CN115040656B - Freeze-drying protective agent for rabies vaccine, application, vaccine and preparation method of vaccine - Google Patents
Freeze-drying protective agent for rabies vaccine, application, vaccine and preparation method of vaccine Download PDFInfo
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Classifications
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- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- 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
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- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F26—DRYING
- F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
- F26B5/00—Drying solid materials or objects by processes not involving the application of heat
- F26B5/04—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum
- F26B5/06—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum the process involving freezing
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- C—CHEMISTRY; METALLURGY
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20111—Lyssavirus, e.g. rabies virus
- C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C12N2770/24011—Flaviviridae
- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to the field of biological products, in particular to a rabies vaccine freeze-drying protective agent, application and vaccine and a preparation method thereof. The freeze-drying protective agent provided by the invention comprises the following components: one or more than two of sucrose, polyvinylpyrrolidone, sodium glutamate, L-arginine or human serum albumin, and the lyoprotectant does not contain gelatin and dextran. The freeze-dried human rabies vaccine prepared by using the freeze-drying protective agent has the advantages of good appearance, good re-solubility, good stability and strong heat resistance, and can be stored for 3 months at 37 ℃ and 48 months at 4 ℃.
Description
Technical Field
The invention relates to the field of biological products, in particular to a rabies vaccine freeze-drying protective agent, application and vaccine and a preparation method thereof.
Background
Rabies is a disease which is common to human and animals, all warm-blooded animals can be infected, the disease is a severe lethal infectious disease, the death rate is up to one hundred percent after the disease is generated, the human disease is mainly caused by the bite of the diseased animal, or the disease is closely contacted and related to the disease, the disease can be transmitted through the non-dominant skin or mucous membrane, and the diseased animal contacts the mucous membrane of the human, the skin with wounds and the like to cause the infection. The rabies virus is a pathogen of rabies, belongs to the genus rabies of the Rhabdoviridae family, is enveloped single negative strand RNA virus, and the surface bulge on the protein shell of the rabies virus consists of rabies virus glycoprotein GP, which is the only antigen protein in the rabies virus structural protein capable of stimulating the organism to produce neutralizing antibodies.
Vaccination is the only effective means for preventing rabies virus, can reduce the incidence and death rate of rabies, and is an automatic immune preparation for preventing or treating infectious diseases, which is the most common type of biological preparation, and is prepared by artificially attenuating, inactivating or utilizing transgenic methods of pathogenic microorganisms and metabolites thereof. The common dosage forms of the vaccine are oral administration and injection, and the current vaccine dosage form research is mainly focused on the injection due to the molecular activity requirement of the vaccine, and the common vaccine injection is mainly composed of two kinds of injection solution and freeze-dried powder injection. The vacuum freeze-drying technology is a technology of vacuum drying medicines at low temperature, can reduce protein denaturation, reduce loss of microbial activity, reduce loss of volatile components and thermal denaturation components, reduce loss of substances which are easy to oxidize, and inhibit growth of microorganisms, and the prepared freeze-dried product has the advantages of stable property, mild preservation condition, long preservation period and the like.
In the vacuum freeze-drying process, the freeze and the drying inevitably cause the damage and death of partial microbial cells, so that the denaturation of proteins is caused, a great deal of researches are carried out, and various freeze-drying protective agents are added in the freeze-drying process of the vaccine so as to improve the bioactivity of the freeze-dried vaccine, improve the stability of the freeze-dried vaccine, improve the storage temperature and prolong the storage time. Lyoprotectants have become an integral part of vaccine lyoprotectant formulations.
Along with the wide application of vaccine products and the rapid development of molecular biology and cell biology, researches find that many adverse reaction problems occurring after vaccination are directly related to protective agents of vaccine products, in particular to freeze-drying protective agents in freeze-dried vaccine products, wherein gelatin or gelatin derivatives can directly cause systemic urticaria, angioneurotic edema, asthma and other systemic allergic reactions of vaccinators to different degrees, dextran can cause systemic urticaria, skin itch, fever, chest distress, anaphylactic shock and other sporadic allergic reactions of the vaccinators, certain safety risks exist, and along with the continuous improvement of people on vaccine safety awareness, in order to alleviate or avoid adverse reactions caused by gelatin and dextran in the use process of vaccines, the freeze-drying protective agents of freeze-dried human canine vaccines with stability, safety, less side effects, high storage temperature and long storage period are urgently needed in the field.
