protein irradiation sterilization, comprising the following steps: S1: Dissolve trehalose, mannitol and the polysaccharide complex in a partial solvent to obtain a polysaccharide mixture; S2: Add the crosslinking agent to the solution obtained in step S1 and stir gently; S3: Add the free radical scavenger to the solution obtained in step S2 and stir to dissolve; S4: Adjust the pH and make up the volume with solvent, filter and sterilize to obtain a composite protective agent for protein irradiation sterilization.

[0016] Furthermore, in step S2, the gentle stirring speed is 100-300 rpm and the time is 10-30 minutes; in step S4, the pH is adjusted to 6.0-7.5; and the filtration and sterilization uses a 0.22-0.25 pore filter membrane.

[0017] This invention provides an application of a composite protective agent for protein irradiation sterilization, wherein the protein irradiation sterilization method includes the following steps: a) Mix the active protein with a composite protective agent used for protein irradiation sterilization to form a mixture; b) The mixture is sterilized by membrane filtration; c) The mixture after membrane filtration is sterilized by electron beam radiation at a dose of 5-50 kGy. In step b), the membrane filtration uses a 0.22-0.25 filter membrane; in step c), the radiation dose is 15-30 kGy; and the active protein is recombinant mussel adhesive protein or collagen.

[0018] This invention constructs a multi-mechanism, multi-target synergistic protection network by scientifically combining trehalose (osmotic protection), mannitol (free radical scavenging), chitosan (film formation and physical barrier), yeast β-glucan / mannan (efficient free radical scavenging and immune regulation), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (mild cross-linking stability), and vitamin C (efficient free radical scavenging).

[0019] This protective agent not only scavenge free radicals but also stabilizes proteins through multiple pathways: Physical barrier: Chitosan and yeast polysaccharides form a dynamic physical protective film around protein molecules, reducing the direct impact of high-energy radiation and the proximity of free radicals. Chemical scavenging: Mannitol, vitamin C, and yeast polysaccharides with excellent antioxidant activity together constitute a highly efficient free radical scavenging system, which can rapidly quench reactive oxygen species such as ·OH generated by radiation, reducing oxidative damage at its source. Conformational stabilization: Trehalose forms a glassy protective layer on the protein surface through the "water replacement" theory, stabilizing its native conformation. Structural reinforcement: The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide crosslinking agent can moderately stabilize the structure between protein molecules or between protein and polysaccharides, enhancing their resistance to radiation-induced depolymerization or mis-aggregation. DSC test results (heat denaturation temperature increased by 7°C) directly demonstrate the excellent stabilizing effect of this protective agent on the higher-order structure of proteins.

[0020] Beneficial effects: The composite protective agent for protein irradiation sterilization provided by this invention and its application have the following significant beneficial effects: 1. Synergistic effect and excellent protective effect: The components work synergistically through multiple mechanisms to increase the protein irradiation activity retention rate from less than 30% to more than 85%, which is significantly better than single or simple compound protective agents. 2. Comprehensive protein structure stabilization: It has the functions of physical barrier, efficient free radical scavenging and structural stabilization, and the thermal stability of the protected protein is significantly enhanced (the thermal denaturation temperature is increased by about 7°C). 3. Excellent long-term stability: In accelerated stability tests, the protein activity retention rate after treatment was much higher than that of the control group, effectively extending the product's shelf life; 4. Safe and compatible: All components are biocompatible and can be seamlessly integrated with existing aseptic production processes. It is suitable for a variety of protein formulations and provides a reliable solution for industrialization. Detailed Implementation

[0021] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0022] Chitosan was purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd., CAS: 83512-85-0; Saccharomyces cerevisiae cell wall was purchased from Wuhan Kabuda Chemical Co., Ltd., model M011.

[0023] Preparation of yeast β-glucan: a. Take 100g of Saccharomyces cerevisiae cell wall, place it in a 2L flask, then add 2000mL of 1mol / L NaOH solution (1:20, w / v), place it in an 80℃ water bath, and stir at 200rpm for 2 hours. b. After the reaction is complete, transfer the mixture to a centrifuge tube and centrifuge at 8000g for 15 minutes. Carefully discard the supernatant. Collect the precipitate, resuspend it in deionized water and wash it. Centrifuge again and repeat this process until the supernatant is neutral (pH=7.0). c. The washed precipitate was dehydrated with anhydrous ethanol, then dried in a vacuum drying oven at 50°C to constant weight, ground, and passed through a 100-mesh sieve to obtain 35g of grayish-white powdery yeast β-glucan.

