CN116440282B - Freeze-drying protective agent for live vaccine of infectious bronchitis and preparation method thereof - Google Patents
Freeze-drying protective agent for live vaccine of infectious bronchitis and preparation method thereof Download PDFInfo
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- CN116440282B CN116440282B CN202310514007.4A CN202310514007A CN116440282B CN 116440282 B CN116440282 B CN 116440282B CN 202310514007 A CN202310514007 A CN 202310514007A CN 116440282 B CN116440282 B CN 116440282B
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- infectious bronchitis
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 3
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a freeze-drying protective agent for a live vaccine of infectious bronchitis and a preparation method thereof, which belong to the technical field of biology, and specifically comprise liquid A and liquid B which are uniformly mixed in equal volume by mass and volume percent, wherein the liquid A comprises 4.0-8.0% of glucan, 4.0-8.0% of enzymolysis casein, 2.0-6.0% of carrageenan and 2.0-6.0% of chondroitin sulfate; the solution B comprises 2.0 to 6.0 percent of hydrolyzed milk protein and 2.0 to 6.0 percent of sorbitol. The invention also discloses a preparation method of the freeze-drying protective agent and an application method for preparing a freeze-drying product of the live vaccine. The protective agent is simple to prepare, is environment-friendly, ensures that the prepared vaccine product has smoother and smoother appearance, is easy to dissolve, has prolonged heat resistance and retention time, and is convenient for mass production.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a freeze-drying protective agent for a live vaccine of infectious bronchitis and a preparation method thereof.
Background
Infectious bronchitis (Avian infectious bronchitis, IB), a second type of infectious disease, is an acute, highly contagious, respiratory infectious disease in chickens caused by infectious bronchitis virus. The clinical features are dyspnea, royalty, cough, open mouth breathing and sneeze. The disease is widely spread around the world and is an important epidemic disease in chicken industry. The infectious bronchitis virus has strong variability, more than 30 serotypes are separated in the world, the cross immunity protection effect between different serotypes is poor, and the infectious bronchitis virus QX (Qingdao Xianwei) type infectious bronchitis virus can cause the adeno-gastrosis of chickens, so that the development of the QX type attenuated vaccine and the freeze-drying protective agent thereof has important significance for preventing and controlling the occurrence and the epidemic of IB in China.
In order to maintain the immunoreactivity of viruses or bacteria in the freeze-dried vaccine and reduce the damage of freeze-drying to the viruses or bacteria, a freeze-drying protective agent is required to be added when the live vaccine is freeze-dried, but the existing freeze-dried vaccine for the infectious bronchitis prepared by adopting the conventional freeze-drying protective agent such as milk or gelatin protective agent exists: uneven surface with depressions and cracks; when the vaccine is used, the dissolution speed is slower; the freeze-drying toxic price loss is large; can only be stored under the freezing condition of minus 15 ℃, has poor storage effect under the refrigerating condition (2-8 ℃), and can only reach 18 months generally; also has a partial refrigeration condition (2-8 ℃) which can be stored for more than 24 months, but the vaccine titer is seriously reduced after the vaccine is diluted; or has long preservation time, high cost, toxic substances, inconvenient operation, environmental pollution and other problems.
The patent ZL201210543549.6 discloses a freeze-drying protective agent for HI antigen of infectious bronchitis virus and application thereof in preparation of HI antigen, and the protective agent is prepared by uniformly mixing solution A and solution B according to equal volume, wherein solution A: comprises trehalose with mass and volume percentage of 5% and gelatin with mass and volume percentage of 2%, and deionized water for constant volume, and sterilizing at 116 ℃ under high pressure for 30min; and (2) liquid B: contains 0.1 percent of sodium azide and 1 percent of sodium thiosulfate in mass and volume percent, and the deionized water is used for constant volume and 0.22 mu m filter membrane sterilization. However, if sodium azide is usually used as a preservative, the sodium azide has strong toxicity, has strong toxic and side effects on products, personnel and environment, has very strict control measures in the processes of purchasing, transporting and storing, and has very high requirements on operators during use.
Patent No. ZL201510835120.8 discloses a heat-resistant protective agent for live vaccines, a preparation method and application thereof, and the protective agent is prepared from the following components: trehalose, glucose, gelatin, thiourea, polyvinylpyrrolidone K30 and pure water; the content of each component in each 100ml of the heat-resistant protective agent for the live vaccine is as follows: 5g of trehalose, 5g of glucose, 2g of gelatin, 1g of thiourea, 306g of polyvinylpyrrolidone and the balance of pure water. However, thiourea has carcinogenic, teratogenic and mutagenic properties, has strong toxic and side effects and irritation to immunized animals, and influences the safety of products.
Disclosure of Invention
In order to solve the problems, the inventor provides a freeze-drying protective agent for live vaccine of infectious bronchitis and a preparation method thereof on the basis of the prior art, which can realize complete appearance of the vaccine without concave cracks; the dissolution speed is high; the operation is safe and simple, and the heat resistance is good, the preservation time is long, the effect of diluting and using the vaccine is not affected, and the like.
