A method for optimizing the production process of a freeze-dried preparation of deer gua polypeptide for injection

Abstract

This invention relates to the field of biomedical technology and discloses an optimized production process for lyophilized deer bone and melon seed polypeptides for injection, comprising the following steps: raw material pretreatment, enzymatic extraction, multi-stage membrane separation and purification, mixing and decolorization / deodorization, preparation of lyophilization solution, and freeze-drying; a complex protease composed of neutral protease and trypsin is used to perform stepwise enzymatic hydrolysis of deer bone and melon seed raw materials. The different optimal action sites and enzymatic digestion characteristics of the two enzymes form a synergistic effect. Compared with single enzymatic hydrolysis, this method can degrade proteins in the raw materials into polypeptides within the target molecular weight range, improving the yield of the target active polypeptides and making the molecular weight distribution of the product more concentrated. Subsequent targeted membrane separation and purification can remove impurities of different molecular weight ranges. While improving product purity, the gentle physical process ensures the bioactivity of the polypeptides, thereby guaranteeing the high quality and batch-to-batch consistency of the final product.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an optimized method for the production process of lyophilized deer antler polypeptide for injection. Background Technology

[0002] Deer-melon polypeptide is a sterilized aqueous solution prepared from the bones of the sika deer (a member of the Cervidae family) and the dried, mature seeds of the melon (a member of the Cucurbitaceae family), after separate extraction. Deer bone is sweet and slightly warm in nature, and is used to replenish deficiencies, strengthen muscles and bones, and treat rheumatic pain in the limbs and coldness in the muscles and bones. Melon seeds, on the other hand, have the effects of dispersing nodules and relieving pain. The combination of these two ingredients is used to dispel cold, eliminate wind and dampness, and relieve pain, and is suitable for rheumatism, rheumatoid arthritis, early healing of fractures, osteoarthritis, lower back and leg pain, and wound recovery.

[0003] Currently, in the production process of lyophilized deer antler peptides for injection, a single protease is often used for enzymatic hydrolysis, which cannot achieve optimal synergistic degradation of proteins from different sources. This results in low peptide yield and an excessively wide molecular weight distribution, affecting the enrichment efficiency of the target active peptides and batch-to-batch consistency.

[0004] Therefore, an optimized production process method for lyophilized deer antler polypeptide for injection is proposed to solve the above problems. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an optimized production process for lyophilized deer antler polypeptide for injection, which solves the problems mentioned in the background art that lead to low polypeptide yield and excessively wide molecular weight distribution, affecting the enrichment efficiency and batch-to-batch consistency of the target active polypeptide.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an optimized method for the production process of lyophilized deer antler polypeptide for injection, comprising the following steps: Step 1: Raw material pretreatment. Select deer bone raw material and melon seed raw material respectively. Degrease, demarrow, and crush the deer bone raw material, and shell, screen and crush the melon seed raw material. Step 2: Enzymatic extraction. The pretreated deer bone fragments and melon seeds were placed in different enzymatic reaction tanks, and extraction solvent and compound protease were added for stepwise enzymatic hydrolysis to obtain deer bone hydrolysate and melon seed hydrolysate. Step 3: Multi-stage membrane separation and purification. The deer bone hydrolysate and melon seed hydrolysate obtained in Step 2 are subjected to microfiltration, ultrafiltration and nanofiltration respectively to separate and enrich peptides in the target molecular weight range, so as to obtain deer bone peptide concentrate and melon seed peptide concentrate. Step 4: Mixing and decolorizing / deodorizing. The deer bone polypeptide concentrate obtained in Step 3 and the melon seed polypeptide concentrate are mixed at a ratio of 2:1 to 6:1 dry matter weight of deer bone polypeptide to melon seed polypeptide to obtain a mixed polypeptide solution. The mixed polypeptide solution is then decolorized and deodorized. Step 5: Preparation of lyophilization solution. Add the lyophilization protectant, pH adjuster and water for injection to the mixed peptide solution after step 4 in sequence. Stir to dissolve and make up to volume. Then filter sterile to obtain the lyophilized solution. Step Six: Freeze-drying. The freeze-dried solution obtained in Step Five is dispensed into vials, partially stoppered, and then transferred to a freeze dryer. The freeze dryer performs a pre-freezing, first drying, and second drying process, and finally fully stops the vials to obtain the freeze-dried formulation of deer antler polypeptide for injection.

[0007] Preferably, the raw material pretreatment in step one specifically includes: Deer bone pretreatment: Select long bones of the limbs of healthy sika deer or red deer, remove attached muscles and fascia, rinse with running water, and crush into bone pieces with a particle size of 3-8 mm in a crusher. Put the bone pieces into a defatting tank, add 3-5 times the volume of bone pieces of 90%-98% ethanol, and reflux extract at 45-55℃ for 2-4 hours. Filter, wash the bone pieces with purified water until neutral, and then put the defatted bone pieces into a demedullary tank, add 5-8 times the volume of 1-3% sodium hydroxide solution, and stir at 60-70℃ for 1-2 hours to remove bone marrow fat and impurities. After treatment, wash with purified water until neutral, drain, and obtain pretreated deer bone fragments. Pre-treatment of melon seeds: Select mature melon seeds, remove impurities and shriveled seeds by wind screening, remove the shells mechanically using a shelling machine, collect the melon seed kernels, rinse the melon seed kernels with purified water, and dry them in an oven at 45-55℃ until the moisture content is less than 8%. Crush the dried melon seed kernels with a pulverizer and pass them through a 40-60 mesh sieve to obtain melon seed raw powder.

[0008] Preferably, the enzymatic extraction in step two specifically includes: Deer bone enzymatic hydrolysis: Pretreated deer bone fragments are put into an enzymatic hydrolysis tank, and 8-12 times the weight of the fragments of purified water are added as the extraction solvent. The pH is adjusted to 6.5-7.5 with dilute hydrochloric acid or dilute sodium hydroxide solution, and the temperature is raised to 50-55℃. A complex protease consisting of 0.8%-1.5% of neutral protease and 0.5%-1.0% of trypsin by weight of the deer bone fragments is added. Enzymatic hydrolysis is carried out for 4-8 hours under constant temperature and stirring. After the enzymatic hydrolysis is completed, the temperature is raised to 90-95℃ and maintained for 10-15 minutes to inactivate the enzyme, and the crude deer bone enzymatic hydrolysate is obtained. Melon seed enzymatic hydrolysis: Melon seed powder is put into another enzymatic hydrolysis tank, and phosphate buffer solution with pH 7.0-8.0 (10-15 times the weight of the powder) is added as the extraction solvent. The temperature is raised to 45-50℃, and alkaline protease (1.0%-2.0% of the powder weight) is added. The mixture is enzymatically hydrolyzed for 3-6 hours under constant temperature and stirring. After the enzymatic hydrolysis is completed, the temperature is raised to 85-90℃ and maintained for 10-15 minutes to inactivate the enzyme, thus obtaining the crude enzymatic hydrolysate of melon seeds. The crude enzymatic extracts of deer bone and melon seeds need to be filtered through a 200-mesh sieve to remove large particles and obtain preliminarily clarified deer bone and melon seed enzymatic extracts.

