CN114931557B - Freeze-drying protective agent, vaccine freeze-drying preparation and preparation method thereof - Google Patents

Freeze-drying protective agent, vaccine freeze-drying preparation and preparation method thereof Download PDF

Info

Publication number
CN114931557B
CN114931557B CN202210269607.4A CN202210269607A CN114931557B CN 114931557 B CN114931557 B CN 114931557B CN 202210269607 A CN202210269607 A CN 202210269607A CN 114931557 B CN114931557 B CN 114931557B
Authority
CN
China
Prior art keywords
freeze
vaccine
preparation
component
protective agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210269607.4A
Other languages
Chinese (zh)
Other versions
CN114931557A (en
Inventor
李沛阳
毛昱
刘俊
曹鹏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Liaoning Chengda Biotechnology Co ltd
Original Assignee
Liaoning Chengda Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Liaoning Chengda Biotechnology Co ltd filed Critical Liaoning Chengda Biotechnology Co ltd
Priority to CN202210269607.4A priority Critical patent/CN114931557B/en
Publication of CN114931557A publication Critical patent/CN114931557A/en
Application granted granted Critical
Publication of CN114931557B publication Critical patent/CN114931557B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • 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
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • 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
    • 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
    • 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
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Inorganic Chemistry (AREA)
  • Mycology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The application relates to the technical field of rabies vaccine medicine preparations, and particularly discloses a freeze-drying protective agent, a vaccine freeze-drying preparation and a preparation method thereof. The lyoprotectant comprises a component A; the component A comprises hydroxypropyl-beta-cyclodextrin, N-N-octyl-D-glucosamine and N-acetyl-D-glucosamine; the preparation method of the freeze-drying protective agent; the vaccine freeze-dried preparation prepared by mixing the freeze-dried protective agent and the vaccine and the preparation method thereof; the preparation method of the vaccine freeze-dried preparation comprises annealing treatment. The freeze-drying protective agent, the vaccine freeze-drying preparation and the preparation method thereof can greatly shorten the re-dissolution time of the vaccine freeze-drying preparation and prolong the effective period of the vaccine.