The invention provides a freeze-drying protective agent for a live vaccine of infectious bronchitis, which is formed by mixing liquid A and liquid B in equal volume proportion, wherein the liquid A comprises 4.0-8.0% of glucan, 4.0-8.0% of enzymolysis casein, 2.0-6.0% of carrageenan, 2.0-6.0% of chondroitin sulfate and the balance of sterile water in percentage by mass and volume; the liquid B comprises 2.0% -6.0% of hydrolyzed milk protein, 2.0% -6.0% of sorbitol and the balance of sterile water.
Wherein, the dextran plays a significant role in preventing the secondary structure of the protein from changing, and the extension and aggregation of the polypeptide chains of the protein during the freeze-drying treatment and the storage period. After the dextran is mixed with the virus liquid, the freeze-dried live vaccine is protected in the stages of freeze-drying, transportation, preservation and the like, so that the heat resistance of the freeze-dried live vaccine is improved and the preservation time is prolonged. The hydrolyzed milk protein is an enzymolysis product of whey protein, contains high levels of essential amino acids, has higher nutrition quality, and improves the survival rate and biological activity of viruses. The carrageenan serves as a filling agent and an excipient, plays a role in solubilization and stabilization, and can prevent the freeze-dried product from drying shrinkage deformation. The chondroitin sulfate can be combined to the vaccine virus nucleoprotein, so that the structural stability of the protective agent is further enhanced, and the vaccine activity is better protected. Enzymatic hydrolysis of casein and sorbitol. Sorbitol can promote the protein to be uniformly dispersed in the freeze-dried block, and further improves the heat resistance of the freeze-dried live vaccine.
Further, phosphate Buffer (PBS) is used as a pH regulator in the solution A to adjust the pH of the protective agent to 7.2-7.4.
Further, the PBS is 1.64 percent of dipotassium hydrogen phosphate and 0.52 percent of monopotassium hydrogen phosphate,
further, the total volume of the protective agent contains 3.0% of glucan, 3.0% of enzymolysis casein, 2.0% of carrageenan, 2.0% of chondroitin sulfate, 2.0% of hydrolyzed milk protein and 2.0% of sorbitol.
Further, the protective agent B also comprises 0.4% of glutathione. The glutathione has an antioxidation effect.
The invention also provides an application of the freeze-drying protective agent in preparing a live vaccine product of the avian infectious bronchitis QX87 strain.
The invention also provides a preparation method of the live vaccine product for the infectious bronchitis, which comprises the steps of mixing any one of the freeze-drying protective agents with the QX87 virus liquid of the infectious bronchitis in equal proportion, quantitatively split charging, half plugging, vacuum freeze drying, and then plugging and taking out. The method specifically comprises the following steps:
(1) Taking each component of the solution A according to a proportion, dissolving the components with sterile water to fix the volume, adjusting the pH to 7.2-7.4 with PBS, and sterilizing for 30 minutes at 116 ℃; taking each component of the solution B according to a proportion, dissolving the components with sterile water to a certain volume, and filtering and sterilizing the solution B with the volume of 0.22 mu m;
(2) Uniformly mixing chicken infectious bronchitis QX87 virus liquid and the same amount of freeze-drying protective agent; (3) Quantitatively packaging, half-plugging, and vacuum freeze-drying.
The lyophilization procedure was: pre-freezing to-40.0deg.C for 4 hr, sublimating at-3.0deg.C for 1 hr, sublimating at 0deg.C for 8 hr, sublimating at 5.0deg.C for 4 hr, sublimating at 10.0deg.C for 4 hr, sublimating at 13.0deg.C for 1 hr, sublimating at 15.0deg.C for 1 hr, analyzing at 25.0deg.C for 1 hr, analyzing at 29.0deg.C for 4 hr, pressing in the container, and discharging.
The invention has the beneficial effects that:
(1) The vaccine prepared by the freeze-drying protective agent can be stored for 6 months at room temperature (25 ℃), and can be stored for 36 months under the conditions of refrigeration (2-8 ℃) and freezing (-15 ℃);
(2) The freeze-drying protective agent prepared by the invention does not contain toxic and harmful substances, is environment-friendly, is safe to animals, has no stress, and has convenient material sources;
(3) The freeze-dried vaccine prepared by the invention has good water solubility, and can be quickly dissolved into transparent solution after being added with water, so that the use is convenient; (4) The invention uses the combination of dextran and carrageenan with a certain proportion as a freeze-dried product bracket, so that the freeze-dried vaccine has higher glass transition temperature, low endotoxin and low hygroscopicity; the freeze-dried live vaccine is protected in the stages of freeze-drying, transportation, preservation and the like, so that the heat resistance of the freeze-dried live vaccine is improved and the preservation time is prolonged;
(5) The invention uses hydrolyzed milk protein and enzymatic hydrolysis casein to make the freeze-dried vaccine easier to hydrolyze, and the enzymatic hydrolysis casein hydrolysate is rich and easy to be absorbed;
(6) The sorbitol and carrageenan of the invention make the appearance of the freeze-dried vaccine smoother;
(7) The glutathione can reduce the oxidation of the vaccine in the storage process;
(8) The protective agent provided by the invention has simple formula and preparation process, and the freeze-drying time is 28 hours, so that the heat-resistant protective agent and the preparation of the live vaccine for the infectious bronchitis of chickens using the protective agent can be applied to mass production.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, but the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by a person skilled in the art based on the embodiments of the invention without any inventive effort, fall within the scope of the protection of the invention.