[0009] Preferably, the total amount of the compound protease added accounts for 1.5%-2.5% of the weight of the pretreated deer bone fragments, wherein the mass ratio of the neutral protease to the trypsin is 1.0-1.4:0.4-0.8, the pH value of the deer bone enzymatic hydrolysis is 6.8-7.5, the enzymatic hydrolysis temperature is 50-55℃, and the enzymatic hydrolysis time is 4-8 hours. The amount of the alkaline protease added accounts for 1.0%-2.0% of the weight of the melon seed raw material powder, the pH value of the melon seed enzymatic hydrolysis is 7.0-8.0, the enzymatic hydrolysis temperature is 45-52℃, and the enzymatic hydrolysis time is 3-6 hours.

[0010] Preferably, the multi-stage membrane separation and purification in step three specifically includes: Microfiltration: Deer bone hydrolysate and melon seed hydrolysate are pumped into the microfiltration system respectively. Ceramic membranes or hollow fiber membranes with a molecular weight cutoff of 0.1-0.2 microns are used for filtration under operating conditions of 0.1-0.3 MPa and 20-40℃ to remove suspended particles, colloids and macromolecular impurities. The permeate is collected. Ultrafiltration: The obtained permeate is pumped into an ultrafiltration system, and an ultrafiltration membrane with a molecular weight cutoff of 3000-5000 Daltons is used for separation under operating conditions of 0.2-0.4 MPa and 15-30℃. The retentate is collected and the permeate is discarded. The retentate is the target peptide solution that has been preliminarily enriched. Nanofiltration: The obtained target peptide solution is pumped into a nanofiltration system. A nanofiltration membrane with a molecular weight cutoff of 200-500 Daltons is used for desalination and concentration under operating conditions of 0.5-1.0 MPa and 10-25℃. Most of the water and residual trace small molecule impurities and salts are removed, and finally, deer bone peptide concentrate and melon seed peptide concentrate with a solid content of 15%-25% are obtained.

[0011] Preferably, the mixing, decolorization, and deodorization in step four specifically include: Mixing: Pump the purified deer bone polypeptide concentrate and melon seed polypeptide concentrate from step three into the mixing tank. Under stirring, mix them at a weight ratio of 2:1 to 6:1 for deer bone polypeptide dry matter to melon seed polypeptide dry matter. Control the mixing temperature at 20-30℃, the stirring speed at 50-100 r / min, and the mixing time at 30-60 minutes to obtain a homogeneous mixed polypeptide solution. Decolorization: Add 1%-3% of the volume of activated carbon to the mixed peptide solution, heat to 60-70℃, stir and adsorb for 30-45 minutes, and then filter through a plate and frame filter or filter cartridge to remove the activated carbon and obtain the decolorized mixed peptide solution. Deodorization: The decolorized mixed polypeptide solution is passed through an adsorption column filled with food-grade β-cyclodextrin at a flow rate of 1-2 column volumes per hour. The inclusion effect of β-cyclodextrin is used to adsorb and remove the fishy substances in the polypeptide solution. The effluent is collected, which is the decolorized and deodorized mixed polypeptide solution.

[0012] Preferably, the activated carbon used in the decolorization step is 767 or 777 type powdered activated carbon for injection, and its addition amount is 1.5%-2.5% of the volume of the mixed polypeptide liquid, the adsorption temperature is 65℃±2℃, and the adsorption time is 35-40 minutes.

[0013] Preferably, the preparation of the lyophilization solution in step five specifically includes: Preparation of excipients: The freeze-drying protectant is composed of mannitol, glycine and dextran 40 in a weight ratio of 5-8:1-2:0.5-1, and the pH adjuster is a disodium hydrogen phosphate-citric acid buffer pair, used to stabilize the pH of the solution at 6.0-7.0. Preparation process: Transfer the mixed polypeptide solution after step four to a preparation tank and maintain the temperature at 15-25℃. First, add 80% of the prescribed amount of water for injection. Then, while stirring at 100-200 r / min, add the weighed components of the lyophilization protectant in sequence and stir until completely dissolved. Next, add the pH adjuster, stir evenly, and then bring the volume to the total volume with water for injection. Measure and fine-tune the pH value to 6.4-6.8. Sterile filtration: The drug solution after being brought to a fixed volume is filtered sequentially through a first-stage 0.45μm and a second-stage 0.22μm polyethersulfone sterile filter. The resulting filtrate is a sterile lyophilized solution, and its total peptide concentration should be controlled between 20-50 mg / mL.

[0014] Preferably, in the sterilization filtration process, the operating pressure of the first-stage filtration is controlled at 0.05-0.15 MPa, the operating pressure of the second-stage filtration is controlled at 0.1-0.25 MPa, and the temperature of the filtration solution is maintained at 15-25℃ throughout the filtration process.

[0015] Preferably, the freeze-drying in step six specifically includes the following procedures: Pre-freezing stage: Transfer the dispensed vials to the freeze dryer shelf, start the program, and lower the shelf temperature from room temperature to -45℃±2℃ at a rate of 0.5-1.0℃ / min, and maintain it at this temperature for 2-4 hours; First drying stage: Turn on the freeze dryer vacuum pump to reduce the pressure inside the drying chamber to 10-30Pa, then slowly raise the shelf temperature to -20℃±2℃ at a rate of 0.1-0.2℃ / min, and maintain this temperature for 20-30 hours to remove most of the free water; Secondary drying stage: After the first drying is completed, the shelf temperature is raised to 25℃±2℃ at a rate of 0.1-0.3℃ / min and maintained at this temperature for 8-12 hours, while maintaining a high vacuum of ≤10Pa to remove bound water; Stopping and unpacking: After the secondary drying is completed, the vials are automatically sealed by a hydraulic system under vacuum or nitrogen protection. Then the vacuum is broken and the vials are unpacked to obtain white or off-white freeze-dried block or powder of injectable deer antler polypeptide.

[0016] Compared with the prior art, the present invention provides an optimized production process for lyophilized formulations of injectable deer antler polypeptide, which has the following beneficial effects: 1. In this invention, a complex protease consisting of neutral protease and trypsin is used to perform stepwise enzymatic hydrolysis of deer bone and melon seed raw materials. The different optimal action sites and enzymatic cleavage characteristics of the two enzymes form a synergistic effect. Compared with single enzymatic hydrolysis, the protein in the raw materials can be degraded into peptides within the target molecular weight range, which improves the yield of the target active peptides and makes the molecular weight distribution of the product more concentrated. Subsequent targeted membrane separation and purification can remove impurities of different molecular weight ranges. While improving the purity of the product, the gentle physical process ensures the bioactivity of the peptides, thereby ensuring the high quality and batch-to-batch consistency of the final product.

[0017] 2. In this invention, after mixing, activated carbon adsorption and β-cyclodextrin column adsorption are used sequentially for decolorization and deodorization treatment. Activated carbon removes pigments and some odor substances due to its strong adsorption capacity; β-cyclodextrin selectively encapsulates residual odor substances through its special cavity structure. The two-step treatment works synergistically to improve the solution color, clarity and odor of the final product without introducing new impurities, thereby enhancing the sensory quality and clinical acceptability of the drug.