Description

Freeze-drying protective agent, vaccine freeze-drying preparation and preparation method thereof
Technical Field
The application relates to the technical field of rabies vaccine medicine preparations, in particular to a freeze-drying protective agent, a vaccine freeze-drying preparation and a preparation method thereof.
Background
Rabies is a very deadly disease, and when a human body contacts rabies virus, if the rabies is not found and treated in time, the life safety of the human body is seriously endangered. At present, vaccine immunization is the only way to control the occurrence and spread of rabies for people who are in long-term contact with sick animals.
The rabies vaccine currently used in China comprises a freeze-dried powder injection preparation and a water injection preparation, wherein the freeze-dried powder injection preparation is prepared by adding a freeze-dried protective agent into the vaccine and then processing the vaccine added with the freeze-dried protective agent, so that the vaccine freeze-dried powder injection preparation which is loose in cake shape and low in water content is obtained, and compared with the water injection preparation, the vaccine freeze-dried powder injection preparation is more favorable for stabilizing the physicochemical property of medicines and has a longer storage life.
However, the existing lyoprotectant has some defects, firstly, bovine serum and human serum albumin are adopted in the lyoprotectant, and the safety problem of introducing foreign viruses and the problems of source and supply stability possibly exist; and secondly, dextran is adopted in the freeze-drying protective agent, so that the re-dissolution time is longer. Therefore, shortening the re-dissolution time of the vaccine and further prolonging the effective period of the vaccine become the technical problem to be solved urgently.
Disclosure of Invention
In order to further shorten the re-dissolution time of the freeze-dried preparation and prolong the effective period of the vaccine, the application provides a freeze-dried protective agent, a freeze-dried preparation of the vaccine and a preparation method of the freeze-dried preparation.
In a first aspect, the present application provides a lyoprotectant, which adopts the following technical scheme:
a lyoprotectant comprising component a; the component A comprises hydroxypropyl-beta-cyclodextrin, N-N-octyl-D-glucosamine and N-acetyl-D-glucosamine.
In the freeze-drying protective agent provided by the application, hydroxypropyl-beta-cyclodextrin, N-N-octyl-D-glucosamine and N-acetyl-D-glucosamine are adopted, so that human serum albumin in the related technology is effectively replaced, the risk of introducing foreign viruses is reduced, a good skeleton supporting effect is achieved, and the stability of the vaccine is improved.
The hydroxypropyl-beta-cyclodextrin has low relative surface activity and hemolytic activity, has no irritation to muscle, is often used as an injection solubilizer and a drug excipient, can increase the stability of the drug, and improves the bioavailability of the drug.
Glucosamine is also called glucosamine, is a substance necessary for human body to synthesize proteoglycan, and has antiseptic, antibacterial, antioxidant, and immunity enhancing effects.
Preferably, the lyoprotectant further comprises component B and a surfactant; the component A comprises the following components in parts by weight: 10-20 parts of hydroxypropyl-beta-cyclodextrin and N-N-octyl-D-glucosamine (3-5) multiplied by 10 -6 Parts and said N-acetyl-D-glucosamine (3-5). Times.10 -6 A part(s); the weight part of the component B is 2-6 parts; the weight part of the surfactant is 0.01-0.05 part.
In a specific embodiment, the hydroxypropyl-beta-cyclodextrin may be 10 parts, 15 parts, or 20 parts.
In some specific embodiments, the hydroxypropyl-beta-cyclodextrin may also be 10-15 parts or 15-20 parts.
In a specific embodiment, the N-N-octyl-D-glucosamine may be 3X 10 -6 Parts, 4×10 -6 Parts or 5X 10 -6 Parts by weight.
In some specific embodiments, the N-N-octyl-D-glucosamine may also be (3-4). Times.10 -6 Parts or (4-5) x 10 -6 Parts by weight.
In a specific embodiment, the N-acetyl-D-glucosamine may be 3X 10 -6 Parts, 4×10 -6 Parts or 5X 10 -6 Parts by weight.
In some specific embodiments, the N-acetyl-D-glucosamine may also be (3-4). Times.10 -6 Parts or (4-5) x 10 -6 Parts by weight.
In a specific embodiment, the weight part of component B may be 2 parts, 4 parts, or 6 parts.
In some specific embodiments, the parts by weight of component B may also be 2-4 parts or 4-6 parts.
In a specific embodiment, the surfactant may be 0.01 parts, 0.03 parts, or 0.05 parts by weight.
In some specific embodiments, the surfactant may also be present in an amount of 0.01 to 0.03 parts by weight or 0.03 to 0.05 parts by weight.
The hydroxypropyl-beta-cyclodextrin, N-N-octyl-D-glucosamine, N-acetyl-D-glucosamine, the component B and the surfactant are matched for use, so that the freeze-drying protective agent is prepared, the protective force of the freeze-drying protective agent on the serum-free rabies vaccine can be remarkably improved, the dropping rate of the potency in the vaccine storage process is reduced, and the effective period of the vaccine is prolonged.
Preferably, the component B is selected from trehalose, lactose or maltose.
Preferably, the surfactant is tween 20 or tween 80.
In the freeze-drying protective agent provided by the application, the re-dissolution time of the vaccine can be effectively shortened by adding trehalose, lactose and maltose, and when the freeze-drying protective agent is used for preparing the freeze-drying protective agent for the vaccine, the vaccine can be rapidly and fully dissolved, so that the freeze-drying protective agent has the advantages of convenience and quickness.