1. Material source
The experimental methods in the following examples, unless otherwise indicated, are all conventional methods, and the materials and reagents, unless otherwise indicated, are commercially available.
2. Experimental method
1. Preparing a protective agent: the heat-resistant freeze-drying protective agent for the chicken infectious bronchitis live vaccine QX87 strain is prepared according to the following formula, and meanwhile, the conventional sucrose skim milk protective agent and the gelatin sucrose protective agent are prepared. When in seedling preparation, the protective agent is respectively mixed with the virus liquid of the avian infectious bronchitis QX87 strain in equal quantity, quantitatively packaged and vacuum freeze-dried.
Example 1:
and (3) solution A: dissolving 20g of glucan, 20g of enzymolysis casein, 10g of carrageenan, 10g of chondroitin sulfate, 8.2g of dipotassium hydrogen phosphate and 0.26g of potassium dihydrogen phosphate with sterile water, fixing the volume to 500ml, and sterilizing at 116 ℃ for 30 minutes; and (2) liquid B: dissolving 10g of hydrolyzed milk protein and 10g of sorbitol with sterile water respectively, fixing the volume to 500ml, and filtering and sterilizing with 0.22 μm;
when in use, the solution A and the solution B are mixed uniformly, 1000ml of infectious bronchitis QX87 strain virus solution is added, and the mixture is mixed uniformly. The components and parts by weight of the lyoprotectant are shown in table 1.
Example 2:
and (3) solution A: 30g of glucan, 30g of enzymolysis casein, 20g of carrageenan, 20g of chondroitin sulfate, 8.2g of dipotassium hydrogen phosphate and 0.26g of potassium dihydrogen phosphate are respectively dissolved by sterile water, the volume is fixed to 500ml, and the mixture is sterilized for 30 minutes at 116 ℃; and (2) liquid B: dissolving hydrolyzed milk protein 20g and sorbitol 20g with sterile water respectively, fixing volume to 500ml, and filtering for sterilization at 0.22 μm;
when in use, the solution A and the solution B are mixed uniformly, 1000ml of infectious bronchitis QX87 strain virus solution is added, and the mixture is mixed uniformly. The components and parts by weight of the lyoprotectant are shown in table 1.
Example 3:
and (3) solution A: dissolving 40g of glucan, 40g of enzymolysis casein, 30g of carrageenan, 30g of chondroitin sulfate, 8.2g of dipotassium hydrogen phosphate and 0.26g of potassium dihydrogen phosphate with sterile water, fixing the volume to 500ml, and sterilizing at 116 ℃ for 30 minutes; and (2) liquid B: 30g of hydrolyzed milk protein and 30g of sorbitol are respectively dissolved by sterile water, the volume is fixed to 500ml, and filtration and sterilization are carried out on the milk protein and the sorbitol at 0.22 mu m;
when in use, the solution A and the solution B are mixed uniformly, 1000ml of infectious bronchitis QX87 strain virus solution is added, and the mixture is mixed uniformly. The components and parts by weight of the lyoprotectant are shown in table 1.
Example 4:
and (3) solution A: 30g of glucan, 30g of enzymolysis casein, 20g of carrageenan, 10g of chondroitin sulfate, 8.2g of dipotassium hydrogen phosphate and 0.26g of potassium dihydrogen phosphate are respectively dissolved by sterile water, the volume is fixed to 500ml, and the mixture is sterilized for 30 minutes at 116 ℃; and (2) liquid B: dissolving hydrolyzed milk protein 20g and sorbitol 20g with sterile water respectively, fixing volume to 500ml, and filtering for sterilization at 0.22 μm;
when in use, the solution A and the solution B are mixed uniformly, 1000ml of infectious bronchitis QX87 strain virus solution is added, and the mixture is mixed uniformly. The components and parts by weight of the lyoprotectant are shown in table 1.
Example 5:
and (3) solution A: 30g of glucan, 30g of enzymolysis casein, 20g of carrageenan, 30g of chondroitin sulfate, 8.2g of dipotassium hydrogen phosphate and 0.26g of potassium dihydrogen phosphate are respectively dissolved by sterile water, the volume is fixed to 500ml, and the mixture is sterilized for 30 minutes at 116 ℃; and (2) liquid B: dissolving hydrolyzed milk protein 20g and sorbitol 20g with sterile water respectively, fixing volume to 500ml, and filtering for sterilization at 0.22 μm;
when in use, the solution A and the solution B are mixed uniformly, 1000ml of infectious bronchitis QX87 strain virus solution is added, and the mixture is mixed uniformly. The components and parts by weight of the lyoprotectant are shown in table 1.
Example 6:
and (3) solution A: dissolving 20g of glucan, 30g of enzymolysis casein, 20g of carrageenan, 20g of chondroitin sulfate, 8.2g of dipotassium hydrogen phosphate and 0.26g of potassium dihydrogen phosphate with sterile water, fixing the volume to 500ml, and sterilizing at 116 ℃ for 30 minutes; and (2) liquid B: dissolving hydrolyzed milk protein 20g, sorbitol 20g and glutathione 2g with sterile water respectively, fixing volume to 500ml, and filtering and sterilizing with 0.22 μm;
when in use, the solution A and the solution B are mixed uniformly, 1000ml of infectious bronchitis QX87 strain virus solution is added, and the mixture is mixed uniformly. The components and parts by weight of the lyoprotectant are shown in table 1.