The vacuum freeze-drying process of the vaccine has great influence on the appearance property, the re-solubility, the stability and the heat resistance of a freeze-dried product of the vaccine, and the conventional vacuum freeze-drying process has the defects that the size of ice crystals formed is uneven due to the ultralow-temperature freezing, the uniformity of drying of the product is influenced, the stability of viruses and the re-solubility of the freeze-dried product are influenced, so that in the conventional freeze-drying process, although the formula of the freeze-drying protective agent is improved, the heat resistance of the product is limited.
Disclosure of Invention
In view of this, the invention provides a rabies vaccine lyoprotectant, application and vaccine and a preparation method thereof. The invention aims to provide a freeze-drying protective agent of a heat-resistant freeze-dried human rabies vaccine without gelatin and dextran and a freeze-drying method thereof, and the prepared freeze-dried human rabies vaccine has the advantages of good appearance, good re-solubility, good stability and strong heat resistance, and can be stored for 3 months at 37 ℃ and 48 months at 4 ℃.
In order to achieve the above object, the present invention provides the following technical solutions:
the present invention provides a lyoprotectant comprising: one or more of sucrose, polyvinylpyrrolidone, sodium glutamate, L-arginine or human serum albumin;
The lyoprotectant is free of gelatin and dextran.
In some embodiments of the present invention, the lyoprotectant comprises, in parts by mass:
in some embodiments of the invention, the lyoprotectant comprises, in concentration:
in some embodiments of the invention, the pH of the solution form of the lyoprotectant described above comprises 7.6.
The invention also provides application of the lyoprotectant in reducing vaccine freeze-drying damage.
The invention also provides application of the freeze-drying protective agent in improving heat resistance and stability of the vaccine in freeze-drying and storage processes, improving safety of the freeze-dried vaccine to human bodies and/or reducing adverse reactions of the vaccine.
The invention also provides application of the freeze-dried protective agent in preparing rabies vaccine, japanese encephalitis inactivated vaccine and/or pseudorabies vaccine.
The invention also provides a preparation method of the vaccine, which comprises the steps of mixing the vaccine stock solution with the freeze-drying protective agent to obtain a semi-finished product; lyophilizing the semi-finished product to obtain vaccine;
The lyophilization procedure included: the temperature in the pre-freezing stage is-55 to-45 ℃ and is maintained for 2 to 4 hours; heating to-25 to-15 ℃ and then maintaining for 5-12 hours; cooling to-55 to-45 ℃ again, and maintaining for 2-4 hours; heating for 10-25 hours to-30 to-20 ℃ in the sublimation drying stage, wherein the vacuum pressure is 0.10-0.30 mbar; heating for 6-15 hours to 20-30 ℃ in the analysis drying stage, wherein the vacuum pressure is 0.001-0.01 mbar;
the vaccine stock solution comprises rabies vaccine stock solution, japanese encephalitis inactivated vaccine stock solution and/or pseudorabies vaccine stock solution.
In some embodiments of the invention, the ratio of the vaccine stock solution to the lyoprotectant in the semi-finished product in the above preparation method is 1:1.5; the pH value of the semi-finished product is 7.3-7.7.
The invention also provides a vaccine, which comprises the freeze-drying protective agent; and/or the preparation method for preparing the vaccine.