By adopting the technical scheme, the freeze-drying protective agent can be prepared, and the freeze-drying protective agent does not have potential risks caused by adopting bovine serum or human serum albumin in the related technology, can also reduce the dropping rate of potency in the vaccine preservation process, has a good protective effect on the vaccine, and further prolongs the validity period of the vaccine.
In a second aspect, the present application further provides a preparation method of a lyoprotectant, which adopts the following technical scheme:
a method for preparing a lyoprotectant, comprising the following steps: after the component A, the component B and the surfactant are dissolved in PBS solution, a microporous filter membrane with the pore diameter of 0.22 mu m is adopted for sterilization and filtration.
Preferably, the weight ratio of the total weight of the component A, the component B and the surfactant to the PBS solution is 1: (1.2-2.5).
According to the preparation method of the freeze-drying protective agent, the freeze-drying protective agent can be obtained by uniformly mixing all the components and dissolving in Phosphate Buffer Solution (PBS) and then performing sterilization and filtration.
In a third aspect, the present application provides a vaccine freeze-dried formulation made from the lyoprotectant described above.
In a fourth aspect, the present application provides a method for preparing a vaccine freeze-dried formulation, comprising the steps of:
mixing the vaccine with the freeze-drying protective agent to obtain a semi-finished product solution; and then cooling, annealing, vacuumizing and heating the semi-finished product solution in sequence to obtain the vaccine freeze-dried preparation.
The preparation method of the vaccine freeze-dried preparation provided by the application adopts the steps of cooling, annealing, vacuumizing, heating and the like, and the prepared vaccine freeze-dried preparation is loose cake-shaped, good in stability and high in glass transition temperature.
The freeze-drying protective agent removes the traditional protective agent, namely human serum albumin, so that the freeze-drying type of the vaccine freeze-drying preparation is poor; therefore, an annealing treatment step is added in the preparation process of the vaccine freeze-dried preparation, so that the glass transition temperature of the vaccine freeze-dried preparation is increased, and the drying speed of the vaccine freeze-dried preparation is accelerated without collapsing.
Preferably, the weight ratio of the vaccine to the lyoprotectant is 1: (2-5).
In a specific embodiment, the weight ratio of the vaccine to the lyoprotectant may be 1:2. 1:3 or 1:5.
in some specific embodiments, the weight ratio of the vaccine to the lyoprotectant may also be 1: (2-3) or 1: (3-5).
Preferably, the annealing time is 13-17min.
In a specific embodiment, the time of the annealing may be 13min, 15min, or 17min.
In some specific embodiments, the time of the annealing may also be 13-15min or 15-17min.
In the preparation process of the vaccine freeze-dried preparation, the drying rate of the vaccine freeze-dried preparation can be accelerated and the glass transition temperature and the stability of the vaccine freeze-dried preparation can be improved by controlling the adding proportion of the vaccine and the freeze-dried protective agent and the annealing time.
In a fifth aspect, the present application provides a serum-free rabies vaccine lyophilized formulation prepared by the above vaccine lyophilized formulation preparation method.
Compared with the related technology, the vaccine freeze-dried preparation provided by the application has no risk of potential virus pollution; in the preservation process, the titer is reduced slowly; the medicine is dissolved fast and fully when in use, and is convenient and quick.
In summary, the present application has the following beneficial effects:
1. the application firstly provides a freeze-drying protective agent and a preparation method thereof, the freeze-drying protective agent has no potential risk brought by adopting bovine serum or human serum albumin in the related technology, and can also reduce the dropping rate of potency in the vaccine preservation process, and can play a good role in protecting the vaccine, thereby further prolonging the effective period of the vaccine. The preparation method of the freeze-drying protective agent is simple, low in cost and good in practical prospect.
2. The application also provides a preparation method of the vaccine freeze-dried preparation, which adopts an annealing process, so that the glass transition temperature of the vaccine freeze-dried preparation is increased, the drying speed of the vaccine freeze-dried preparation is accelerated, the vaccine freeze-dried preparation does not collapse, and the stability of the vaccine freeze-dried preparation is further improved.
3. The vaccine freeze-dried preparation is prepared by using the vaccine freeze-dried preparation, and compared with the related technology, the vaccine freeze-dried preparation has no risk of potential virus pollution; in the preservation process, the titer is reduced slowly, so that the shelf life is longer; meanwhile, when the vaccine freeze-dried preparation is used, the medicine is dissolved fast and fully, and the vaccine freeze-dried preparation has the advantage of convenience and rapidness.
Drawings
Fig. 1 is a flow chart of a preparation method of a vaccine freeze-dried preparation provided by the application.
Detailed Description
The application provides a lyoprotectant comprising component A; the component A comprises hydroxypropyl-beta-cyclodextrin, N-N-octyl-D-glucosamine and N-acetyl-D-glucosamine.
Further, the lyoprotectant further comprises a component B and a surfactant; the component A comprises the following components in parts by weight: 10-20 parts of hydroxypropyl-beta-cyclodextrin and N-N-octyl-D-glucosamine (3-5) multiplied by 10 -6 Parts and said N-acetyl-D-glucosamine (3-5). Times.10 -6 A part(s); the weight part of the component B is 2-6 parts; the weight part of the surfactant is 0.01-0.05 part. The component B is selected from trehalose, lactose or maltose; the surfactant is Tween 20 or Tween 80.