[0018] 3. In this invention, a freeze-drying protectant composed of mannitol, glycine, and dextran 40 is added to the mixed polypeptide solution before freeze-drying, and a programmed freeze-drying process is matched. This composite protectant system can form a stable protective layer around the polypeptide molecules, maintain their natural spatial conformation, and prevent denaturation, aggregation, and activity reduction caused by dehydration and phase change during freeze-drying and storage, thereby ensuring that the freeze-dried formulation has good resolubility and long-term storage stability. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: An optimized production process for a lyophilized formulation of injectable deer antler polypeptide, comprising the following steps: Step 1: Raw material pretreatment. Select deer bone raw material and melon seed raw material respectively. Degrease, demarrow, and crush the deer bone raw material, and shell, screen and crush the melon seed raw material. Step 2: Enzymatic extraction. The pretreated deer bone fragments and melon seeds were placed in different enzymatic reaction tanks, and extraction solvent and compound protease were added for stepwise enzymatic hydrolysis to obtain deer bone hydrolysate and melon seed hydrolysate. Step 3: Multi-stage membrane separation and purification. The deer bone hydrolysate and melon seed hydrolysate obtained in Step 2 are subjected to microfiltration, ultrafiltration and nanofiltration respectively to separate and enrich peptides in the target molecular weight range, so as to obtain deer bone peptide concentrate and melon seed peptide concentrate. Step 4: Mixing and decolorizing / deodorizing. The deer bone polypeptide concentrate obtained in Step 3 and the melon seed polypeptide concentrate are mixed at a ratio of 2:1 (dry matter weight of deer bone polypeptide to melon seed polypeptide) to obtain a mixed polypeptide solution. The mixed polypeptide solution is then decolorized and deodorized. Step 5: Preparation of lyophilization solution. Add the lyophilization protectant, pH adjuster and water for injection to the mixed peptide solution after step 4 in sequence. Stir to dissolve and make up to volume. Then filter sterile to obtain the lyophilized solution. Step Six: Freeze-drying. The freeze-dried solution obtained in Step Five is dispensed into vials, partially stoppered, and then transferred to a freeze dryer. The freeze dryer performs a pre-freezing, first drying, and second drying process, and finally fully stops the vials to obtain the freeze-dried formulation of deer antler polypeptide for injection.

[0021] The raw material pretreatment in step one specifically includes: Deer bone pretreatment: Select long bones of the limbs of healthy sika deer, remove attached muscles and fascia, rinse with running water, and crush into bone pieces with a particle size of 3mm in a crusher. Put the bone pieces into a defatting tank, add 3 times the volume of 90% ethanol, and reflux extract at 45℃ for 2 hours. Filter, wash the bone pieces with purified water until neutral, and then put the defatted bone pieces into a demedullary tank, add 5 times the volume of 1% sodium hydroxide solution, and stir at 60℃ for 1 hour to remove bone marrow fat and impurities. After treatment, wash with purified water until neutral, drain, and obtain pretreated deer bone fragments. Pretreatment of melon seeds: Select mature melon seeds, remove impurities and shriveled seeds by wind screening, remove the shells mechanically by shelling machine, collect melon seed kernels, rinse the melon seed kernels with purified water, dry them in a 45℃ oven until the moisture content is less than 8%, crush the dried melon seed kernels with a pulverizer, and pass them through a 40-mesh sieve to obtain melon seed raw powder.

[0022] Step two, enzymatic extraction, specifically includes: Deer bone enzymatic hydrolysis: Pretreated deer bone fragments were put into an enzymatic hydrolysis tank, and purified water of 8 times the weight of the fragments was added as the extraction solvent. The pH was adjusted to 6.5 with dilute hydrochloric acid solution, the temperature was raised to 50°C, and a complex protease consisting of 0.8% neutral protease and 0.5% trypsin by weight of the deer bone fragments was added. Enzymatic hydrolysis was carried out for 4 hours under constant temperature and stirring. After the enzymatic hydrolysis was completed, the temperature was raised to 90°C and maintained for 10 minutes to inactivate the enzyme, and the crude deer bone enzymatic hydrolysate was obtained. Melon seed enzymatic hydrolysis: Melon seed powder was put into another enzymatic hydrolysis tank, and phosphate buffer with pH 7.0 was added as the extraction solvent at 10 times the weight of the raw powder. The temperature was raised to 45°C, and alkaline protease at 1.0% of the weight of the melon seed powder was added. The mixture was enzymatically hydrolyzed for 3 hours under constant temperature and stirring. After the enzymatic hydrolysis was completed, the temperature was raised to 85°C and maintained for 10 minutes to inactivate the enzyme, and the crude extract of melon seed enzymatic hydrolysis was obtained. The crude enzymatic hydrolysate of deer bone and melon seed needs to be filtered through a 200-mesh sieve to remove large particles of residue, resulting in preliminarily clarified deer bone and melon seed enzymatic hydrolysate.

[0023] The total amount of compound protease added accounted for 1.5% of the weight of pretreated deer bone fragments, of which the mass ratio of neutral protease to trypsin was 1.0:0.4. The pH value of deer bone enzymatic hydrolysis was 6.8, the enzymatic hydrolysis temperature was 50℃, and the enzymatic hydrolysis time was 4 hours. The amount of alkaline protease added accounted for 1.0% of the weight of melon seed raw material powder. The pH value of melon seed enzymatic hydrolysis was 7.0, the enzymatic hydrolysis temperature was 45℃, and the enzymatic hydrolysis time was 3 hours.

[0024] Step three, multi-stage membrane separation and purification, specifically includes: Microfiltration: Deer bone hydrolysate and melon seed hydrolysate were pumped into the microfiltration system, respectively. A ceramic membrane with a molecular weight cutoff of 0.1 micrometers was used for filtration at an operating pressure of 0.1 MPa and a temperature of 20°C to remove suspended particles, colloids and macromolecular impurities. The permeate was collected. Ultrafiltration: The obtained permeate is pumped into the ultrafiltration system, and an ultrafiltration membrane with a molecular weight cutoff of 3000 Daltons is used for separation under the conditions of operating pressure of 0.2 MPa and temperature of 15℃. The retentate is collected and the permeate is discarded. The retentate is the target peptide solution that has been preliminarily enriched. Nanofiltration: The obtained target peptide solution is pumped into a nanofiltration system. A nanofiltration membrane with a molecular weight cutoff of 200 Daltons is used to desalinate and concentrate the solution under the conditions of operating pressure of 0.5 MPa and temperature of 10℃. Most of the water and residual trace small molecule impurities and salts are removed, and finally, deer bone peptide concentrate and melon seed peptide concentrate with a solid content of 15% are obtained.

[0025] Step four, mixing and decolorization / deodorization, specifically includes: Mixing: Pump the purified deer bone polypeptide concentrate and melon seed polypeptide concentrate from step three into the mixing tank. Under stirring, mix them at a weight ratio of 2:1 for deer bone polypeptide dry matter to melon seed polypeptide dry matter. Control the mixing temperature at 20℃, the stirring speed at 50r / min, and the mixing time at 30 minutes to obtain a homogeneous mixed polypeptide solution. Decolorization: Add 1% by volume of activated carbon to the mixed peptide solution, heat to 60°C, stir and adsorb for 30 minutes, then filter through a plate and frame filter to remove the activated carbon and obtain the decolorized mixed peptide solution. Deodorization: The decolorized mixed polypeptide solution is passed through an adsorption column filled with food-grade β-cyclodextrin at a flow rate of 1 column volume / hour. The inclusion effect of β-cyclodextrin is used to adsorb and remove the fishy substances in the polypeptide solution. The effluent is collected, which is the decolorized and deodorized mixed polypeptide solution.

[0026] The activated carbon used in the decolorization step was 767 type powdered activated carbon for injection, and its addition amount was 1.5% of the volume of the mixed polypeptide liquid. The adsorption temperature was 63℃ and the adsorption time was 35 minutes.