The invention has the following effects:
the freeze-drying protective agent and the freeze-drying process can improve the heat resistance and the stability of the vaccine in the freeze-drying and storage processes, greatly improve the safety of the freeze-dried vaccine to human bodies and reduce the occurrence of adverse reactions of the vaccine. In particular
(1) The long-term preservation experiment result and the stability result at 37 ℃ show that the product titer is more than 2.5 IU/dose after the long-term preservation experiment result and the stability result at 37 ℃ are stored for 90 days at 37 ℃; after being preserved for 48 months in a long-term preservation experiment at 2-8 ℃, the product titer is more than 2.5 IU/dose, and meets the standards of Chinese pharmacopoeia 2020 edition.
(2) The vaccine products in the current market have the product titer of more than 2.5 IU/dose after being stored for 28 days at 37 ℃; after being preserved for 36 months in a long-term preservation experiment at 2-8 ℃, the potency of the disease products is more than 2.5 IU/dose, and the disease products all accord with the standards of Chinese pharmacopoeia 2020 edition. But after 90 days of preservation at 37 ℃, the product titer is less than 2.5 IU/dose; after being preserved for 48 months in a long-term preservation experiment at 2-8 ℃, the product titer is less than 2.5 IU/dose and does not accord with the 2020 edition standard of Chinese pharmacopoeia.
Detailed Description
The invention discloses a rabies vaccine freeze-drying protective agent, application, vaccine and a preparation method thereof, and a person skilled in the art can refer to the content of the vaccine freeze-drying protective agent, the application, the vaccine and the preparation method, and the person skilled in the art can properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
Aiming at the defects of the conventional freeze-drying process, the annealing technology is introduced, so that the glass transition temperature of the product is improved, the sublimation temperature is improved, the influence of the vacuum freeze-drying process on the vaccine activity is reduced, and the heat resistance of the product is improved.
The vaccine freeze-drying protective agent comprises the following components in a semi-finished product:
The solvent is PBS buffer solution with pH of 7.6, and the vaccine semi-finished product is liquid vaccine before freeze-drying.
Preparing a freeze-drying protective agent: sucrose, polyvinylpyrrolidone, sodium glutamate, L-arginine and human serum albumin were dissolved in PBS solution and then sterilized by filtration through a 0.22 μm microporous filter.
Preparing a semi-finished product: mixing the rabies vaccine stock solution with a freeze-drying protective agent 1:1.5, regulating the pH to 7.3-7.7, and determining the potency according to the method of Chinese pharmacopoeia 2020 edition, wherein the potency of a vaccine semi-finished product is more than 5IU/mL.
In the preparation method, the vaccine semi-finished product is sub-packaged according to 1 mL/branch.
Lyophilization procedure: the lowest temperature in the pre-freezing stage is-55 to-45 ℃, the temperature is raised to-25 to-15 ℃ when the lowest temperature is reached for 2 to 4 hours, the temperature is maintained for 5 to 12 hours, the temperature is lowered to-55 to-45 ℃ again, and the temperature is maintained for 2 to 4 hours when the lowest temperature is reached; the final temperature of sublimation drying is-30 to-20 ℃, the time for reaching the final temperature is 10-25 hours, and the vacuum pressure is controlled to be 0.10-0.30 mbar; the final temperature of the analytical drying is 20-30 ℃, the vacuum pressure is controlled to be 0.001-0.01 mbar, and the operation is carried out for 6-15 hours.
The freeze-drying protective agent and the freeze-drying method thereof aim at freeze-drying a rabies vaccine for human, and realize that the freeze-drying preparation of the freeze-drying rabies vaccine has good re-solubility, long storage period and wide storage condition by providing the heat-resistant freeze-drying protective agent and the improved freeze-drying process under the premise of not adding gelatin and dextran.
The raw materials and reagents used in the present invention are commercially available.