The preparation method of the freeze-drying protective agent comprises the following steps: after the component A, the component B and the surfactant are dissolved in PBS solution, a microporous filter membrane with the pore diameter of 0.22 mu m is adopted for sterilization and filtration.
The weight ratio of the total weight of the component A, the component B and the surfactant to the PBS solution is 1: (1.5-2.5).
The application also provides a vaccine freeze-dried preparation prepared by using the freeze-dried protective agent.
The preparation method of the vaccine freeze-dried preparation comprises the following steps:
(1) The vaccine and the freeze-drying protective agent are mixed according to the weight ratio of 1: (2-5) mixing to obtain a semi-finished product solution.
(2) And rapidly cooling the semi-finished product solution to the temperature of (35-45) DEG C, maintaining the temperature for 4-5h, and then heating to the temperature of (10-15) DEG C for annealing for 13-17min.
(3) The temperature was then reduced to- (35-45) ℃and maintained at this temperature for 4-6 hours, after which a vacuum was applied to (0.1-0.2) mbar.
(4) Then heating to the temperature of- (20-30) DEG C, and preserving heat for 15-17h at the temperature; finally, heating to 20-30 ℃, and preserving heat for 6-8 hours at the temperature to obtain the vaccine freeze-dried preparation.
The serum-free rabies vaccine freeze-dried preparation prepared by the preparation method of the vaccine freeze-dried preparation is provided.
The starting materials, reagents, solvents and other test materials used in each of the following preparations and examples are commercially available.
The following preparations 1 to 22, examples 1 to 30, comparative examples 1 to 4 and performance test are described in further detail.
Preparation example
Preparation examples 1 to 20
Preparation examples 1-20 provide a lyoprotectant.
The above preparation example is different in that: the addition amounts of the components in the lyoprotectant are shown in table 1.
The preparation method of the freeze-drying protective agent comprises the following steps:
according to the addition amount of each component in the table 1, hydroxypropyl-beta-cyclodextrin, N-N-octyl-D-glucosamine, N-acetyl-D-glucosamine, trehalose and tween 20 are uniformly mixed and dissolved in PBS solution to obtain PBS mixed solution; and then sterilizing and filtering the PBS mixed solution by adopting a microporous filter membrane with the pore diameter of 0.22 mu m, thus obtaining the freeze-drying protective agent. Wherein the weight ratio of the total weight of the hydroxypropyl-beta-cyclodextrin, the N-N-octyl-D-glucosamine, the N-acetyl-D-glucosamine, the trehalose and the Tween 20 to the PBS solution is 1:2.
TABLE 1 addition amount of each component in lyoprotectants provided in preparation examples 1-20
Figure BDA0003554088790000051
Figure BDA0003554088790000061
Preparation example 21
Preparation 21 provides a lyoprotectant.
The above preparation example differs from preparation example 3 in that: component B in the lyoprotectant was 4g lactose.
PREPARATION EXAMPLE 22
Preparation 22 provides a lyoprotectant.
The above preparation example differs from preparation example 3 in that: component B in the lyoprotectant was 4g maltose.
Examples
Examples 1 to 22
Examples 1-22 each provide a serum-free rabies vaccine lyophilized formulation.
The above-described embodiments differ in that: lyoprotectants in serum-free rabies vaccine lyophilized formulations the lyoprotectants provided in preparation examples 1-22 were applied to examples 1-22, respectively, as shown in table 2.
The preparation method of the serum-free rabies vaccine freeze-dried preparation comprises the following steps:
(1) The serum-free rabies inactivated vaccine strain is cultured for 20 days by a microcarrier at 37 ℃, and then the virus liquid is harvested; and (3) concentrating and inactivating the virus liquid, and finally purifying through a molecular sieve to obtain the serum-free rabies vaccine.
Taking 10mL of serum-free rabies vaccine and 30mL of freeze-drying protective agent, fully mixing the two, and bottling to obtain a vaccine-protective agent mixture.
(2) Placing the vaccine-protective agent mixture in a freeze-drying machine box, pre-freezing at-40deg.C for 4 hr, and annealing at-10deg.C for 15min.
(3) The temperature was again lowered to-40℃and maintained at this temperature for 5 hours, after which a vacuum was applied to 0.1mbar.
(4) Continuously heating to-25 ℃, and preserving heat for 16h at the temperature; finally, the temperature is raised to 25 ℃, and the temperature is kept for 7 hours at the temperature, so that the serum-free rabies vaccine freeze-dried preparation can be obtained.
Table 2 examples 1-22 provide lyoprotectants in serum-free rabies vaccine lyophilized formulations
Examples Freeze-drying protective agent source Examples Freeze-drying protective agent source
1 Preparation example 1 12 Preparation example 12
2 Preparation example 2 13 Preparation example 13
3 Preparation example 3 14 PREPARATION EXAMPLE 14
4 Preparation example 4 15 Preparation example 15
5 Preparation example 5 16 PREPARATION EXAMPLE 16
6 Preparation example 6 17 Preparation example 17
7 Preparation example 7 18 PREPARATION EXAMPLE 18
8 Preparation example 8 19 Preparation example 19
9 Preparation example 9 20 Preparation example 20
10 Preparation example 10 21 Preparation example 21
11 PREPARATION EXAMPLE 11 22 PREPARATION EXAMPLE 22
Examples 23 to 26
Examples 23-26 each provide a serum-free rabies vaccine lyophilized formulation.