Example 7:
and (3) solution A: 30g of glucan, 30g of enzymolysis casein, 20g of carrageenan, 20g of chondroitin sulfate, 8.2g of dipotassium hydrogen phosphate and 0.26g of potassium dihydrogen phosphate are respectively dissolved by sterile water, the volume is fixed to 500ml, and the mixture is sterilized for 30 minutes at 116 ℃; and (2) liquid B: dissolving hydrolyzed milk protein 20g, sorbitol 20g and glutathione 2g with sterile water respectively, fixing volume to 500ml, and filtering and sterilizing with 0.22 μm;
when in use, the solution A and the solution B are mixed uniformly, 1000ml of infectious bronchitis QX87 strain virus solution is added, and the mixture is mixed uniformly. The components and parts by weight of the lyoprotectant are shown in table 1.
Example 8:
and (3) solution A: dissolving 40g of glucan, 30g of enzymolysis casein, 30g of carrageenan, 20g of chondroitin sulfate, 8.2g of dipotassium hydrogen phosphate and 0.26g of potassium dihydrogen phosphate with sterile water, fixing the volume to 500ml, and sterilizing at 116 ℃ for 30 minutes; and (2) liquid B: dissolving hydrolyzed milk protein 20g, sorbitol 20g and glutathione 2g with sterile water respectively, fixing volume to 500ml, and filtering and sterilizing with 0.22 μm;
when in use, the solution A and the solution B are mixed uniformly, 1000ml of infectious bronchitis QX87 strain virus solution is added, and the mixture is mixed uniformly. The components and parts by weight of the lyoprotectant are shown in table 1.
Example 9:
and (3) solution A: 30g of glucan, 30g of enzymolysis casein, 20g of carrageenan, 10g of chondroitin sulfate, 8.2g of dipotassium hydrogen phosphate and 0.26g of potassium dihydrogen phosphate are respectively dissolved by sterile water, the volume is fixed to 500ml, and the mixture is sterilized for 30 minutes at 116 ℃; and (2) liquid B: dissolving hydrolyzed milk protein 20g, sorbitol 20g and glutathione 2g with sterile water respectively, fixing volume to 500ml, and filtering and sterilizing with 0.22 μm;
when in use, the solution A and the solution B are mixed uniformly, 1000ml of infectious bronchitis QX87 strain virus solution is added, and the mixture is mixed uniformly. The components and parts by weight of the lyoprotectant are shown in table 1.
Example 10:
and (3) solution A: 30g of glucan, 30g of enzymolysis casein, 20g of carrageenan, 30g of chondroitin sulfate, 8.2g of dipotassium hydrogen phosphate and 0.26g of potassium dihydrogen phosphate are respectively dissolved by sterile water, the volume is fixed to 500ml, and the mixture is sterilized for 30 minutes at 116 ℃; and (2) liquid B: dissolving hydrolyzed milk protein 20g, sorbitol 20g and glutathione 2g with sterile water respectively, fixing volume to 500ml, and filtering and sterilizing with 0.22 μm;
when in use, the solution A and the solution B are mixed uniformly, 1000ml of infectious bronchitis QX87 strain virus solution is added, and the mixture is mixed uniformly. The components and parts by weight of the lyoprotectant are shown in table 1.
Comparative example 1 sucrose skim milk protectant (fixed formulation)
50g of sucrose, 100g of skimmed milk powder, adding 1000ml of sterile water, mixing uniformly, sterilizing at 116 ℃ for 30 minutes, and preserving at 2-8 ℃. When in use, the same amount of sucrose skim milk protective agent is added into the virus liquid of the avian infectious bronchitis QX87 strain, and the mixture is uniformly mixed. The components and parts by weight of the lyoprotectant are shown in table 1.
Comparative example 2 gelatin sucrose protectant (fixed formulation)
25g of gelatin, 100g of sucrose and 1000ml of sterile water are added to be mixed uniformly, sterilized for 30 minutes at 116 ℃ and stored at room temperature. When in use, the equal amount of gelatin sucrose protective agent is added into the virus liquid of the avian infectious bronchitis QX87 strain, and the mixture is uniformly mixed. The components and parts by weight of the lyoprotectant are shown in table 1.
Table 1 lyoprotectant composition and parts by weight
When in use, the protective agent A liquid and the protective agent B liquid are uniformly mixed in equal quantity, and then the infectious bronchitis QX87 strain virus liquid which is equal to the protective agent is added and uniformly mixed.
2. Mixing the virus liquid with the same amount of protective agent, stirring, packaging with 2.0 ml/bottle, namely 1000 parts per bottle, half adding plug, and vacuum freeze drying.
The lyophilization procedure was: pre-freezing to-40.0deg.C for 4 hr, sublimating at-3.0deg.C for 1 hr, sublimating at 0deg.C for 8 hr, sublimating at 5.0deg.C for 4 hr, sublimating at 10.0deg.C for 4 hr, sublimating at 13.0deg.C for 1 hr, sublimating at 15.0deg.C for 1 hr, analyzing at 25.0deg.C for 1 hr, analyzing at 29.0deg.C for 4 hr, pressing in the container, and discharging. The entire lyophilization procedure was about 28 hours.