The invention is further illustrated by the following examples:
example 1: preparation of vaccine stock solution
The preparation method of the rabies vaccine (human diploid cells) stock solution for freeze-drying human comprises the following steps:
1) PM rabies virus is adopted as the strain, and human diploid cells are adopted as the culture medium;
2) Digesting and dispersing the recovered human diploid cells uniformly by using 0.25% trypsin, adding 199 culture solution, mixing uniformly, adding into a cell factory, and culturing at 37 ℃ until the single-layer human diploid cells are uniform;
3) PBS solution in the bioreactor is washed and replaced by 199 culture solution containing 10% of new born calf serum, and parameters of the bioreactor are set: the temperature is 37 ℃, the rotating speed is 50rpm, the pH is 7.0, and the dissolved oxygen is 50 percent. After each parameter is stable, inoculating single-layer human diploid cells which are digested and dispersed uniformly, wherein the number of inoculated cells, namely the working volume, is controlled within the range of 2X 10 5～5×105/mL;
4) Adjusting the filling amount of the bioreactor according to the growth and propagation conditions of the human diploid cells, and inoculating PM strain rabies virus when the density of the human diploid cells is in the range of 2X 10 7～4×107 per mL;
5) Discarding cell growth solution for 5-10 days in the bioreactor, adding 199 culture solution of 3% new born calf serum of virus growth solution, inoculating working seed batch into the bioreactor according to the MOI of 0.01-0.003 MOI, continuously pouring and harvesting virus solution, and setting parameters of the bioreactor: the temperature is 35 ℃, the rotating speed is 50rpm, the pH value is 7.4, and the dissolved oxygen is 50%;
6) Continuously harvesting virus liquid by using a maintenance solution of 199 culture solution without new-born calf serum and with 1% human serum albumin for 17 days after inoculating PM strain rabies virus, sampling for aseptic examination and virus titration experiments, and storing at 2-8 ℃;
7) Combining multiple virus solutions produced by the same batch of cells under aseptic condition, clarifying and filtering with 0.65 μm filter membrane, washing with 300kD ultrafiltration membrane, concentrating, diluting if the protein content in the concentrated virus solution is not more than 60mg/mL, and performing aseptic inspection;
8) Adding beta-propiolactone with the concentration of 1:4000 into the concentrated virus liquid for inactivation, continuously shaking for 24 hours at the temperature of 2-8 ℃, heating in water bath to the temperature of 37 ℃ and keeping for 2 hours to ensure that the residual beta-propiolactone is completely hydrolyzed, and sampling for an inactivation verification test after the virus liquid is inactivated;
9) Purifying the inactivated virus liquid by adopting a column chromatography, wherein the chromatographic medium is agarose gel Sepharose 4FF, eluting by adopting PBS (phosphate buffer solution) with the pH of 7.6 and the concentration of 0.01mol/L and containing NaCl of 0.14mol/L, wherein the sample loading amount of each time is not more than 7 percent of the volume of the chromatographic column, the linear flow rate is 50cm/h, the first peak is collected by ultraviolet monitoring at 280nm, the collection is started when the ultraviolet monitoring value rises to 50AU after the peak, the collection is ended when the ultraviolet monitoring value falls to 70AU after the peak falls back, the human serum albumin with the final concentration of 1 percent is added, and the rabies vaccine stock solution for freeze-dried human is obtained after the sample is sampled and is qualified as the stock solution and is stored at 2-8 ℃.
Example 2
The components, content (final concentration in vaccine semi-finished product) of the lyoprotectant of this example:
The protective agent solvent is PBS with pH of 7.6, filtering and sterilizing with microporous filter membrane with 0.22 μm, mixing rabies vaccine stock solution with freeze-drying protective agent 1:1.5, adjusting pH to 7.3, and measuring titer according to Chinese pharmacopoeia 2020 edition method, wherein the titer of vaccine semi-finished product is greater than 5.1IU/mL.
And (3) sub-packaging the semi-finished vaccine product into penicillin bottles, and freeze-drying 1 mL/bottle to obtain a freeze-dried rabies vaccine finished product for human.
The freeze-drying method comprises the following steps: the lowest temperature in the pre-freezing stage is minus 45 ℃, the temperature reaches the lowest temperature and is maintained for 4 hours, the temperature is raised to minus 25 ℃ and is maintained for 12 hours, the temperature is reduced to minus 45 ℃ again, and the temperature reaches the lowest temperature and is maintained for 4 hours; the final temperature of sublimation drying is-30 ℃, the time for reaching the final temperature is 25 hours, and the vacuum pressure is controlled to be 0.30mbar; the final temperature of the analytical drying was 30℃and the vacuum pressure was controlled at 0.001mbar and the operation was continued for 15 hours.