[0024] Preparation of yeast mannan: a. Take 100g of Saccharomyces cerevisiae cell wall, add 1500mL of deionized water (1:15w / v), and extract at 125℃ for 0.5 hours; b. Centrifuge the extract at 8000g for 20 minutes, take the supernatant and concentrate it to 1 / 5 of the original volume, add 3 times the volume of anhydrous ethanol (final concentration 75%), and let it stand overnight at 4℃; c. The precipitate was washed twice with 75% ethanol and once with anhydrous ethanol, dried under vacuum at 50°C to constant weight, and ground through a 100-mesh sieve to obtain a white powdery yeast mannan.

[0025] Example 1 Raw material formula (based on 1000mL): Trehalose: 40g (4% w / v) Mannitol: 30g (3% w / v) Polysaccharide complex system: 20g (2% w / v, including 12g chitosan, 4g yeast β-glucan, and 4g yeast mannan, in a mass ratio of 3:1:1) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide: 0.8 g (0.08% w / v) Vitamin C: 8g (0.8% w / v) Water for injection: bring the volume up to 1000 mL Preparation method of composite protective agent for protein irradiation sterilization: S1: In a clean preparation container, add 800 mL of water for injection, weigh trehalose, mannitol, chitosan and yeast β-glucan, and add them to the water in sequence under magnetic stirring (room temperature, 500 rpm) until all components are completely dissolved to obtain a clear polysaccharide mixture; S2: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the solution obtained in step S1, adjust the stirring speed to 200 rpm, and stir gently for 15 minutes. S3: Add vitamin C to the solution obtained in step S2 and continue stirring until completely dissolved; S4: Slowly adjust the pH to 6.8 with 0.1 mol / L dilute hydrochloric acid solution, bring the volume to 1000 mL with water for injection, stir well, filter the solution through a 0.22 μm PTFE filter membrane for sterilization, and aseptically dispense it into vials to obtain the composite protective agent for protein irradiation sterilization.

[0026] Example 2 Raw material formula (based on 1000mL): Trehalose: 10g (1% w / v) Mannitol: 80g (8% w / v) Polysaccharide complex system: 50g (5% w / v, including chitosan: 35.7g, yeast β-glucan: 7.15g, yeast mannan: 7.15g, mass ratio 5:1:1) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide: 5g (0.5% w / v) Vitamin C: 20g (0.8% w / v) Water for injection: bring the volume up to 1000 mL Preparation method of composite protective agent for protein irradiation sterilization: S1: In a clean preparation container, add 800 mL of water for injection, weigh trehalose, mannitol, chitosan and yeast β-glucan, and add them to the water in sequence under magnetic stirring (room temperature, 500 rpm) until all components are completely dissolved to obtain a clear polysaccharide mixture; S2: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the solution obtained in step S1, adjust the stirring speed to 200 rpm, and stir gently for 30 minutes. S3: Add vitamin C to the solution obtained in step S2 and continue stirring until completely dissolved; S4: Slowly adjust the pH to 6.0 with 0.1 mol / L dilute hydrochloric acid solution, bring the volume to 1000 mL with water for injection, stir well, filter the solution through a 0.25 μm PTFE filter membrane for sterilization, and aseptically dispense it into vials to obtain the composite protective agent for protein irradiation sterilization.

[0027] Example 3 Raw material formula (based on 1000mL): Trehalose: 100g (1% w / v) Mannitol: 10g (8% w / v) Polysaccharide complex system: 5g (5% w / v, including 1.8g chitosan, 1.6g yeast β-glucan, and 1.6g yeast mannan, in a mass ratio of 1.125:1:1) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide: 0.1 g (0.01% w / v) Vitamin C: 1g (0.8% w / v) Water for injection: bring the volume up to 1000 mL Preparation method of composite protective agent for protein irradiation sterilization: S1: In a clean preparation container, add 800 mL of water for injection, weigh trehalose, mannitol, chitosan and yeast mannan, and add them to the water in sequence under magnetic stirring (room temperature, 500 rpm) until all components are completely dissolved to obtain a clear polysaccharide mixture. S2: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the solution obtained in step S1, adjust the stirring speed to 300 rpm, and stir gently for 10 minutes. S3: Add vitamin C to the solution obtained in step S2 and continue stirring until completely dissolved; S4: Slowly adjust the pH to 7.5 with 0.1 mol / L dilute hydrochloric acid solution, bring the volume to 1000 mL with water for injection, stir well, filter the solution through a 0.22 μm PTFE filter membrane for sterilization, and aseptically dispense it into vials to obtain the composite protective agent for protein irradiation sterilization.