3. And (3) checking a finished product: after the freeze-drying of the product is finished, the container is filled with plugs, and the samples are respectively sampled for properties, sterility test, mycoplasma test, exogenous virus test, identification test, safety test, efficacy test, residual moisture measurement, vacuum degree measurement and the like.
3.1 traits: the yellowish spongy loose agglomerate is easy to separate from the bottle wall and can be dissolved quickly after the diluent is added.
3.2 sterility testing: the test is carried out according to the annex of the current Chinese animal pharmacopoeia, and the bacteria should grow aseptically.
3.3 mycoplasma assay: the test is carried out according to the annex of the current Chinese animal pharmacopoeia, and mycoplasma growth should not be caused.
3.4 exogenous virus assay: the test is carried out according to the annex of the current Chinese animal pharmacopoeia, and no exogenous virus pollution is needed. 3.5 authentication test: diluting vaccine preparation with sterile physiological saline or PBS to 200EID 50 0.1ml, mixing with equal amount of specific serum for resisting avian infectious bronchitis virus QX87 strain, neutralizing at room temperature for 1 hr, inoculating 10-11 in allantoic cavity10 SPF chick embryos of day-age, 0.2ml per embryo, should not cause specific death or chick embryo pathological changes when being placed at 36-37 ℃ for 144 hours, and at least more than 8 chick embryos are healthy and alive.
3.6 safety inspection: after diluting the vaccine preparation with 3ml of sterile physiological saline or PBS, 10 SPF chickens with 1-10 days of age are inoculated by nasal drip, each 0.03ml (containing 10 doses) is observed for 14 days, and the vaccine preparation is fully healthy (normal spirit, feeding, drinking water and feces and no abnormal clinical symptoms). If the non-specific death exists, the number of the non-specific death should not exceed 1, otherwise, the detection should be repeated for 1 time.
3.7 efficacy test: diluting vaccine product with sterilized normal saline to 1 feather content of 0.1ml, serial diluting 10 times, and collecting 10 times -2 、10 -3 、10 -4 Three dilutions, 5 SPF chick embryos of 10-11 days old are inoculated in each allantoic cavity, 0.1ml of each embryo, 5 chick embryos of the same day old without SPF are set as a control, the chick embryos are incubated at 37 ℃ for 144 hours, dead chick embryos within 24 hours are discarded, and EID is calculated according to total number of chick embryos with specific lesions such as fetal loss, curling, small development (the inoculated fetus is more than 2g lower than the lightest fetus weight of a control) in the dead chick embryos 24-144 hours after inoculation and the live chick embryos 144 hours after inoculation 50 . Each feather should be not less than 10 3.5 EID 50 。
3.8 residual moisture determination: each batch of products is randomly sampled for 2 bottles, and the content of the samples is not higher than 4.0 percent according to the annex of the current Chinese animal pharmacopoeia.
3.9 vacuum measurement: the white, pink or purple glow should appear as measured in accordance with the annex of the current "Chinese animal pharmacopoeia".
4. Ageing resistance test: after the freeze-drying of the samples is finished, taking 4 bottles of samples from each group, respectively storing at 37 ℃, taking out after 10 days, and randomly selecting 2 bottles for efficacy test; and also compared with samples stored at-15 ℃.
5. Shelf life test at different temperatures
After the freeze-drying of the product is finished, taking 60 bottles for each group, respectively placing the bottles at room temperature (25 ℃), refrigerating (2-8 ℃) and freezing (-15 ℃) for 20 bottles, taking 2 bottles for efficacy test every 1 month under the room temperature preservation condition, and finishing 6 months; 2 bottles were sampled every 6 months for efficacy testing under refrigerated and frozen storage conditions to the end of 36 months.
3. Experimental results
1. The batch numbers of 10 products freeze-dried by the formula protective agent are respectively S180601, S180602, S180603, S180604, S180605, S180606, S180607, S180608, S180609 and S180610, 1 batch of products are freeze-dried by the sucrose fat emulsion protective agent and the gelatin sucrose protective agent, the batch number of the freeze-dried product of the sucrose skim emulsion protective agent is M180611, the batch number of the freeze-dried product of the gelatin sucrose protective agent is A180612, and all detection indexes of all batches of products accord with the regulations. The production and inspection data of each batch of products are shown in tables 2 and 3, and the titer of the virus liquid of the avian infectious bronchitis QX87 strain is 10 6.50 EID 50 /0.1ml。
Table 2 production of lyophilized products from each batch
TABLE 3 examination of lyophilized products for each batch (efficacy unit: 10 x EID 50 Feather/feather)
2. After each batch of samples are stored for 10 days at 37 ℃ in the aging resistance test, the titer of the vaccine of the test formula is reduced by 0.12-0.54 titer, and no titer exceeds 1 titer, so that the vaccine meets the requirements of the heat-resistant protective agent of biological products; glutathione is added into the protective agent, so that the aging resistance is better, and the titer loss amplitude can be as low as 0.12 titer. The virus titers of the sucrose skim milk protectant and the gelatin sucrose protectant vaccine respectively drop by 2.00 titer and 1.49 titer, and the titers are far beyond the standard of 1 titer. See table 4.