The above embodiment differs from embodiment 3 in that: the weight ratio of the serum-free rabies vaccine to the lyoprotectant in the serum-free rabies vaccine lyophilized preparation is specifically shown in table 3.
Table 3 weight ratio of serum-free rabies vaccine to lyoprotectant in example 3, examples 23-26
Figure BDA0003554088790000071
Figure BDA0003554088790000081
Examples 27 to 30
Examples 27-30 provide a serum-free rabies vaccine lyophilized formulation, respectively.
The above embodiment differs from embodiment 3 in that: the annealing time of the preparation process of the serum-free rabies vaccine freeze-dried preparation is shown in table 4.
TABLE 4 annealing time during preparation of formulations provided in example 3, examples 27-30
Figure BDA0003554088790000082
Comparative example
Comparative example 1
Comparative example 1 provides a serum-free rabies vaccine lyophilized formulation.
The above comparative example is different from example 3 in that: no surfactant is added into the lyoprotectant in the serum-free rabies vaccine lyophilized preparation.
Comparative example 2
Comparative example 2 provides a serum-free rabies vaccine lyophilized formulation.
The above comparative example is different from example 3 in that: in the freeze-dried preparation of the serum-free rabies vaccine, the component B in the freeze-dried protective agent adopts dextran.
Comparative example 3
Comparative example 3 provides a serum-free rabies vaccine lyophilized formulation.
The above comparative example is different from example 3 in that: the preparation process of the serum-free rabies vaccine freeze-dried preparation does not comprise an annealing step.
The preparation method of the serum-free rabies vaccine freeze-dried preparation provided in the comparative example 1 is as follows:
(1) The serum-free rabies inactivated vaccine strain is cultured for 20 days by a microcarrier at 37 ℃, and then the virus liquid is harvested; and (3) concentrating and inactivating the virus liquid, and finally purifying through a molecular sieve to obtain the serum-free rabies vaccine.
Taking 10mL of serum-free rabies vaccine and 30mL of freeze-drying protective agent, fully mixing the two, and bottling to obtain a vaccine-protective agent mixture.
(2) The loaded vaccine-protectant mixture was placed in a lyophilizer housing, pre-frozen first at-40 ℃ and maintained at this temperature for 4 hours, then evacuated to 0.1mbar.
(3) Continuously heating to-25 ℃, and preserving heat for 16h at the temperature; finally, the temperature is raised to 25 ℃, and the temperature is kept for 7 hours at the temperature, so that the serum-free rabies vaccine freeze-dried preparation can be obtained.
Comparative example 4
Comparative example 4 provides a rabies vaccine freeze-dried formulation.
The preparation method of the rabies vaccine freeze-dried preparation comprises the following steps:
firstly, mixing 2g of human serum albumin and 5g of sucrose, and dissolving the mixture by using 50mL of PBS solution with pH of 7.5 to prepare a vaccine stabilizer; purifying the rabies virus antigen stock solution to obtain rabies virus antigen; then 50mL of vaccine stabilizer and 50mL of rabies virus antigen are mixed to prepare a semi-finished product, and split charging is carried out by using a tube bottle; and finally, placing the vial into a small freeze dryer for freeze drying to obtain the rabies vaccine freeze-dried preparation.
Safety test
The serum-free rabies vaccine lyophilized formulation provided in example 3 was subjected to drug safety evaluation.
Taking 5 mice, and respectively carrying out an intracerebral injection experiment, an allergy experiment and an injection site irritation experiment on the mice, and observing the states of the mice within 10 days. The results show that the mice have no abnormal performance and grow healthily; the mice were then intraperitoneally injected with the serum-free rabies vaccine lyophilized formulation provided in example 3 and observed for 7 consecutive days. The results show that mice are healthy and have normal weight increase.
Therefore, the serum-free rabies vaccine freeze-dried preparation provided by the application has no acute toxic or side effect, and has no influence on weight growth, growth and development of animals and human bodies.
Performance test
The freeze-dried effect, reconstitution time and potency of the serum-free rabies vaccine freeze-dried preparations provided in examples 1 to 30 and comparative examples 1 to 4 and the rabies vaccine freeze-dried preparation provided in comparative example 3 were tested, and accelerated thermal stability tests were performed, and the test results are shown in table 5.
The potency is measured according to the method 3503 of Sanfeng Tongsu of 2015 of Chinese pharmacopoeia.
Accelerated thermal stability test: the freeze-dried preparation of the serum-free rabies vaccine is respectively stored at the temperature of 5 ℃ and the temperature of 37 ℃ for 28 days, and then the potency detection is carried out on the serum-free rabies vaccine before and after 28 days of storage; and the rate of decline in potency of the serum-free rabies vaccine was calculated according to the following formula.
Potency-lowering efficiency (%) = (potency at 5 ℃ minus potency at 37 ℃) per potency at 5 °c
TABLE 5 results of testing lyophilized vaccine formulations provided in examples 1-30, comparative examples 1-4
Figure BDA0003554088790000101
Figure BDA0003554088790000111
As can be seen from Table 5, the serum-free rabies vaccine freeze-dried formulations provided in examples 1-30 have good safety and freeze-drying effect, short reconstitution time and low potency reduction rate compared with comparative example 4. The serum-free rabies vaccine freeze-dried preparation provided by the application has the advantages of good stability, more convenience and rapidness in use and long shelf life. In particular, the serum-free rabies vaccine freeze-dried preparation provided in example 3 has a reconstitution time of only 7s, and the titer reduction rate of the freeze-dried preparation is only 15.1% as detected by a 28-day thermal stability test.