Example 3
The components, content (final concentration in vaccine semi-finished product) of the lyoprotectant of this example:
The protective agent solvent is PBS with pH of 7.6, filtering and sterilizing with microporous filter membrane with 0.22 μm, mixing rabies vaccine stock solution with freeze-drying protective agent 1:1.5, adjusting pH to 7.7, and measuring potency according to Chinese pharmacopoeia 2020 edition method, wherein potency of vaccine semi-finished product is 5.1IU/mL.
And (3) sub-packaging the semi-finished vaccine product into penicillin bottles, and freeze-drying 1 mL/vial to obtain a freeze-dried rabies vaccine finished product for human use.
The freeze-drying method comprises the following steps: the lowest temperature in the pre-freezing stage is minus 50 ℃, the lowest temperature is reached and maintained for 3 hours, the temperature is raised to minus 20 ℃, the temperature is maintained for 10 hours, the temperature is reduced to minus 50 ℃ again, and the lowest temperature is reached and maintained for 3 hours; the final temperature of sublimation drying is-20 ℃, the time for reaching the final temperature is 10 hours, and the vacuum pressure is controlled to be 0.15mbar; the final temperature of the analytical drying was 25℃and the vacuum pressure was controlled at 0.005mbar and the operation was continued for 12 hours.
Example 4
The components, content (final concentration in vaccine semi-finished product) of the lyoprotectant of this example:
The protective agent solvent is PBS with pH of 7.6, filtering and sterilizing the solution by using a microporous filter membrane with the thickness of 0.22 mu m, mixing a rabies vaccine stock solution with a freeze-drying protective agent with the ratio of 1:1.5, and adjusting the pH to 7.5 by using the stock solution as in the example 1 so that the efficacy of a vaccine semi-finished product is 5.1IU/mL.
And (3) sub-packaging the semi-finished vaccine product into penicillin bottles, and freeze-drying 1 mL/vial to obtain a freeze-dried rabies vaccine finished product for human use.
The freeze-drying method comprises the following steps: the lowest temperature in the pre-freezing stage is minus 55 ℃, the lowest temperature is reached and maintained for 2 hours, the temperature is raised to minus 15 ℃, the temperature is maintained for 5 hours, the temperature is reduced to minus 55 ℃ again, and the lowest temperature is reached and maintained for 2 hours; the final temperature of sublimation drying is-30 ℃, the time for reaching the final temperature is 20 hours, and the vacuum pressure is controlled to be 0.10mbar; the final temperature of the analytical drying was 20℃and the vacuum pressure was controlled at 0.01mbar and the operation was continued for 6 hours.
Comparative example 1: freeze-drying process control
The components, content (final concentration in vaccine semi-finished product) of the lyoprotectant of this comparative example:
The protective agent solvent is PBS with pH of 7.6, filtering and sterilizing with microporous filter membrane with 0.22 μm, mixing rabies vaccine stock solution with freeze-drying protective agent 1:1.5, adjusting pH to 7.5, and measuring titer according to Chinese pharmacopoeia 2020 edition method, wherein the titer of vaccine semi-finished product is 5.1IU/mL.
And (3) sub-packaging the semi-finished vaccine product into penicillin bottles, and freeze-drying 1 mL/vial to obtain a freeze-dried rabies vaccine finished product for human use.
The freeze-drying method comprises the following steps: the lowest temperature in the pre-freezing stage is minus 55 ℃, the lowest temperature is reached and maintained for 4 hours, the final temperature of sublimation drying is minus 30 ℃, the time for reaching the final temperature is 30 hours, and the vacuum pressure is controlled at 0.10mbar; the final temperature of the analytical drying was 20℃and the vacuum pressure was controlled at 0.01mbar and the operation was continued for 12 hours.