Table 4 results of aging resistance test at 37 ℃ (unit: 10 x EID 50 Feather/feather)
| Lot number | Initial potency | Titers after 10 days of standing at 37 DEG C | Loss of potency |
| S180601 | 4.17 | 3.63 | 0.54 |
| S180602 | 4.32 | 4.03 | 0.29 |
| S180603 | 4.32 | 3.96 | 0.36 |
| S180604 | 4.17 | 3.68 | 0.49 |
| S180605 | 4.38 | 3.96 | 0.42 |
| S180606 | 4.32 | 4.08 | 0.24 |
| S180607 | 4.32 | 4.20 | 0.12 |
| S180608 | 4.38 | 4.21 | 0.17 |
| S180609 | 4.17 | 3.85 | 0.32 |
| S180610 | 4.32 | 4.11 | 0.21 |
| M180611 | 4.38 | 2.38 | 2.00 |
| A180612 | 4.17 | 2.68 | 1.49 |
3. Shelf life test the vaccine is preserved for 6 months under room temperature, the vaccine titer of the protective agent of the test formula is reduced by 0.49-0.67 titer, the titer loss of the added glutathione group in the protective agent is lower, the virus titer of the vaccine of the sucrose skim milk protective agent is reduced by 1.75 titer, and the virus titer of the vaccine of the gelatin sucrose protective agent is reduced by 1.49 titer; after the vaccine is preserved for 36 months under the refrigerating condition, the virus titer of the vaccine of the protective agent of the test formula is reduced by 0.32-0.67 titer, the titer loss of the glutathione group added into the protective agent is lower, the virus titer of the vaccine of the sucrose skim milk protective agent is reduced by 1.88 titer, and the virus titer of the vaccine of the gelatin sucrose protective agent is reduced by 1.39 titer; after 36 months of preservation under the freezing condition, the virus titer of the vaccine of the protective agent of the test formula is reduced by 0.10-0.40 titer, the titer loss of the glutathione group added into the protective agent is lower, the virus titer of the vaccine of the sucrose skim milk protective agent is reduced by 1.00 titer, and the virus titer of the vaccine of the gelatin sucrose protective agent is reduced by 0.95 titer. The results are shown in tables 5, 6 and 7.
TABLE 5 results of the storage tests at room temperature (25 ℃ C.) (unit: 10) x EID 50 Feather/feather)
| Lot number | Initial potency | 1 month | 2 months of | For 3 months | For 4 months | For 5 months | 6 months of | Decrease in amplitude |
| S180601 | 4.17 | 4.17 | 4.17 | 4.0 | 4.0 | 3.78 | 3.50 | 0.67 |
| S180602 | 4.32 | 4.17 | 4.17 | 4.0 | 3.83 | 3.75 | 3.75 | 0.57 |
| S180603 | 4.32 | 4.32 | 4.22 | 4.0 | 3.78 | 3.68 | 3.68 | 0.64 |
| S180604 | 4.17 | 4.17 | 4.0 | 3.83 | 3.78 | 3.50 | 3.50 | 0.67 |
| S180605 | 4.38 | 4.32 | 4.22 | 4.17 | 3.83 | 3.83 | 3.78 | 0.60 |
| S180606 | 4.32 | 4.22 | 4.22 | 4.0 | 4.0 | 3.83 | 3.78 | 0.54 |
| S180607 | 4.32 | 4.0 | 4.0 | 4.0 | 3.83 | 3.83 | 3.83 | 0.49 |
| S180608 | 4.38 | 4.22 | 4.22 | 4.0 | 4.0 | 3.83 | 3.83 | 0.55 |
| S180609 | 4.17 | 4.17 | 4.0 | 4.0 | 3.83 | 3.68 | 3.63 | 0.54 |
| S180610 | 4.32 | 4.22 | 4.22 | 4.0 | 3.83 | 3.83 | 3.78 | 0.54 |
| M180611 | 4.38 | 4.00 | 3.75 | 3.75 | 3.22 | 2.83 | 2.63 | 1.75 |
| A180612 | 4.17 | 3.83 | 3.38 | 3.32 | 3.17 | 2.83 | 2.68 | 1.49 |
TABLE 6 preservation of test results (unit: 10) by refrigeration (2-8deg.C) x EID 50 Feather/feather)
TABLE 7 freezing (-15 ℃ C.) preservation of the test results (unit: 10) x EID 50 Feather/feather)
| Lot number | Initial potency | 6 months of | For 12 months | 18 months of | 24 months of | For 30 months | For 36 months | Decrease in amplitude |
| S180601 | 4.17 | 4.17 | 4.16 | 4.14 | 4.09 | 3.88 | 3.79 | 0.38 |
| S180602 | 4.32 | 4.32 | 4.30 | 4.27 | 4.25 | 4.18 | 4.08 | 0.24 |
| S180603 | 4.32 | 4.22 | 4.22 | 4.20 | 4.17 | 4.11 | 4.00 | 0.32 |
| S180604 | 4.17 | 4.17 | 4.17 | 4.15 | 4.08 | 3.75 | 3.77 | 0.40 |
| S180605 | 4.38 | 4.37 | 4.37 | 4.33 | 4.30 | 4.12 | 4.02 | 0.36 |
| S180606 | 4.32 | 4.32 | 4.32 | 4.29 | 4.27 | 4.18 | 4.12 | 0.20 |
| S180607 | 4.32 | 4.32 | 4.32 | 4.32 | 4.32 | 4.22 | 4.22 | 0.10 |
| S180608 | 4.38 | 4.38 | 4.38 | 4.36 | 4.35 | 4.25 | 4.23 | 0.15 |
| S180609 | 4.17 | 4.17 | 4.17 | 4.15 | 4.13 | 4.09 | 4.03 | 0.14 |
| S180610 | 4.32 | 4.32 | 4.32 | 4.30 | 4.29 | 4.19 | 4.16 | 0.16 |
| M180611 | 4.38 | 4.17 | 4.0 | 3.83 | 3.68 | 3.50 | 3.38 | 1.00 |