In combination with tables 1 and 5, it is understood from the results of the examination of examples 1 to 13 that the components in component A of the lyoprotectant were controlled to be: 10-20 parts of hydroxypropyl-beta-cyclodextrin and N-N-octyl-D-glucosamine (3-5). Times.10 -6 Parts, N-acetyl-D-glucosamine (3-5). Times.10 -6 When the preparation is used, the obtained serum-free rabies vaccine freeze-dried preparation is white loose cake-shaped, fluffy and capable of shaking; the re-dissolution time is less than or equal to 8s; the titer reduction rate is less than or equal to 18.0 percent. In this regard, the present application controls the components of component a in the lyoprotectant to: 10-20 parts of hydroxypropyl-beta-cyclodextrin and N-N-octyl-D-glucosamine (3-5). Times.10 -6 Parts, N-acetyl-D-glucosamine (3-5). Times.10 -6 In the part range, the serum-free rabies vaccine freeze-dried preparation with good stability, short redissolution time and low titer loss can be obtained.
The test results of comparative example 3 and examples 14-17 show that when the amount of trehalose in the lyoprotectant is less than 2 parts, the re-dissolution time of the serum-free rabies vaccine lyophilized preparation is longer and the titer reduction rate is high; when the dosage of trehalose in the freeze-drying protective agent is controlled within the range of 2-6 parts, the re-dissolution time of the serum-free rabies vaccine freeze-drying preparation is shorter, and the titer reduction rate is low; when the dosage of trehalose in the freeze-drying protective agent is more than 6 parts, the freeze-drying preparation of the serum-free rabies vaccine has short re-dissolution time, but has high titer reduction rate; therefore, the application controls the dosage of the trehalose in the freeze-drying protective agent within the range of 2-6 parts, and the prepared serum-free rabies vaccine freeze-drying preparation has the advantages of fluffy appearance, short re-dissolution time and low titer reduction rate.
The detection results of the embodiment 3, the embodiment 18-20 and the comparative example 1 show that the reconstitution time and the potency reduction rate of the freeze-dried preparation of the serum-free rabies vaccine all show the trend of increasing and then decreasing along with the increase of the amount of Tween 20 in the freeze-dried protective agent; further comparing, the re-dissolution time and the titer reduction rate of the serum-free rabies vaccine freeze-dried preparation provided in the example 3 and the examples 18-19 are superior to those of the serum-free rabies vaccine freeze-dried preparation provided in the example 20 and the comparative example 1. Therefore, the application shows that the dosage of Tween 20 in the freeze-dried protective agent is controlled within the range of 0.01-0.05 part, and the serum-free rabies vaccine freeze-dried preparation with good appearance and stability, short reconstitution time and long shelf life can be prepared.
As shown by the detection results of comparative example 3, examples 21-22 and comparative example 3, when the component B in the freeze-drying protective agent is trehalose, lactose or maltose respectively, the re-dissolution time of the obtained serum-free rabies vaccine freeze-dried preparation is 7s, and the titer reduction rate is less than 17.5%; and the re-dissolution time of the serum-free rabies vaccine freeze-dried preparation prepared by adopting the dextran provided in the comparative example 3 is 25s, and the titer reduction rate is 23.1%. Therefore, in the freeze-drying protective agent provided by the application, when the component B is trehalose, lactose or maltose, the serum-free rabies vaccine freeze-drying preparation with short re-dissolution time and low titer reduction rate can be obtained.
As can be seen from the test results of examples 3 and 23 to 26 in combination with tables 3 and 5, the reconstitution time and the potency of the serum-free rabies vaccine freeze-dried preparation provided in examples 3 and 24 to 25 were smaller than those provided in examples 23 and 26. Therefore, the weight ratio of the serum-free rabies vaccine to the lyoprotectant is controlled to be 1: in the range of (2-5), the prepared serum-free rabies vaccine freeze-dried preparation has good stability, short reconstitution time and long shelf life.
The test results of examples 3, 27-30 and comparative example 2 in tables 4 and 5 show that the serum-free rabies vaccine freeze-dried preparation prepared by the annealing step provided in examples 3 and 27-30 has fluffy appearance, the re-dissolution time is less than or equal to 8s and the titer reduction rate is low; the serum-free rabies vaccine freeze-dried preparation prepared by the annealing-free step provided in the comparative example 2 is not fluffy, is easy to collapse and has high titer reduction rate. Therefore, the application adopts an annealing step, and the annealing time of the annealing step is controlled to be 13-17min, so that the serum-free rabies vaccine freeze-dried preparation which is white and loose cake-shaped and can be shaken can be prepared, and the serum-free rabies vaccine freeze-dried preparation has the advantages of short re-dissolution time, low titer reduction rate and long shelf life.
In conclusion, the serum-free rabies vaccine freeze-dried preparation provided by the application has no potential risk caused by bovine serum or human serum albumin in the prior art, and the potency reduction rate is reduced in the storage process, so that the freeze-dried preparation can be stored for a longer time. In addition, the freeze-dried preparation of the serum-free rabies vaccine has short re-dissolution time and is convenient and quick to use.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (6)