[0028] Comparative Example 1 Raw material formula (based on 1000mL): Trehalose: 40g (4% w / v) Mannitol: 30g (3% w / v) Chitosan: 20g (2% w / v) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide: 0.8 g (0.08% w / v) Vitamin C: 8g (0.8% w / v) Water for injection: bring the volume up to 1000 mL The preparation method of the composite protective agent for protein irradiation sterilization is the same as in Example 1.

[0029] Comparative Example 2 Raw material formula (based on 1000mL): Trehalose: 40g (4% w / v) Mannitol: 30g (3% w / v) Yeast beta-glucan: 20g (2% w / v) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide: 0.8 g (0.08% w / v) Vitamin C: 8g (0.8% w / v) Water for injection: bring the volume up to 1000 mL The preparation method of the composite protective agent for protein irradiation sterilization is the same as in Example 1.

[0030] Comparative Example 3 Raw material formula (based on 1000mL): Trehalose: 40g (4% w / v) Mannitol: 30g (3% w / v) Polysaccharide complex system: 20g (2% w / v, including 12g chitosan, 4g yeast β-glucan, and 4g yeast mannan, in a mass ratio of 3:1:1) 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU): 0.8g (0.08% w / v) Vitamin C: 8g (0.8% w / v) Water for injection: Add to 1000 mL, as its raw material formulation is missing a cross-linking agent; The preparation method of the composite protective agent for protein irradiation sterilization is the same as in Example 1.

[0031] The composite protective agents for protein irradiation sterilization prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to the following tests: Test subject: The recombinant mussel adhesive protein solution (concentration 1 mg / mL) was mixed with each protective agent at a volume ratio of 1:1, stirred for 10 minutes to make the system homogeneous, and allowed to stand at room temperature for 20 minutes before use for testing; 1. Irradiation treatment test: Electron beam radiation was used at a dose of 25 kGy, a temperature of 25℃, a humidity of 40% RH, and an irradiation time of 15 seconds. After irradiation, the protein activity retention rate was calculated using a double antibody sandwich ELISA method.

[0032] 2. Thermal denaturation temperature (T) m Take 10 mg of test sample, use differential scanning calorimetry (DSC), set the heating rate to 10 °C / min, and the temperature range to 25 °C → 90 °C. Measure T. m The temperature corresponding to the endothermic peak in the DSC curve reflects the stability of the protein's native conformation. 3. Stability test: The protein activity retention rate was tested by using a constant temperature and humidity chamber, 60℃ and 75%RH accelerated storage for 60 days. 4. Sterility pass rate: Tested according to the "Sterility Test Method for Medical Devices" (GB / T 14233.2), and the percentage of qualified samples was calculated.

[0033] Table 1: Test Results Table

[0034] As shown in Table 1, the composite protective agents for protein irradiation sterilization prepared in Examples 1-3 of this invention exhibit excellent performance in terms of protein activity retention after irradiation, thermal denaturation temperature, long-term storage stability, and aseptic protection. The synergistic effect of each component demonstrates significant technical advantages, as detailed below: A comparison between Comparative Example 1 and Example 1 shows that replacing the "chitosan + yeast β-glucan + yeast mannan" composite system with only chitosan reduces the protein activity retention rate and thermal denaturation temperature after irradiation, and decreases the activity retention rate after 60 days of storage. The decrease in activity retention rate indicates a significant synergistic effect in the ternary polysaccharide complex. Chitosan provides interfacial binding force, yeast β-glucan constructs a rigid network, and yeast mannan enhances the system's homogeneity. A single polysaccharide cannot simultaneously achieve all three functions; the lack of yeast polysaccharide leads to gaps in the physical barrier, making it easier for high-energy rays and free radicals to attack protein active sites. Comparing Comparative Example 2 with Example 1, it is evident that replacing the ternary complex system with single yeast β-glucan resulted in a decrease in activity retention rate after irradiation and a decrease in thermal denaturation temperature. This is because the absence of chitosan leads to a decrease in the polysaccharide retention rate. The electrostatic binding with proteins disappears, making it difficult for yeast β-glucan to form a continuous protective film on the protein surface. Simultaneously, the lack of water solubility regulation by yeast mannan makes the system prone to local aggregation, further weakening the protective effect. This demonstrates that chitosan is key to maintaining the homogeneity and binding stability of the protective agent in the compound system. A comparison between Comparative Example 3 and Example 1 shows that replacing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) with HATU as the crosslinking agent resulted in a sharp decrease in the activity retention rate after irradiation and a drop in the thermal denaturation temperature (60°C). The activity decreased after 60 days of storage because HATU's cross-linking activity was too strong, causing irreversible aggregation between protein molecules and directly destroying the active sites. EDC, on the other hand, only promoted mild cross-linking between polysaccharides and proteins without affecting the protein's native conformation. This fully demonstrates that the "mildness" of the cross-linking agent is the core prerequisite for ensuring protein activity, and highly active cross-linking agents are completely unsuitable for this scenario. Comparison between the blank control group and Example 1 shows that without the protectant, the protein activity after irradiation was only 28.6%, and after 60 days of storage, the activity decreased to 15.3%, with a sterility rate of only 92%. This contrasts sharply with the excellent performance of Example 1, directly verifying that the composite protectant of this invention completely solves the technical problem of protein activity loss during irradiation sterilization through a synergistic mechanism of "free radical scavenging (vitamin C + mannitol) + structural stabilization (ternary polysaccharide compound) + mild cross-linking (EDC)," while ensuring sterility and long-term stability.

[0035] This invention can also be implemented in various other ways. Without departing from the spirit and essence of this invention, those skilled in the art can make various corresponding changes and modifications according to this invention, but these corresponding changes and modifications should all fall within the protection scope of the appended claims.

Claims

1. A composite protective agent for protein irradiation sterilization, characterized in that, The composition comprises the following components by weight / volume percentage (w / v): trehalose 1-10%, mannitol 1-8%, polysaccharide complex system 0.5-5%, cross-linking agent 0.01-0.5%, free radical scavenger 0.1-2%, and the remainder is solvent.

2. The composite protective agent for protein irradiation sterilization according to claim 1, characterized in that, The polysaccharide complex system is composed of chitosan, yeast β-glucan, and yeast mannan. The mass ratio of chitosan to yeast β-glucan and yeast mannan in the polysaccharide complex system is (1:1:1) to (5:1:1).

3. The composite protective agent for protein irradiation sterilization according to claim 1, characterized in that, The yeast β-glucan is a glucan with β-1,3-glycosidic bonds as the main chain and β-1,6-glycosidic bonds as the branch chain, prepared by alkaline extraction or irradiation degradation.

4. The composite protective agent for protein irradiation sterilization according to claim 2, characterized in that, The yeast β-glucan was prepared by treating yeast cell wall material with 1-2 mol / L alkaline solution at 70-90℃ for 1-3 hours, collecting the insoluble matter, washing and drying it to obtain yeast β-glucan.

5. The composite protective agent for protein irradiation sterilization according to claim 2, characterized in that, The yeast mannan is a mannan with a main chain consisting of α-1,6-mannoglycosidic bonds and side chains containing α-1,2 and α-1,3-mannoglycosidic bonds, prepared by autolysis or hot water extraction.

6. The composite protective agent for protein irradiation sterilization according to claim 5, characterized in that, The hot water extraction method includes: extracting yeast cell wall raw material with water at 110-125℃ for 0.5-2 hours, collecting the supernatant, concentrating it, and then precipitating it with alcohol to obtain yeast mannan.

7. The composite protective agent for protein irradiation sterilization according to claim 1, characterized in that, The crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; the free radical scavenger is vitamin C.

8. The method for preparing the composite protective agent for protein irradiation sterilization according to any one of claims 1-7, characterized in that, Includes the following steps: S1: Dissolve trehalose, mannitol and the polysaccharide complex in a partial solvent to obtain a polysaccharide mixture; S2: Add the crosslinking agent to the solution obtained in step S1 and stir gently; S3: Add the free radical scavenger to the solution obtained in step S2 and stir to dissolve; S4: Adjust the pH and make up the volume with solvent, filter and sterilize to obtain a composite protective agent for protein irradiation sterilization.

9. The method for preparing the composite protective agent for protein irradiation sterilization according to claim 8, characterized in that, In step S2, the gentle stirring speed is 100-300 rpm and the time is 10-30 minutes; in step S4, the pH is adjusted to 6.0-7.5; the filtration and sterilization uses a 0.22-0.25 pore filter membrane.

10. The application of the composite protective agent for protein irradiation sterilization according to any one of claims 1-7, characterized in that, Includes the following steps: a) Mix the active protein with a composite protective agent used for protein irradiation sterilization to form a mixture; b) The mixture is sterilized by membrane filtration; c) The mixture after membrane filtration is sterilized by electron beam radiation at a dose of 5-50 kGy. In step b), the membrane filtration uses a 0.22-0.25 filter membrane; in step c), the radiation dose is 15-30 kGy; and the active protein is recombinant mussel adhesive protein or collagen.

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