| A180612 | 4.17 | 4.0 | 3.83 | 3.63 | 3.50 | 3.50 | 3.22 | 0.95 |
3. Conclusion(s)
The live vaccine of the avian infectious bronchitis QX87 strain prepared by the test formula protective agent is preserved for 10 days at 37 ℃, the virus titer is reduced by 0.12 to 0.54 titer, and no titer exceeds 1 titer, so that the requirement of the biological product heat-resistant protective agent is met; glutathione is added into the protective agent, so that the aging resistance is better, and the titer loss amplitude can be as low as 0.12 titer. The virus titers of the sucrose skim milk protectant and the gelatin sucrose protectant vaccine respectively drop by 2.00 titer and 1.49 titer, and the titers are far beyond the standard of 1 titer.
The vaccine is preserved for 6 months at room temperature, the vaccine titer of the protective agent of the test formula is reduced by 0.49-0.67 titer, the vaccine virus titer of the protective agent of the sucrose skim milk is reduced by 1.75 titer, and the vaccine virus titer of the protective agent of the gelatin sucrose is reduced by 1.49 titer.
Preserving for 24 months under refrigeration condition, and reducing the virus titer of the vaccine of the test formula protective agent by 0.1-0.27 titer; after 36 months, the virus titer of the vaccine of the protective agent of the test formula is reduced by 0.32-0.67 titer, the virus titer of the vaccine of the protective agent of the sucrose skim milk is reduced by 1.88 titer, and the virus titer of the vaccine of the gelatin sucrose protective agent is reduced by 1.39 titer.
After 24 months of preservation under the freezing condition, the virus titer of the vaccine of the test formula protective agent is reduced by 0-0.09 titer; after 36 months, the virus titer of the vaccine of the protective agent of the test formula is reduced by 0.10-0.40 titer, the virus titer of the vaccine of the protective agent of the sucrose skim milk is reduced by 1.00 titer, and the virus titer of the vaccine of the gelatin sucrose protective agent is reduced by 0.95 titer. The glutathione is added into the protective agent to have better preservation effect and lower vaccine potency loss.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. The freeze-drying protective agent for the live vaccine of the infectious bronchitis is characterized by being prepared by mixing liquid A and liquid B in an equal volume ratio according to the mass volume percentage, wherein the liquid A comprises 4.0-8.0% of glucan, 4.0-8.0% of enzymolysis casein, 2.0-6.0% of carrageenan, 2.0-6.0% of chondroitin sulfate and the balance of sterile water; the liquid B comprises 2.0% -6.0% of hydrolyzed milk protein, 2.0% -6.0% of sorbitol and the balance of sterile water.
2. The lyoprotectant of claim 1, wherein the protectant solution a is adjusted to a pH of 7.2-7.4 using phosphate buffer.
3. The lyoprotectant of claim 2, wherein the phosphate buffer is 1.64% dipotassium hydrogen phosphate and 0.52% potassium dihydrogen phosphate.
4. A lyoprotectant according to any one of claims 1-3 wherein the solution B further comprises 0.4% glutathione.
5. A method for preparing the lyoprotectant according to any one of claims 1-4, wherein the components of solution a are taken in proportion, dissolved in sterile water to a certain volume, adjusted in pH, and sterilized at 116 ℃ for 30 minutes; the components of the solution B are taken according to the proportion, dissolved by sterile water to fix the volume, filtered and sterilized by 0.22 mu m, and when in use, the solution A and the solution B are taken and mixed with equal volumes.
6. Use of a lyoprotectant according to any one of claims 1-4 for the preparation of a live vaccine preparation of strain QX87 for infectious bronchitis in chickens.
7. A preparation method of a live vaccine product for infectious bronchitis, which is characterized in that the freeze-drying protective agent in any one of claims 1-4 is mixed with infectious bronchitis virus liquid in equal proportion, quantitatively packaged, half-plugged, vacuum freeze-dried and then plugged out.
8. The method according to claim 7, wherein the vacuum freeze-drying process is performed by pre-freezing to-40.0 ℃ for 4 hours, -3.0 ℃ for 1 hour, 0 ℃ for 8 hours, 5.0 ℃ for 4 hours, 10.0 ℃ for 4 hours, 13.0 ℃ for 1 hour, 15.0 ℃ for 1 hour, 25.0 ℃ for analysis and drying, 29.0 ℃ for 4 hours, performing in-tank pressure plugging, and ending out of the tank.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003264166A1 (en) * | 2002-09-23 | 2004-04-08 | Pfizer Products Inc. | Improvements in or relating to vaccines |
| CN101612404A (en) * | 2009-07-29 | 2009-12-30 | 哈药集团生物疫苗有限公司 | Newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent |
| CN102341105A (en) * | 2009-02-03 | 2012-02-01 | 微生物公司 | Bismuth-thiols as antiseptics for epithelial tissues, acute and chronic wounds, bacterial biofilms and other indications |
| CN102908625A (en) * | 2011-08-01 | 2013-02-06 | 普莱柯生物工程股份有限公司 | Chicken Newcastle disease and avian infectious bronchitis bivalent live vaccine heat-resistant protective agent and preparation process thereof |
| CN105999288A (en) * | 2016-07-20 | 2016-10-12 | 安徽东方帝维生物制品股份有限公司 | Swine erysipelas live vaccine heat-resistant protective agent, preparation method and application |
| CN110801436A (en) * | 2019-11-19 | 2020-02-18 | 畜科生物工程有限公司 | Porcine transmissible gastroenteritis and porcine epidemic diarrhea bivalent live vaccine freeze-drying protective agent and bivalent live vaccine |
| CN112533946A (en) * | 2018-05-15 | 2021-03-19 | 旗舰创业创新六公司 | Pathogen control composition and use thereof |
| CN113652404A (en) * | 2021-08-25 | 2021-11-16 | 江苏省农业科学院 | Freeze-drying protective agent for porcine circovirus and application of freeze-drying protective agent in preservation |
| CN113730592A (en) * | 2021-09-03 | 2021-12-03 | 吉林正业生物制品股份有限公司 | Duck tembusu virus live vaccine freeze-drying protective agent, preparation method and application |
| CN114042167A (en) * | 2021-12-08 | 2022-02-15 | 山东滨州博莱威生物技术有限公司 | Newcastle disease and infectious bronchitis bivalent live vaccine heat-resistant protective agent and preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2532232A1 (en) * | 2011-06-10 | 2012-12-12 | InterMed Discovery GmbH | Long chain glycolipids useful to avoid perishing or microbial contamination of materials |
-
2023
- 2023-05-09 CN CN202310514007.4A patent/CN116440282B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003264166A1 (en) * | 2002-09-23 | 2004-04-08 | Pfizer Products Inc. | Improvements in or relating to vaccines |
| CN102341105A (en) * | 2009-02-03 | 2012-02-01 | 微生物公司 | Bismuth-thiols as antiseptics for epithelial tissues, acute and chronic wounds, bacterial biofilms and other indications |
| CN101612404A (en) * | 2009-07-29 | 2009-12-30 | 哈药集团生物疫苗有限公司 | Newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent |
| CN102908625A (en) * | 2011-08-01 | 2013-02-06 | 普莱柯生物工程股份有限公司 | Chicken Newcastle disease and avian infectious bronchitis bivalent live vaccine heat-resistant protective agent and preparation process thereof |
| CN105999288A (en) * | 2016-07-20 | 2016-10-12 | 安徽东方帝维生物制品股份有限公司 | Swine erysipelas live vaccine heat-resistant protective agent, preparation method and application |
| CN112533946A (en) * | 2018-05-15 | 2021-03-19 | 旗舰创业创新六公司 | Pathogen control composition and use thereof |
| CN110801436A (en) * | 2019-11-19 | 2020-02-18 | 畜科生物工程有限公司 | Porcine transmissible gastroenteritis and porcine epidemic diarrhea bivalent live vaccine freeze-drying protective agent and bivalent live vaccine |
| CN113652404A (en) * | 2021-08-25 | 2021-11-16 | 江苏省农业科学院 | Freeze-drying protective agent for porcine circovirus and application of freeze-drying protective agent in preservation |
| CN113730592A (en) * | 2021-09-03 | 2021-12-03 | 吉林正业生物制品股份有限公司 | Duck tembusu virus live vaccine freeze-drying protective agent, preparation method and application |
| CN114042167A (en) * | 2021-12-08 | 2022-02-15 | 山东滨州博莱威生物技术有限公司 | Newcastle disease and infectious bronchitis bivalent live vaccine heat-resistant protective agent and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Horst G等.Effects of beta-1,3-glucan (AletaTM) on vaccination response in broiler chickens.Poult Sci.2019,第98卷(第4期),1643-1647. * |
| 兽用冻干疫苗耐热保护剂的研究――鸡传染性支气管炎冻干活疫苗耐热保护剂试验;张云浦, 冯丽秋, 于作利, 崔京春, 董雅文, 张君, 李宝臣, 陈培军, 胡瑛瑛, 邬立权, 丁国义, 杨晓丽;中国兽药杂志(第06期);全文 * |
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Denomination of invention: A freeze-dried protective agent for a live vaccine against infectious bronchitis in chickens and its preparation method Effective date of registration: 20231221 Granted publication date: 20231020 Pledgee: Jiangsu Changshu Rural Commercial Bank Co.,Ltd. Taizhou Branch Pledgor: Zhonghai biopharmaceutical (Taizhou) Co.,Ltd. Registration number: Y2023980072958 |