1. The freeze-dried preparation of the serum-free rabies vaccine comprises the serum-free rabies vaccine and a freeze-dried protective agent, and is characterized in that the freeze-dried protective agent comprises a component A, a component B and a surfactant;
the component A is selected from hydroxypropyl-beta-cyclodextrin, N-N-octyl-D-glucosamine and N-acetyl-D-glucosamine;
the component A comprises the following components in parts by weight: 10-20 parts of hydroxypropyl-beta-cyclodextrin and 3-5 multiplied by 10 of N-N-octyl-D-glucosamine -6 Parts and said N-acetyl-D-glucosamine (3-5). Times.10 -6 A part(s);
the component B is selected from trehalose, lactose or maltose; the weight part of the component B is 2-6 parts;
the surfactant is Tween 20 or Tween 80; the weight part of the surfactant is 0.01-0.05 part.
2. The method of preparing a serum-free rabies vaccine lyophilized formulation according to claim 1, comprising the steps of:
after dissolving the component A, the component B and the surfactant in PBS solution, adopting a microporous filter membrane with the pore diameter of 0.22 mu m to perform sterilization and filtration to obtain a freeze-drying protective agent;
mixing the vaccine with the freeze-drying protective agent to obtain a semi-finished product solution; and then cooling, annealing, vacuumizing and heating the semi-finished product solution in sequence to obtain the vaccine freeze-dried preparation.
3. The method for preparing a vaccine freeze-dried preparation according to claim 2, wherein the weight ratio of the vaccine to the lyoprotectant is 1: (2-5).
4. The method of preparing a lyophilized vaccine formulation according to claim 2, wherein the annealing time is 13-17min.
5. The method for preparing a vaccine freeze-dried preparation according to claim 2, wherein the weight ratio of the vaccine to the lyoprotectant is 1: (3-5).
6. The method of preparing a lyophilized vaccine formulation according to claim 2, wherein the weight ratio of the total weight of component a, component B and surfactant to PBS solution is 1: (1.2-2.5).
CN202210269607.4A 2022-03-18 2022-03-18 Freeze-drying protective agent, vaccine freeze-drying preparation and preparation method thereof Active CN114931557B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210269607.4A CN114931557B (en) 2022-03-18 2022-03-18 Freeze-drying protective agent, vaccine freeze-drying preparation and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210269607.4A CN114931557B (en) 2022-03-18 2022-03-18 Freeze-drying protective agent, vaccine freeze-drying preparation and preparation method thereof