Comparative example 2: recipe control
The components, content (final concentration in vaccine semi-finished product) of the lyoprotectant of this comparative example:
The protective agent solvent is PBS with pH of 7.6, filtering and sterilizing with microporous filter membrane with 0.22 μm, mixing rabies vaccine stock solution with freeze-drying protective agent 1:1.5, adjusting pH to 7.5, and measuring titer according to Chinese pharmacopoeia 2020 edition method, wherein the titer of vaccine semi-finished product is 5.1IU/mL.
And (3) sub-packaging the semi-finished vaccine product into penicillin bottles, and freeze-drying 1 mL/vial to obtain a freeze-dried rabies vaccine finished product for human use.
The freeze-drying method comprises the following steps: the lowest temperature in the pre-freezing stage is minus 55 ℃, the lowest temperature is reached and maintained for 2 hours, the temperature is raised to minus 15 ℃, the temperature is maintained for 5 hours, the temperature is reduced to minus 55 ℃ again, and the lowest temperature is reached and maintained for 2 hours; the final temperature of sublimation drying is-30 ℃, the time for reaching the final temperature is 20 hours, and the vacuum pressure is controlled to be 0.10mbar; the final temperature of the analytical drying was 20℃and the vacuum pressure was controlled at 0.01mbar and the operation was continued for 6 hours.
Comparative example 3: recipe control
The components, content (final concentration in vaccine semi-finished product) of the lyoprotectant of this comparative example:
Sucrose pharmaceutical grade 20g/L
Pharmaceutical grade 5g/L sodium glutamate
Human serum albumin pharmaceutical grade 20g/L
The protective agent solvent is PBS with pH of 7.6, filtering and sterilizing with microporous filter membrane with 0.22 μm, mixing rabies vaccine stock solution with freeze-drying protective agent 1:1.5, adjusting pH to 7.5, and measuring titer according to Chinese pharmacopoeia 2020 edition method, wherein the titer of vaccine semi-finished product is 5.1IU/mL.
And (3) sub-packaging the semi-finished vaccine product into penicillin bottles, and freeze-drying 1 mL/vial to obtain a freeze-dried rabies vaccine finished product for human use.
The freeze-drying method comprises the following steps: the lowest temperature in the pre-freezing stage is minus 60 ℃, the lowest temperature is reached and maintained for 4 hours, the final temperature of sublimation drying is minus 30 ℃, the time for reaching the final temperature is 40 hours, and the vacuum pressure is controlled at 0.10mbar; the final temperature of the analytical drying was 20℃and the vacuum pressure was controlled at 0.01mbar and the operation was continued for 15 hours.
Example 5
The freeze-dried human rabies vaccine finished products of the examples 2, 3 and 4 and the comparative examples 1,2 and 3 are respectively placed at 37 ℃ and 4 ℃ for different times, sampled and subjected to appearance, moisture and potency measurement, and the quality standard is according to the standard of the 2020 edition of Chinese pharmacopoeia.
Table 1 results of the thermal stability experiments (IU/dose) at 37℃of the lyophilized products
Table 2 results of experiments (IU/dose) of freeze-dried products stored at 4 ℃ for a long period of time
The above result data are integrated:
(1) The long-term preservation test results and the stability results of the examples 2,3 and 4 at 37 ℃ show that the product titer is more than 2.5 IU/dose after the long-term preservation test results and the stability results are stored at 37 ℃ for 90 days; after being preserved for 48 months in a long-term preservation experiment at 2-8 ℃, the product titer is more than 2.5 IU/dose, and meets the standards of Chinese pharmacopoeia 2020 edition.
(2) The vaccine products of the comparative example and the current market, which have the product titer of more than 2.5 IU/dose after being stored for 28 days at 37 ℃; after being preserved for 36 months in a long-term preservation experiment at 2-8 ℃, the potency of the disease products is more than 2.5 IU/dose, and the disease products all accord with the standards of Chinese pharmacopoeia 2020 edition. But after 90 days of preservation at 37 ℃, the product titer is less than 2.5 IU/dose; after being preserved for 48 months in a long-term preservation experiment at 2-8 ℃, the product titer is less than 2.5 IU/dose and does not accord with the 2020 edition standard of Chinese pharmacopoeia.
All the results prove that the freeze-dried product has the advantages of good safety, good re-solubility, long storage period, wide storage condition and the like compared with other existing freeze-dried protective agent formulas.
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 (9)
1. The freeze-drying protective agent is characterized by comprising the following components in parts by mass:
10-100 parts of sucrose;
1-20 parts of polyvinylpyrrolidone;
2-10 parts of sodium glutamate;
1-10 parts of L-arginine;
1-20 parts of human serum albumin.
2. The lyoprotectant of claim 1, comprising the following components in terms of concentration:
10-100 g/L of sucrose;
1-20 g/L of polyvinylpyrrolidone;
2-10 g/L of sodium glutamate;
l-arginine 1-10 g/L;
1-20 g/L of human serum albumin.
3. Lyoprotectant according to claim 1 or 2, wherein the pH of the solution form thereof comprises 7.6.
4. Use of a lyoprotectant according to claim 1 or 2 for reducing vaccine lyophilization damage.
5. Use of a lyoprotectant according to claim 1 or 2 to improve the heat resistance and stability of a vaccine during lyophilization and storage.
6. Use of a lyoprotectant according to claim 1 or 2 in the preparation of a rabies vaccine.
7. The preparation method of the vaccine is characterized by comprising the steps of mixing a vaccine stock solution with the freeze-drying protective agent according to claim 1 or 2 to obtain a semi-finished product; lyophilizing the semi-finished product to obtain vaccine;
The lyophilization procedure included: the temperature in the pre-freezing stage is-55 to-45 ℃ and is maintained for 2 to 4 hours; heating to-25 to-15 ℃, and maintaining for 5-12 hours; cooling to-55 to-45 ℃ again, and maintaining for 2-4 hours; heating for 10-25 hours to-30 to-20 ℃ in the sublimation drying stage, wherein the vacuum pressure is 0.10-0.30 mbar; heating for 6-15 hours to 20-30 ℃ in the analysis drying stage, wherein the vacuum pressure is 0.001-0.01 mbar;
The vaccine stock solution comprises rabies vaccine stock solution.
8. The method of claim 7, wherein the ratio of the vaccine stock solution to the lyoprotectant in the semi-finished product is 1:1.5; the pH value of the semi-finished product is 7.3-7.7.
9. Vaccine comprising a lyoprotectant according to claim 1 or 2; and/or
The vaccine made by the method of claim 7 or 8.
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| CN103656660A (en) * | 2013-11-08 | 2014-03-26 | 中牧实业股份有限公司 | Heat-resistant freeze-drying protective agent for animal live vaccine, preparation method and application thereof |
| CN110101864A (en) * | 2019-05-31 | 2019-08-09 | 辽宁茂康源生物科技有限公司 | The protective agent of serum-free Antirabic Vaccine a kind of and its application |
| CN113144209A (en) * | 2021-01-19 | 2021-07-23 | 上海荣盛生物药业有限公司 | Rabies vaccine freeze-drying protective agent |
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| CN103463640A (en) * | 2013-09-17 | 2013-12-25 | 中国农业科学院特产研究所 | Canine distemper virus live vaccine heat-resisting cryoprotectant and preparation method thereof |
| CN103656660A (en) * | 2013-11-08 | 2014-03-26 | 中牧实业股份有限公司 | Heat-resistant freeze-drying protective agent for animal live vaccine, preparation method and application thereof |
| CN110101864A (en) * | 2019-05-31 | 2019-08-09 | 辽宁茂康源生物科技有限公司 | The protective agent of serum-free Antirabic Vaccine a kind of and its application |
| CN113144209A (en) * | 2021-01-19 | 2021-07-23 | 上海荣盛生物药业有限公司 | Rabies vaccine freeze-drying protective agent |
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