[0027] Step five, the preparation of the lyophilization solution, specifically includes: Excipient preparation: The freeze-drying protectant is composed of mannitol, glycine and dextran 40 in a weight ratio of 5:1:0.5. The pH adjuster is a disodium hydrogen phosphate-citric acid buffer pair, which is used to stabilize the pH of the solution at 6.0. Preparation process: Transfer the mixed polypeptide solution after step four to the preparation tank and keep the temperature at 15℃. First, add 80% of the prescribed amount of water for injection. Then, add the weighed components of the lyophilization protectant in sequence while stirring at 100 r / min. Stir until completely dissolved. Then, add the pH adjuster and stir evenly. Finally, add water for injection to bring the volume to the total volume. Measure and fine-tune the pH value to 6.4. Sterile filtration: The drug solution after being brought to a fixed volume is filtered sequentially through a first-stage 0.45μm and a second-stage 0.22μm polyethersulfone sterile filter. The resulting filtrate is a sterile lyophilized solution, and its total peptide concentration should be controlled at 20mg / mL.

[0028] During the sterilization filtration process, the operating pressure of the first-stage filtration is controlled at 0.05 MPa, the operating pressure of the second-stage filtration is controlled at 0.1 MPa, and the temperature of the filtration solution is maintained at 15℃ throughout the filtration process.

[0029] Step six, freeze drying, specifically includes the following procedures: Pre-freezing stage: Transfer the dispensed vials to the freeze dryer shelf, start the program, and lower the shelf temperature from room temperature to -47°C at a rate of 0.5°C / minute, and maintain it at this temperature for 2 hours; First drying stage: Turn on the freeze dryer vacuum pump to reduce the pressure inside the drying chamber to 10Pa, then slowly raise the shelf temperature to -22℃ at a rate of 0.1℃ / min, and maintain this temperature for 20 hours to remove most of the free water; Secondary drying stage: After the first drying is completed, the shelf temperature is raised to 23°C at a rate of 0.1°C / min and maintained at this temperature for 8 hours, while maintaining a high vacuum of ≤10Pa to remove bound water; Stopping and unpacking: After the secondary drying is completed, the vials are automatically sealed by a hydraulic system under vacuum protection. Then the vacuum is broken and the vials are unpacked to obtain white freeze-dried blocky injectable deer antler polypeptide.

[0030] Example 2: An optimized production process for a lyophilized formulation of injectable deer antler polypeptide, comprising the following steps: Step 1: Raw material pretreatment. Select deer bone raw material and melon seed raw material respectively. Degrease, demarrow, and crush the deer bone raw material, and shell, screen and crush the melon seed raw material. Step 2: Enzymatic extraction. The pretreated deer bone fragments and melon seeds were placed in different enzymatic reaction tanks, and extraction solvent and compound protease were added for stepwise enzymatic hydrolysis to obtain deer bone hydrolysate and melon seed hydrolysate. Step 3: Multi-stage membrane separation and purification. The deer bone hydrolysate and melon seed hydrolysate obtained in Step 2 are subjected to microfiltration, ultrafiltration and nanofiltration respectively to separate and enrich peptides in the target molecular weight range, so as to obtain deer bone peptide concentrate and melon seed peptide concentrate. Step 4: Mixing and decolorizing / deodorizing. The deer bone polypeptide concentrate obtained in Step 3 and the melon seed polypeptide concentrate are mixed at a dry matter weight ratio of 4:1 to obtain a mixed polypeptide solution. The mixed polypeptide solution is then decolorized and deodorized. Step 5: Preparation of lyophilization solution. Add the lyophilization protectant, pH adjuster and water for injection to the mixed peptide solution after step 4 in sequence. Stir to dissolve and make up to volume. Then filter sterile to obtain the lyophilized solution. Step Six: Freeze-drying. The freeze-dried solution obtained in Step Five is dispensed into vials, partially stoppered, and then transferred to a freeze dryer. The freeze dryer performs a pre-freezing, first drying, and second drying process, and finally fully stops the vials to obtain the freeze-dried formulation of deer antler polypeptide for injection.

[0031] The raw material pretreatment in step one specifically includes: Deer bone pretreatment: Select long bones of the limbs of healthy sika deer, remove attached muscles and fascia, rinse with running water, and crush into bone pieces with a particle size of 5mm in a crusher. Put the bone pieces into a defatting tank, add 4 times the volume of 95% ethanol, and reflux extract at 50℃ for 3 hours. Filter, wash the bone pieces with purified water until neutral, and then put the defatted bone pieces into a demedullary tank, add 6 times the volume of 2% sodium hydroxide solution, and stir at 65℃ for 1.5 hours to remove bone marrow fat and impurities. After treatment, wash with purified water until neutral, drain, and obtain pretreated deer bone fragments. Pretreatment of melon seeds: Select mature melon seeds, remove impurities and shriveled seeds by wind screening, remove the shells mechanically by shelling machine, collect melon seed kernels, rinse the melon seed kernels with purified water, dry them in a 50℃ oven until the moisture content is less than 8%, crush the dried melon seed kernels with a pulverizer, and pass them through a 50-mesh sieve to obtain melon seed raw powder.

[0032] Step two, enzymatic extraction, specifically includes: Deer bone enzymatic hydrolysis: Pretreated deer bone fragments were put into an enzymatic hydrolysis tank, and purified water of 10 times the weight of the fragments was added as the extraction solvent. The pH was adjusted to 7 with dilute hydrochloric acid solution, the temperature was raised to 52°C, and a complex protease consisting of 1.2% neutral protease and 0.7% trypsin by weight of the deer bone fragments was added. Enzymatic hydrolysis was carried out for 6 hours under constant temperature and stirring. After the enzymatic hydrolysis was completed, the temperature was raised to 93°C and maintained for 12 minutes to inactivate the enzyme, and the crude deer bone enzymatic hydrolysate was obtained. Melon seed enzymatic hydrolysis: Melon seed powder was put into another enzymatic hydrolysis tank, and phosphate buffer with pH 7.5 was added as the extraction solvent at 12 times the weight of the powder. The temperature was raised to 47°C, and alkaline protease at 1.5% of the weight of the melon seed powder was added. The mixture was enzymatically hydrolyzed for 4 hours under constant temperature and stirring. After the enzymatic hydrolysis was completed, the temperature was raised to 88°C and maintained for 12 minutes to inactivate the enzyme, and the crude extract of melon seed enzymatic hydrolysis was obtained. The crude enzymatic hydrolysate of deer bone and melon seed needs to be filtered through a 200-mesh sieve to remove large particles of residue, resulting in preliminarily clarified deer bone and melon seed enzymatic hydrolysate.

[0033] The total amount of compound protease added accounted for 2% of the weight of pretreated deer bone fragments. The mass ratio of neutral protease to trypsin was 1.2:0.6. The pH value of deer bone enzymatic hydrolysis was 7.2, the enzymatic hydrolysis temperature was 52℃, and the enzymatic hydrolysis time was 6 hours. The amount of alkaline protease added accounted for 1.5% of the weight of melon seed raw powder. The pH value of melon seed enzymatic hydrolysis was 7.5, the enzymatic hydrolysis temperature was 48℃, and the enzymatic hydrolysis time was 5 hours.

[0034] Step three, multi-stage membrane separation and purification, specifically includes: Microfiltration: Deer bone hydrolysate and melon seed hydrolysate were pumped into the microfiltration system, respectively. A ceramic membrane with a molecular weight cutoff of 0.15 micrometers was used for filtration at an operating pressure of 0.2 MPa and a temperature of 30°C to remove suspended particles, colloids and macromolecular impurities. The permeate was collected. Ultrafiltration: The obtained permeate is pumped into the ultrafiltration system, and an ultrafiltration membrane with a molecular weight cutoff of 4000 Daltons is used for separation under the conditions of operating pressure of 0.3 MPa and temperature of 20℃. The retentate is collected and the permeate is discarded. The retentate is the target peptide solution that has been preliminarily enriched. Nanofiltration: The obtained target peptide solution is pumped into a nanofiltration system. A nanofiltration membrane with a molecular weight cutoff of 350 Daltons is used to desalinate and concentrate the solution under the conditions of operating pressure of 0.7 MPa and temperature of 20°C. This removes most of the water and residual trace small molecule impurities and salts, and finally obtains deer bone peptide concentrate and melon seed peptide concentrate with a solid content of 20%.

[0035] Step four, mixing and decolorization / deodorization, specifically includes: Mixing: Pump the purified deer bone polypeptide concentrate and melon seed polypeptide concentrate from step three into the mixing tank. Under stirring, mix them at a weight ratio of 4:1 for deer bone polypeptide dry matter to melon seed polypeptide dry matter. Control the mixing temperature at 25℃, the stirring speed at 70r / min, and the mixing time at 45 minutes to obtain a homogeneous mixed polypeptide solution. Decolorization: Add 2% by volume of activated carbon to the mixed peptide solution, heat to 65°C, stir and adsorb for 40 minutes, then filter through a plate and frame filter to remove the activated carbon and obtain the decolorized mixed peptide solution. Deodorization: The decolorized mixed polypeptide solution is passed through an adsorption column filled with food-grade β-cyclodextrin at a flow rate of 1.5 column volumes per hour. The inclusion effect of β-cyclodextrin is used to adsorb and remove the fishy substances in the polypeptide solution. The effluent is collected, which is the decolorized and deodorized mixed polypeptide solution.

[0036] The activated carbon used in the decolorization step is 767 type powdered activated carbon for injection, and its addition amount is 2% of the volume of the mixed polypeptide liquid. The adsorption temperature is 65℃ and the adsorption time is 35 minutes.

[0037] Step five, the preparation of the lyophilization solution, specifically includes: Excipient preparation: The freeze-drying protectant is composed of mannitol, glycine and dextran 40 in a weight ratio of 6:1.5:0.7. The pH adjuster is a disodium hydrogen phosphate-citric acid buffer pair, which is used to stabilize the pH of the solution at 6.5. Preparation process: Transfer the mixed polypeptide solution after step four to the preparation tank and keep the temperature at 20℃. First, add 80% of the prescribed amount of water for injection. Then, add the weighed components of the lyophilization protectant in sequence while stirring at 150 r / min. Stir until completely dissolved. Then, add the pH adjuster and stir evenly. Finally, add water for injection to bring the volume to the total volume. Measure and fine-tune the pH value to 6.6. Sterile filtration: The drug solution after being brought to a fixed volume is filtered sequentially through a first-stage 0.45μm and a second-stage 0.22μm polyethersulfone sterile filter. The resulting filtrate is a sterile lyophilized solution, and its total peptide concentration should be controlled at 35mg / mL.

[0038] During the sterilization filtration process, the operating pressure of the first-stage filtration is controlled at 0.1 MPa, the operating pressure of the second-stage filtration is controlled at 0.2 MPa, and the temperature of the filtration solution is maintained at 20℃ throughout the filtration process.

[0039] Step six, freeze drying, specifically includes the following procedures: Pre-freezing stage: Transfer the dispensed vials to the freeze dryer shelf, start the program, and lower the shelf temperature from room temperature to -45°C at a rate of 0.7°C / minute, and maintain this temperature for 3 hours; First drying stage: Turn on the freeze dryer vacuum pump to reduce the pressure inside the drying chamber to 20Pa, then slowly raise the shelf temperature to -20℃ at a rate of 0.15℃ / min, and maintain this temperature for 25 hours to remove most of the free water; Secondary drying stage: After the first drying is completed, the shelf temperature is raised to 25°C at a rate of 0.2°C / min and maintained at this temperature for 10 hours, while maintaining a high vacuum of ≤10Pa to remove bound water; Stopping and unpacking: After the secondary drying is completed, the vials are automatically sealed by a hydraulic system under vacuum protection. Then the vacuum is broken and the vials are unpacked to obtain white freeze-dried blocky injectable deer antler polypeptide.

[0040] Example 3: An optimized production process for a lyophilized formulation of injectable deer antler polypeptide, comprising the following steps: Step 1: Raw material pretreatment. Select deer bone raw material and melon seed raw material respectively. Degrease, demarrow, and crush the deer bone raw material, and shell, screen and crush the melon seed raw material. Step 2: Enzymatic extraction. The pretreated deer bone fragments and melon seeds were placed in different enzymatic reaction tanks, and extraction solvent and compound protease were added for stepwise enzymatic hydrolysis to obtain deer bone hydrolysate and melon seed hydrolysate. Step 3: Multi-stage membrane separation and purification. The deer bone hydrolysate and melon seed hydrolysate obtained in Step 2 are subjected to microfiltration, ultrafiltration and nanofiltration respectively to separate and enrich peptides in the target molecular weight range, so as to obtain deer bone peptide concentrate and melon seed peptide concentrate. Step 4: Mixing and decolorizing / deodorizing. The deer bone polypeptide concentrate obtained in Step 3 and the melon seed polypeptide concentrate are mixed at a ratio of 6:1 (dry matter weight of deer bone polypeptide to melon seed polypeptide) to obtain a mixed polypeptide solution. The mixed polypeptide solution is then subjected to decolorization and deodorization treatment. Step 5: Preparation of lyophilization solution. Add the lyophilization protectant, pH adjuster and water for injection to the mixed peptide solution after step 4 in sequence. Stir to dissolve and make up to volume. Then filter sterile to obtain the lyophilized solution. Step Six: Freeze-drying. The freeze-dried solution obtained in Step Five is dispensed into vials, partially stoppered, and then transferred to a freeze dryer. The freeze dryer performs a pre-freezing, first drying, and second drying process, and finally fully stops the vials to obtain the freeze-dried formulation of deer antler polypeptide for injection.

[0041] The raw material pretreatment in step one specifically includes: Deer bone pretreatment: Select long bones of the limbs of healthy sika deer, remove attached muscles and fascia, rinse with running water, and crush into bone pieces with a particle size of 8mm in a crusher. Put the bone pieces into a defatting tank, add 5 times the volume of 98% ethanol, and reflux extract at 55℃ for 4 hours. Filter, wash the bone pieces with purified water until neutral, and then put the defatted bone pieces into a demedullary tank, add 8 times the volume of 3% sodium hydroxide solution, and stir at 70℃ for 2 hours to remove bone marrow fat and impurities. After treatment, wash with purified water until neutral, drain, and obtain pretreated deer bone fragments. Pretreatment of melon seeds: Select mature melon seeds, remove impurities and shriveled seeds by wind screening, remove the shells mechanically by shelling machine, collect melon seed kernels, rinse the melon seed kernels with purified water, dry them in a 55℃ oven until the moisture content is less than 8%, crush the dried melon seed kernels with a pulverizer, and pass them through a 60-mesh sieve to obtain melon seed raw powder.

[0042] Step two, enzymatic extraction, specifically includes: Deer bone enzymatic hydrolysis: Pretreated deer bone fragments were put into an enzymatic hydrolysis tank, and 12 times the weight of the fragments of purified water were added as the extraction solvent. The pH was adjusted to 7.5 with dilute hydrochloric acid solution, the temperature was raised to 55°C, and a complex protease consisting of 1.5% neutral protease and 1.0% trypsin by weight of the deer bone fragments was added. The enzymatic hydrolysis was carried out for 8 hours under constant temperature and stirring. After the enzymatic hydrolysis was completed, the temperature was raised to 95°C and maintained for 15 minutes to inactivate the enzyme, and the crude deer bone enzymatic hydrolysate was obtained. Melon seed enzymatic hydrolysis: Melon seed powder was put into another enzymatic hydrolysis tank, and phosphate buffer with pH 8.0 was added as the extraction solvent at 15 times the weight of the raw powder. The temperature was raised to 50°C, and alkaline protease at 2.0% of the weight of the melon seed powder was added. The mixture was enzymatically hydrolyzed for 6 hours under constant temperature and stirring. After the enzymatic hydrolysis was completed, the temperature was raised to 90°C and maintained for 15 minutes to inactivate the enzyme, and the crude extract of melon seed enzymatic hydrolysis was obtained. The crude enzymatic hydrolysate of deer bone and melon seed needs to be filtered through a 200-mesh sieve to remove large particles of residue, resulting in preliminarily clarified deer bone and melon seed enzymatic hydrolysate.

[0043] The total amount of compound protease added accounted for 2.5% of the weight of pretreated deer bone fragments, of which the mass ratio of neutral protease to trypsin was 1.4:0.8. The pH value of deer bone enzymatic hydrolysis was 7.5, the enzymatic hydrolysis temperature was 55℃, and the enzymatic hydrolysis time was 8 hours. The amount of alkaline protease added accounted for 2.0% of the weight of melon seed raw powder, the pH value of melon seed enzymatic hydrolysis was 8.0, the enzymatic hydrolysis temperature was 52℃, and the enzymatic hydrolysis time was 6 hours.

[0044] Step three, multi-stage membrane separation and purification, specifically includes: Microfiltration: Deer bone hydrolysate and melon seed hydrolysate were pumped into the microfiltration system, respectively. A ceramic membrane with a molecular weight cutoff of 0.2 microns was used for filtration at an operating pressure of 0.3 MPa and a temperature of 40°C to remove suspended particles, colloids and macromolecular impurities. The permeate was collected. Ultrafiltration: The obtained permeate is pumped into the ultrafiltration system, and an ultrafiltration membrane with a molecular weight cutoff of 5000 Daltons is used for separation under the conditions of operating pressure of 0.4 MPa and temperature of 30°C. The retentate is collected and the permeate is discarded. The retentate is the target peptide solution that has been preliminarily enriched. Nanofiltration: The obtained target peptide solution is pumped into a nanofiltration system. A nanofiltration membrane with a molecular weight cutoff of 500 Daltons is used to desalinate and concentrate the solution under the conditions of operating pressure of 1.0 MPa and temperature of 25°C. This removes most of the water and residual trace small molecule impurities and salts, and finally obtains deer bone peptide concentrate and melon seed peptide concentrate with a solid content of 25%.

[0045] Step four, mixing and decolorization / deodorization, specifically includes: Mixing: Pump the purified deer bone polypeptide concentrate and melon seed polypeptide concentrate from step three into the mixing tank. Under stirring, mix them at a weight ratio of 6:1 for deer bone polypeptide dry matter to melon seed polypeptide dry matter. Control the mixing temperature at 30℃, the stirring speed at 100r / min, and the mixing time at 60 minutes to obtain a homogeneous mixed polypeptide solution. Decolorization: Add 3% by volume of activated carbon to the mixed peptide solution, heat to 70°C, stir and adsorb for 45 minutes, then filter through a plate and frame filter to remove the activated carbon and obtain the decolorized mixed peptide solution. Deodorization: The decolorized mixed polypeptide solution is passed through an adsorption column filled with food-grade β-cyclodextrin at a flow rate of 2 column volumes per hour. The inclusion effect of β-cyclodextrin is used to adsorb and remove the fishy substances in the polypeptide solution. The effluent is collected, which is the decolorized and deodorized mixed polypeptide solution.

[0046] The activated carbon used in the decolorization step was 767 type powdered activated carbon for injection, and its addition amount was 2.5% of the volume of the mixed polypeptide liquid. The adsorption temperature was 67℃ and the adsorption time was 40 minutes.

[0047] Step five, the preparation of the lyophilization solution, specifically includes: Preparation of excipients: The freeze-drying protectant is composed of mannitol, glycine and dextran 40 in a weight ratio of 8:2:1. The pH adjuster is a disodium hydrogen phosphate-citric acid buffer pair, which is used to stabilize the pH of the solution at 7.0. Preparation process: Transfer the mixed polypeptide solution after step four to the preparation tank and keep the temperature at 25℃. First, add 80% of the prescribed amount of water for injection. Then, add the weighed components of the lyophilization protectant in sequence while stirring at 200 r / min. Stir until completely dissolved. Then, add the pH adjuster and stir evenly. Finally, bring the volume to the total volume with water for injection. Measure and fine-tune the pH value to 6.8. Sterile filtration: The drug solution after being brought to a fixed volume is filtered sequentially through a first-stage 0.45μm and a second-stage 0.22μm polyethersulfone sterile filter. The resulting filtrate is a sterile lyophilized solution, and its total peptide concentration should be controlled at 50mg / mL.

[0048] During the sterilization filtration process, the operating pressure of the first-stage filtration is controlled at 0.15 MPa, the operating pressure of the second-stage filtration is controlled at 0.25 MPa, and the temperature of the filtration solution is maintained at 25℃ throughout the filtration process.

[0049] Step six, freeze drying, specifically includes the following procedures: Pre-freezing stage: Transfer the dispensed vials to the freeze dryer shelf, start the program, and lower the shelf temperature from room temperature to -43°C at a rate of 1.0°C / minute, and maintain it at this temperature for 4 hours; First drying stage: Turn on the freeze dryer vacuum pump to reduce the pressure inside the drying chamber to 30Pa, then slowly raise the shelf temperature to -18℃ at a rate of 0.2℃ / min, and maintain this temperature for 30 hours to remove most of the free water; Secondary drying stage: After the first drying is completed, the shelf temperature is raised to 27°C at a rate of 0.3°C / min and maintained at this temperature for 12 hours, while maintaining a high vacuum of ≤10Pa to remove bound water; Stopping and unpacking: After the secondary drying is completed, the vials are automatically sealed by a hydraulic system under vacuum protection. Then the vacuum is broken and the vials are unpacked to obtain white freeze-dried blocky injectable deer antler polypeptide.

[0050] Comparative Example 1: The difference between this comparative example and Example 1 is that this comparative example uses only a single neutral protease for enzymatic extraction, and does not use a complex protease for enzymatic extraction.

[0051] Comparative Example 2 differs from Example 1 in that: this comparative example did not undergo multi-stage membrane separation and purification; the enzyme hydrolysate was directly concentrated after simple filtration.

[0052] Comparative Example 3 differs from Example 1 in that: this comparative example did not use a β-cyclodextrin adsorption column to deodorize the mixed polypeptide solution.

[0053] Comparative Example 4 differs from Example 1 in that no freeze-drying protectant was added to the mixed polypeptide solution during the preparation of the freeze-drying solution in this comparative example.

[0054] The quality and performance of the injectable deer antler polypeptide lyophilized formulations prepared in Examples 1-3 and Comparative Examples 1-4 were tested. The test items and methods are as follows: The peptide content and molecular weight distribution were determined by high performance liquid chromatography (HPLC) using a reversed-phase C18 column and a gradient elution of acetonitrile-0.1% trifluoroacetic acid aqueous solution. The detection wavelength was 220 nm. The total peptide content was calculated, and the molecular weight distribution of the peptides was determined by gel permeation chromatography. For the determination of the substance and the clarity of the solution, high performance liquid chromatography was used. The chromatogram was recorded at the impurity detection wavelength, and the maximum single impurity and total impurity content were calculated. The odor and acceptability of the solution were assessed by a trained sensory evaluation team through blind evaluation of the reconstituted drug solution. The odor and off-odor were scored on a scale of 0 to 5 based on their intensity, and the average score was calculated to evaluate the acceptability of the formulation. The appearance, reconstitution time and stability of the lyophilized formulation were tested. The appearance of the lyophilized cake was observed to be full, the color was uniform and there was no shrinkage. The complete reconstitution time after adding water for injection was recorded. The moisture content was determined by Karl Fischer moisture determination method. Finally, the sample was placed under the conditions of 40℃±2℃ and 75%±5% relative humidity for one month to accelerate the test and examine the changes in appearance, moisture and content.

[0055] The test data of the injectable deer antler polypeptide lyophilized formulations prepared in Examples 1-3 and Comparative Examples 1-4 are recorded in the table below: By comparing and analyzing the data in the table, it can be seen that the lyophilized injectable deer antler polypeptide formulations prepared using the processes in Examples 1-3 exhibit superior performance in all key quality attributes compared to the formulations prepared using the processes in Comparative Examples 1-4. This indicates that... A complex protease consisting of neutral protease and trypsin was used to perform stepwise enzymatic hydrolysis of deer bone and melon seed raw materials. Utilizing the different optimal action sites and enzymatic digestion characteristics of the two enzymes, a synergistic effect was achieved. Compared to single enzymatic hydrolysis, this method degraded proteins in the raw materials into peptides within the target molecular weight range, increasing the yield of the target active peptides and resulting in a more concentrated molecular weight distribution of the products. Subsequent targeted membrane separation and purification removed impurities of different molecular weight ranges. While improving product purity, the gentle physical process ensured the bioactivity of the peptides, thus guaranteeing the high quality and batch-to-batch consistency of the final product. After mixing, decolorization and deodorization were performed sequentially using activated carbon adsorption and β-cyclodextrin column adsorption. Activated carbon, with its strong adsorption capacity, removed pigments and some odor substances; β-cyclodextrin, through its unique cavity structure, selectively encapsulated residual odor substances. The synergistic effect of these two treatments improved the solution color, clarity, and odor of the final product without introducing new impurities, enhancing the sensory quality and clinical acceptability of the drug. Before lyophilization, a lyophilization protectant composed of mannitol, glycine, and dextran 40 is added to the mixed peptide solution, and a programmed lyophilization process is followed. This composite protectant system can form a stable protective layer around the peptide molecules, maintain their natural spatial conformation, and prevent denaturation, aggregation, and activity reduction caused by dehydration and phase transition during lyophilization and storage, thereby ensuring that the lyophilized formulation has good resolubility and long-term storage stability.

[0056] By comparing and analyzing the relevant data in the table, it can be seen that the freeze-dried deer antler polypeptide for injection prepared by the optimized process of this invention has advantages in core quality indicators such as effective ingredient content, purity, stability, and medication compliance, and has excellent comprehensive performance.

[0057] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An optimized production process for lyophilized formulations of injectable deer antler polypeptide, characterized in that: Includes the following steps: Step 1: Raw material pretreatment. Select deer bone raw material and melon seed raw material respectively. Degrease, demarrow, and crush the deer bone raw material, and shell, screen and crush the melon seed raw material. Step 2: Enzymatic extraction. The pretreated deer bone fragments and melon seeds were placed in different enzymatic reaction tanks, and extraction solvent and compound protease were added for stepwise enzymatic hydrolysis to obtain deer bone hydrolysate and melon seed hydrolysate. Step 3: Multi-stage membrane separation and purification. The deer bone hydrolysate and melon seed hydrolysate obtained in Step 2 are subjected to microfiltration, ultrafiltration and nanofiltration respectively to separate and enrich peptides in the target molecular weight range, so as to obtain deer bone peptide concentrate and melon seed peptide concentrate. Step 4: Mixing and decolorizing / deodorizing. The deer bone polypeptide concentrate obtained in Step 3 and the melon seed polypeptide concentrate are mixed at a ratio of 2:1 to 6:1 dry matter weight of deer bone polypeptide to melon seed polypeptide to obtain a mixed polypeptide solution. The mixed polypeptide solution is then decolorized and deodorized. Step 5: Preparation of lyophilization solution. Add the lyophilization protectant, pH adjuster and water for injection to the mixed peptide solution after step 4 in sequence. Stir to dissolve and make up to volume. Then filter sterile to obtain the lyophilized solution. Step Six: Freeze-drying. The freeze-dried solution obtained in Step Five is dispensed into vials, partially stoppered, and then transferred to a freeze dryer. The freeze dryer performs a pre-freezing, first drying, and second drying process, and finally fully stops the vials to obtain the freeze-dried formulation of deer antler polypeptide for injection.

2. The optimized production process of the lyophilized formulation of injectable deer antler polypeptide according to claim 1, characterized in that: The raw material pretreatment in step one specifically includes: Deer bone pretreatment: Select long bones of the limbs of healthy sika deer or red deer, remove attached muscles and fascia, rinse with running water, and crush into bone pieces with a particle size of 3-8 mm in a crusher. Put the bone pieces into a defatting tank, add 3-5 times the volume of bone pieces of 90%-98% ethanol, and reflux extract at 45-55℃ for 2-4 hours. Filter, wash the bone pieces with purified water until neutral, and then put the defatted bone pieces into a demedullary tank, add 5-8 times the volume of 1-3% sodium hydroxide solution, and stir at 60-70℃ for 1-2 hours to remove bone marrow fat and impurities. After treatment, wash with purified water until neutral, drain, and obtain pretreated deer bone fragments. Pre-treatment of melon seeds: Select mature melon seeds, remove impurities and shriveled seeds by wind screening, remove the shells mechanically using a shelling machine, collect the melon seed kernels, rinse the melon seed kernels with purified water, and dry them in an oven at 45-55℃ until the moisture content is less than 8%. Crush the dried melon seed kernels with a pulverizer and pass them through a 40-60 mesh sieve to obtain melon seed raw powder.

3. The optimized production process of the lyophilized formulation of injectable deer antler polypeptide according to claim 1, characterized in that: The enzymatic extraction in step two specifically includes: Deer bone enzymatic hydrolysis: Pretreated deer bone fragments are put into an enzymatic hydrolysis tank, and 8-12 times the weight of the fragments of purified water are added as the extraction solvent. The pH is adjusted to 6.5-7.5 with dilute hydrochloric acid or dilute sodium hydroxide solution, and the temperature is raised to 50-55℃. A complex protease consisting of 0.8%-1.5% of neutral protease and 0.5%-1.0% of trypsin by weight of the deer bone fragments is added. Enzymatic hydrolysis is carried out for 4-8 hours under constant temperature and stirring. After the enzymatic hydrolysis is completed, the temperature is raised to 90-95℃ and maintained for 10-15 minutes to inactivate the enzyme, and the crude deer bone enzymatic hydrolysate is obtained. Melon seed enzymatic hydrolysis: Melon seed powder is put into another enzymatic hydrolysis tank, and phosphate buffer solution with pH 7.0-8.0 (10-15 times the weight of the powder) is added as the extraction solvent. The temperature is raised to 45-50℃, and alkaline protease (1.0%-2.0% of the powder weight) is added. The mixture is enzymatically hydrolyzed for 3-6 hours under constant temperature and stirring. After the enzymatic hydrolysis is completed, the temperature is raised to 85-90℃ and maintained for 10-15 minutes to inactivate the enzyme, thus obtaining the crude enzymatic hydrolysate of melon seeds. The crude enzymatic extracts of deer bone and melon seeds need to be filtered through a 200-mesh sieve to remove large particles and obtain preliminarily clarified deer bone and melon seed enzymatic extracts.

4. The optimized production process of the lyophilized formulation of injectable deer antler polypeptide according to claim 3, characterized in that: The total amount of the compound protease added accounts for 1.5%-2.5% of the weight of the pretreated deer bone fragments, wherein the mass ratio of the neutral protease to the trypsin is 1.0-1.4:0.4-0.8, the deer bone enzymatic hydrolysis is performed at a pH of 6.8-7.5, a hydrolysis temperature of 50-55℃, and a hydrolysis time of 4-8 hours. The amount of the alkaline protease added accounts for 1.0%-2.0% of the weight of the melon seed raw material powder, the melon seed enzymatic hydrolysis is performed at a pH of 7.0-8.0, a hydrolysis temperature of 45-52℃, and a hydrolysis time of 3-6 hours.

5. The optimized production process of the lyophilized formulation of injectable deer antler polypeptide according to claim 1, characterized in that: The multi-stage membrane separation and purification in step three specifically includes: Microfiltration: Deer bone hydrolysate and melon seed hydrolysate are pumped into the microfiltration system respectively. Ceramic membranes or hollow fiber membranes with a molecular weight cutoff of 0.1-0.2 microns are used for filtration under operating conditions of 0.1-0.3 MPa and 20-40℃ to remove suspended particles, colloids and macromolecular impurities. The permeate is collected. Ultrafiltration: The obtained permeate is pumped into an ultrafiltration system, and an ultrafiltration membrane with a molecular weight cutoff of 3000-5000 Daltons is used for separation under operating conditions of 0.2-0.4 MPa and 15-30℃. The retentate is collected and the permeate is discarded. The retentate is the target peptide solution that has been preliminarily enriched. Nanofiltration: The obtained target peptide solution is pumped into a nanofiltration system. A nanofiltration membrane with a molecular weight cutoff of 200-500 Daltons is used for desalination and concentration under operating conditions of 0.5-1.0 MPa and 10-25℃. Most of the water and residual trace small molecule impurities and salts are removed, and finally, deer bone peptide concentrate and melon seed peptide concentrate with a solid content of 15%-25% are obtained.

6. The optimized production process of the lyophilized formulation of injectable deer antler polypeptide according to claim 1, characterized in that: The mixing, decolorization, and deodorization in step four specifically include: Mixing: Pump the purified deer bone polypeptide concentrate and melon seed polypeptide concentrate from step three into the mixing tank. Under stirring, mix them at a weight ratio of 2:1 to 6:1 for deer bone polypeptide dry matter to melon seed polypeptide dry matter. Control the mixing temperature at 20-30℃, the stirring speed at 50-100 r / min, and the mixing time at 30-60 minutes to obtain a homogeneous mixed polypeptide solution. Decolorization: Add 1%-3% of the volume of activated carbon to the mixed peptide solution, heat to 60-70℃, stir and adsorb for 30-45 minutes, and then filter through a plate and frame filter or filter cartridge to remove the activated carbon and obtain the decolorized mixed peptide solution. Deodorization: The decolorized mixed polypeptide solution is passed through an adsorption column filled with food-grade β-cyclodextrin at a flow rate of 1-2 column volumes per hour. The inclusion effect of β-cyclodextrin is used to adsorb and remove the fishy substances in the polypeptide solution. The effluent is collected, which is the decolorized and deodorized mixed polypeptide solution.

7. The optimized production process of the lyophilized formulation of injectable deer antler polypeptide according to claim 6, characterized in that: The activated carbon used in the decolorization step is 767 or 777 type powdered activated carbon for injection, and its addition amount is 1.5%-2.5% of the volume of the mixed polypeptide liquid. The adsorption temperature is 65℃±2℃ and the adsorption time is 35-40 minutes.

8. The optimized production process of the lyophilized formulation of injectable deer antler polypeptide according to claim 1, characterized in that: The preparation of the lyophilization solution in step five specifically includes: Preparation of excipients: The freeze-drying protectant is composed of mannitol, glycine and dextran 40 in a weight ratio of 5-8:1-2:0.5-1, and the pH adjuster is a disodium hydrogen phosphate-citric acid buffer pair, used to stabilize the pH of the solution at 6.0-7.

0. Preparation process: Transfer the mixed polypeptide solution after step four to a preparation tank and maintain the temperature at 15-25℃. First, add 80% of the prescribed amount of water for injection. Then, while stirring at 100-200 r / min, add the weighed components of the lyophilization protectant in sequence and stir until completely dissolved. Next, add the pH adjuster, stir evenly, and then bring the volume to the total volume with water for injection. Measure and fine-tune the pH value to 6.4-6.

8. Sterile filtration: The drug solution after being brought to a fixed volume is filtered sequentially through a first-stage 0.45μm and a second-stage 0.22μm polyethersulfone sterile filter. The resulting filtrate is a sterile lyophilized solution, and its total peptide concentration should be controlled between 20-50 mg / mL.

9. The optimized production process of the lyophilized formulation of injectable deer antler polypeptide according to claim 8, characterized in that: In the sterilization filtration process, the operating pressure of the first-stage filtration is controlled at 0.05-0.15 MPa, the operating pressure of the second-stage filtration is controlled at 0.1-0.25 MPa, and the temperature of the filtration solution is maintained at 15-25℃ throughout the filtration process.

10. The optimized production process of the lyophilized formulation of injectable deer antler polypeptide according to claim 1, characterized in that: The freeze-drying process in step six specifically includes the following procedures: Pre-freezing stage: Transfer the dispensed vials to the freeze dryer shelf, start the program, and lower the shelf temperature from room temperature to -45℃±2℃ at a rate of 0.5-1.0℃ / min, and maintain it at this temperature for 2-4 hours; First drying stage: Turn on the freeze dryer vacuum pump to reduce the pressure inside the drying chamber to 10-30Pa, then slowly raise the shelf temperature to -20℃±2℃ at a rate of 0.1-0.2℃ / min, and maintain this temperature for 20-30 hours to remove most of the free water; Secondary drying stage: After the first drying is completed, the shelf temperature is raised to 25℃±2℃ at a rate of 0.1-0.3℃ / min and maintained at this temperature for 8-12 hours, while maintaining a high vacuum of ≤10Pa to remove bound water; Stopping and unpacking: After the secondary drying is completed, the vials are automatically sealed by a hydraulic system under vacuum or nitrogen protection. Then the vacuum is broken and the vials are unpacked to obtain white or off-white freeze-dried block or powder of injectable deer antler polypeptide.