Publications (2)

Publication Number Publication Date
CN114931557A CN114931557A (en) 2022-08-23
CN114931557B true CN114931557B (en) 2023-07-07

Family

ID=82862673

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210269607.4A Active CN114931557B (en) 2022-03-18 2022-03-18 Freeze-drying protective agent, vaccine freeze-drying preparation and preparation method thereof

Country Status (1)

Country Link
CN (1) CN114931557B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101095950A (en) * 2006-06-30 2008-01-02 辽宁成大生物股份有限公司 Hydrophobia vaccine freezing drying preparations for stable human beings and the preparations thereof
CN103505723A (en) * 2013-09-22 2014-01-15 成都康华生物制品有限公司 Method for preparing freeze-dried rabies vaccine preparation
CN109395074A (en) * 2018-12-21 2019-03-01 辽宁成大生物股份有限公司 A kind of encephalitis B inactivated vaccine lyophilized preparation and preparation method thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2801151A1 (en) * 2010-06-01 2011-12-08 Novartis Ag Concentration of vaccine antigens with lyophilization

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101095950A (en) * 2006-06-30 2008-01-02 辽宁成大生物股份有限公司 Hydrophobia vaccine freezing drying preparations for stable human beings and the preparations thereof
CN103505723A (en) * 2013-09-22 2014-01-15 成都康华生物制品有限公司 Method for preparing freeze-dried rabies vaccine preparation
CN109395074A (en) * 2018-12-21 2019-03-01 辽宁成大生物股份有限公司 A kind of encephalitis B inactivated vaccine lyophilized preparation and preparation method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Effect of β-cyclodextrin as a lyoprotectant for freeze-dried actinidin;T. Furuishi等;《Pharmazie》;第70卷;第296-299页 *
重组猪抑制素蛋白冻干保护剂的筛选;李辉等;《江苏农业科学》;第44卷(第4期);第291-293页 *

Also Published As

Publication number Publication date
CN114931557A (en) 2022-08-23

Similar Documents

Publication Publication Date Title
US8067017B2 (en) Preservation of bioactive materials by freeze dried foam
KR20190027878A (en) Stabilizing excipients for therapeutic protein preparations
CN106063933B (en) Universal vaccine freeze-drying protective agent and application thereof
CN108379561B (en) Pegylated urate oxidase freeze-dried powder and preparation method thereof
CN114931557B (en) Freeze-drying protective agent, vaccine freeze-drying preparation and preparation method thereof
WO2010084298A1 (en) Stable vaccine compositions and methods of use
CN111588859B (en) Freeze-drying protective agent and application thereof, freeze-dried seedling and preparation method thereof
CN106668867B (en) A kind of mumps vaccine freeze drying protectant without gelatin and human serum albumin
US20100183675A1 (en) Stable vaccine compositions and methods of use
EP3040067A1 (en) Chlorogenic acid powder-injection and preparation method thereof
CN115040656B (en) Freeze-drying protective agent for rabies vaccine, application, vaccine and preparation method of vaccine
CZ277712B6 (en) Mixture containing alpha-interferon
BR112020021358A2 (en) METHODS OF PROTECTING THE BIOLOGICAL ACTIVITY OF A BIOLOGICALLY ACTIVE MOLECULE, OF A MULTIPLE OF MOLECULES OF A BIOLOGICALLY ACTIVE MOLECULE, AND OF THE BIOLOGICAL ACTIVITY OF A BIOLOGICALLY ACTIVE PROTEIN
US9821064B2 (en) Vaccine stabilizer
CN110585439B (en) Improved freeze-dried live attenuated hepatitis A vaccine stabilizer, vaccine semi-finished product, vaccine finished product and preparation method thereof
CN114652840A (en) Freeze-drying protective agent for canine virus quadruple live vaccine and preparation method and application thereof
CN117018177A (en) Rabies vaccine composition for freeze-drying and preparation method and application thereof
CN109432017B (en) Heat-resistant freeze-drying protective agent for henpox and application thereof
CN117018176A (en) Rabies vaccine composition and preparation method and application thereof
CN109758427B (en) Pseudorabies live vaccine heat-resistant protective agent and preparation method and application thereof
AU2017303975A1 (en) Process for preparing sterile aripiprazole formulation
CN102058546A (en) Freeze-drying preparation for recombinant swine alpha-interferon and preparation method and usage thereof
CN115282285A (en) Rabies vaccine protective agent, rabies vaccine and preparation method thereof
CN112675134A (en) Recombinant hepatitis B antigen freeze-drying protective agent, recombinant hepatitis B antigen freeze-drying vaccine and preparation method thereof
CN116747312A (en) Vaccine freeze-drying protective agent, preparation and preparation method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant