CN117018177A - Rabies vaccine composition for freeze-drying and preparation method and application thereof - Google Patents

Rabies vaccine composition for freeze-drying and preparation method and application thereof Download PDF

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Publication number
CN117018177A
CN117018177A CN202311058374.4A CN202311058374A CN117018177A CN 117018177 A CN117018177 A CN 117018177A CN 202311058374 A CN202311058374 A CN 202311058374A CN 117018177 A CN117018177 A CN 117018177A
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rabies vaccine
rabies
vaccine composition
drying
freeze
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胡美娜
李育蓉
王琳
王瑞新
张晓娇
柳春羽
尚继领
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Sinovac Research & Development Co ltd
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Sinovac Research & Development Co ltd
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    • 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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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

Abstract

The present disclosure provides a rabies vaccine composition for lyophilization, which comprises an inactivated rabies virus stock solution, a saccharide substance and a high molecular material lyoprotectant. The composition can be formed into a loose cake-like lyophilizate, and can be reconstituted for intramuscular administration. The human diploid cell composition of the rabies vaccine for freeze-dried human, which is prepared by the method, has the advantages of simple formula, good appearance of the preparation, good antigen content and potency stability, and is suitable for large-scale industrial production.

Description

Rabies vaccine composition for freeze-drying and preparation method and application thereof
Technical Field
The present disclosure relates to the field of production technology, and in particular to the production of a freeze-dried rabies vaccine composition for preventing human rabies by using human diploid cells, and a preparation method and application thereof.
Background
Rabies is an acute, progressive, viral infectious disease of the central nervous system that is attacked by human and livestock co-morbid rabies virus (RABV), which can invade the human body through damaged skin or mucous membrane, and once the disease occurs, the death rate reaches almost 100%. Over 55000 deaths caused by rabies worldwide each year, rabies vaccine is the only effective means to prevent rabies.
At present, there are four main rabies vaccine varieties on the market in China: the first method is to prepare the vaccine for the Vero passage cells, and the vaccine variety has higher productivity due to the passage cell line, but has higher purification technical requirements, potential tumorigenic risk and other problems. The second vaccine is a primary rat kidney cell vaccine, wherein the consistency of cell quality is difficult to ensure, and the risk of pollution by exogenous pathogenic microorganisms is high. Thirdly, the vaccine is prepared for chicken embryo cells, which has the disadvantages of higher cost and certain sensitization. The fourth is a human diploid cell rabies vaccine, which has the advantages of strong safety, quick effect, no tumorigenicity, long immunization time and the like, is called as a rabies vaccine 'gold standard', and is commonly used by developed countries. However, human diploid cell rabies vaccines are expensive and difficult to mass produce compared to passaged cells, mainly for two reasons: 1. the existing cell culture technology has certain limitation, so that the rabies virus harvesting amount is low, and the stability and uniformity of the vaccine are poor; 2. the virus stock solution has poor stability, and the freeze-drying protective agent has unsuitable prescription components or proportion in the freeze-drying process, does not have optimal protective effect, and causes larger vaccine antigen content and potency loss after freeze-drying.
In order to avoid the reduction of the antigen content and the titer of the virus stock solution in the freeze-drying process, a certain proportion of stabilizing agents such as human serum albumin, saccharide protecting agents and the like are generally added into the rabies virus semi-finished product. The proper freeze-dried vaccine protective agent can promote the physical and chemical stability of the vaccine and ensure the activity of the vaccine while endowing the vaccine dosage form. Chinese patent CN 103505723 discloses a method for preparing stable human diploid rabies vaccine by absorbing rabies virus with aluminum adjuvant and adding phosphate buffer solution containing maltose, human serum albumin, vitamin C, cysteine and urea as stabilizer, and freeze-drying, but the prescription is complex, the method is not used for scale-up production, and the vaccine contains aluminum, so that the method has a certain potential health risk.
Therefore, there is a need to develop a rabies vaccine formulation with simple composition, strong protective efficacy, high safety and few side effects.
Disclosure of Invention
The purpose of the present disclosure is to provide a rabies vaccine (human diploid cell) composition for lyophilization, which can improve the stability of virus stock solution in the lyophilization process and reduce vaccine antigen and potency losses before and after lyophilization.
In a first aspect, a rabies vaccine composition is provided comprising an inactivated rabies virus stock solution, a carbohydrate, and a polymeric material lyoprotectant.
In some embodiments, the carbohydrate substance comprises at least one of glucose, lactose, sucrose, galactose, trehalose, maltose, mannitol, sorbitol, dextran. Preferably, the saccharide substance comprises at least one of sucrose, trehalose, dextran, maltose.
In some embodiments, the saccharide is present in an amount of 3% -10.0%, e.g., 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or any value therebetween, based on the total weight of the rabies vaccine composition.
In some embodiments, the carbohydrate material comprises maltose or a combination of maltose and a second carbohydrate material. In some embodiments, the second carbohydrate is selected from at least one of glucose, lactose, sucrose, galactose, trehalose, mannitol, sorbitol, or dextran.
In some embodiments, the maltose is present in an amount of 1% -6%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, or any value therebetween, based on the total weight of the rabies vaccine composition. The inventors of the present disclosure found that the formulation containing 3% -10% total weight percent of saccharide, more particularly 2% -6% maltose, was good in appearance and moisture, less than 10% potency loss before and after lyophilization, and heat stability was consistent with quality standards.
In some embodiments, the carbohydrate substances include maltose and trehalose. In other embodiments, the carbohydrate substances include maltose and sucrose. In still other embodiments, the carbohydrate substances include maltose, trehalose, and sucrose. In still other embodiments, the carbohydrate includes trehalose and sucrose. In still other embodiments, the carbohydrate substances include trehalose, sucrose, and dextran.
In some embodiments, the content ratio of the maltose to the second carbohydrate is 1: (0.1-10), for example 1:0.1, 1:0.4, 1:0.8, 1:1. 1:1.5, 1:2. 1:2.5, 1: 3. 1: 4. 1: 6. 1: 8. 1:10 or any value therebetween. In some preferred embodiments, the content ratio of the maltose to the second saccharide is 1: (0.4-5).
In some embodiments, the polymeric lyoprotectant comprises at least one of collagen, hyaluronic acid, sodium hyaluronate, whey protein, human serum albumin. Preferably, the polymeric material protectant comprises human serum albumin.
In some embodiments, the polymeric material lyoprotectant is present in an amount of 1.0% -5.0%, e.g., 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or any value therebetween, based on the total weight of the rabies vaccine composition. In some preferred embodiments, the polymeric material lyoprotectant is present in an amount of 2.0% -5.0% based on the total weight of the rabies vaccine composition. The inventor of the present disclosure found that the content of the high polymer material freeze-drying protective agent is controlled within the range of 1% -5%, the preparation appearance is good, the re-dissolution time is shorter, and the potency loss before and after freeze-drying of the preparation is smaller.
In some embodiments, the rabies vaccine composition further comprises a pH buffer solution and a solvent.
In some embodiments, the pH buffered solution comprises at least one of a histidine solution, a glycine solution, a Tris solution, a citric acid buffered solution, a phosphate buffered solution, an acetate buffered solution. Preferably, the pH buffer solution comprises a phosphate buffer solution.
In some embodiments, the solvent is water for injection.
In some embodiments, the inactivated rabies virus stock is an inactivated rabies virus human diploid cell stock.
In some embodiments, the antigen content of the inactivated rabies virus human diploid cell stock is no less than 3.0IU/mL. In some embodiments, the titer of the inactivated rabies virus human diploid cell stock is no less than 4.0IU/ml. In some embodiments, the antigen content and potency loss of the inactivated rabies virus human diploid cell stock solution before and after lyophilization is no more than 10% (the antigen content is determined by ELISA method, and the potency is determined by the rule 3503 method of the third edition of chinese pharmacopoeia 2020).
In some embodiments, the rabies vaccine composition has a pH of 7.2-8.0.
In some embodiments, the rabies vaccine composition forms a pharmaceutically acceptable loose cake after lyophilization, which can be reconstituted for administration as intramuscular injection. The rabies vaccine composition for freeze-drying has the advantages of simple formula, good appearance of the preparation, good antigen content and potency stability, and suitability for large-scale industrial production.
In a second aspect, there is provided the use of a rabies vaccine composition according to the first aspect in the preparation of a rabies vaccine lyophilized formulation.
In a third aspect there is provided a method of preparing a lyophilized formulation of a rabies vaccine comprising lyophilizing a vaccine semi-finished product of the rabies vaccine composition of the first aspect.
In some embodiments, the freeze-drying comprises sublimation drying at a temperature of-30 to-15 ℃, for example, -30 ℃, -25 ℃, -20 ℃, -15 ℃, or any value therebetween. The inventors of the present disclosure found that the freeze-dried state of the sample was relatively good by controlling the temperature of the sublimation drying temperature to a critical temperature of-30 ℃ to-15 ℃.
In some embodiments, the method of making comprises:
a) Forming a vaccine semi-finished product comprising the rabies vaccine composition of the first aspect;
b) The vaccine semi-finished product is packaged and freeze-dried to form a pharmaceutically acceptable loose cake.
In some embodiments, the step of freeze-drying treatment in step (b) comprises: a prefreezing stage, a prefoaming stage, a sublimation drying stage and a resolution drying stage, wherein the temperature of the sublimation drying stage is controlled between-30 ℃ and-15 ℃ and the drying is carried out for 42 hours.
In some embodiments, the freeze-drying process comprises sequential prefreezing, prefoaming, sublimation drying, and analytical drying.
In some embodiments, the pre-frozen temperature is from-55 ℃ to-45 ℃ (e.g., can be-55 ℃, -54 ℃, -53 ℃, -52 ℃, -51 ℃, -50 ℃, -49 ℃, -48 ℃, -47 ℃, -46 ℃, -45 ℃, etc.), for 3.5 to 4.5 hours (e.g., can be 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, 4 hours, 4.1 hours, 4.2 hours, 4.3 hours, 4.4 hours, 4.5 hours, etc.).
In some embodiments, the pre-evacuated vacuum is between 0.05 and 0.15mbar (e.g., 0.05mbar, 0.06mbar, 0.07mbar, 0.08mbar, 0.09mbar, 0.1mbar, 0.11mbar, 0.12mbar, 0.13mbar, 0.14mbar, 0.15mbar, etc.).
In some embodiments, the sublimation drying temperature is from-30 ℃ to-15 ℃ (e.g., can be-30 ℃, -29 ℃, -28 ℃, -27 ℃, -26 ℃, -25 ℃, -24 ℃, -23 ℃, -22 ℃, -21 ℃, -20 ℃, -19 ℃, -18 ℃, -17 ℃, -16 ℃, -15 ℃, etc.), for a period of time of 42 hours or less (e.g., can be 42 hours, 40 hours, 35 hours, 30 hours, 25 hours, 20 hours, 15 hours, 10 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 0.5 hours, etc.).
In some embodiments, the analytical drying comprises a first analytical drying and a second analytical drying, the temperature of the first analytical drying being from 0 ℃ to 10 ℃ (e.g., may be 0 ℃,1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, etc.) for a period of 5-10 hours (e.g., may be 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, etc.); the temperature of the second analytical drying is 20℃to 30 ℃ (for example, 20℃21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃ and the like) and the time is 4 to 8 hours (for example, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours and the like).
The beneficial effects of the present disclosure are:
(1) The freeze-drying protective agent has simple formula, is convenient for large-scale production, has no sensitization component, and reduces side reaction and potential safety risk.
(2) The freeze-drying protective agent disclosed by the disclosure has a good freeze-drying protective effect, is small in potency loss before and after freeze-drying, and has good preparation stability.
(3) The freeze-dried rabies vaccine prepared by the method has good appearance, the redissolution time is within 30 seconds, and the clinical use is convenient.
Detailed Description
In order to make the objects, technical solutions and advantages of the present disclosure more apparent, the present disclosure will be further described in detail with reference to the following examples. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the disclosure in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.
EXAMPLE 1 Effect of sugar species and maltose concentration on vaccine formulations
1) Preparation of vaccine semi-finished products
In the following examples, the rabies vaccine stock solutions were all inactivated rabies virus human diploid cell stock solutions from MRC-5 cells.
Mixing the inactivated virus stock with vaccine protectant according to the amount of 4 IU/dose, and adjusting sugar type and maltose concentration to prepare vaccine semi-finished product, wherein the pH value of the semi-finished product is 7.5. Wherein the composition of the rabies vaccine composition is as shown in table 1.
2) Freeze-drying of vaccine semi-finished products
Lyophilizing the vaccine semi-finished product obtained in step 1) by:
(1) Cleaning and sterilizing by a freeze dryer: the inner cavity of the freeze dryer is cleaned once by water for injection, then pure steam is introduced for sterilization for 30min at 121 ℃, and the freeze dryer is dried and cooled to room temperature.
(2) Split charging and box feeding: the prepared vaccine semi-finished product is respectively filled into 2mL boron-silicon penicillin bottles, 1.0 mL/bottle, half-pressing bottle stoppers are arranged on a plate layer of a freeze dryer, after the box feeding is finished, the box door is closed, and the freeze drying procedure is started.
(3) Pre-freezing: setting the temperature to be quickly reduced to minus 50 ℃ within 1h, pre-freezing for 4h at minus 50 ℃ and controlling the vacuum to be 0bar;
(4) Pre-vacuumizing: the vacuum pump was turned on and a pre-vacuum was applied at-45℃to 0.1mbar.
(5) Primary drying (sublimation drying): adjusting the temperature of the setting plate layer, controlling the temperature of the setting plate layer to minus 30 ℃, maintaining the vacuum degree to be 0.15mbar, and preserving the heat for 20 hours.
(6) And (5) analysis and drying: adjusting the temperature of the plate layer, quickly heating the temperature of the baffle plate to 5 ℃ within 1h, and maintaining the vacuum degree to 0.1mbar for 8h; then the temperature of the separator was raised to 25℃and the vacuum was 0mbar and incubated for 6h. And (5) reducing the vacuum compaction bottle stopper of the plate layer after the freeze-drying is finished, and taking out the rabies vaccine from the box, rolling the cover and sealing the cover.
3) Quality evaluation of vaccine
(1) Vaccine semi-finished product detection: detecting the pH value, the antigen content and the titer of the semi-finished product;
(2) And (3) vaccine finished product detection: the inspection items of the freeze-dried rabies vaccine (human diploid cells) in the Chinese pharmacopoeia are referred to as 'criteria of freeze-dried rabies vaccine (human diploid cells)' and comprise appearance, moisture, antigen content, potency, abnormal toxicity inspection and thermal stability test. And simultaneously, measuring the re-dissolution time of each freeze-drying protective agent prescription finished product, and evaluating the properties of the finished product.
And (3) pH measurement: the measurement is carried out according to the method of the rule 0631 of the three parts of the 2020 edition of Chinese pharmacopoeia;
antigen content determination: the method is measured according to the rule 3429 of three parts of the edition of Chinese pharmacopoeia 2020;
potency determination: the method is measured according to the rule 3503 of Sanfeng Tong of the edition 2020 of Chinese pharmacopoeia;
moisture content: the method is measured according to the rule 0832 of three parts of the edition of Chinese pharmacopoeia 2020;
thermal stability test: placing the vaccine in an incubator at 37+/-2 ℃ for 0 days and 28 days, and measuring the titer by adopting an NIH mouse in-vivo immunization method after the vaccine expires;
the freeze-dried semi-finished product and the finished product detection result and specific test data are shown in table 2. The antigen content and the titer of the vaccine semi-finished product and the freeze-dried finished product are measured, and the loss rate of the vaccine activity in the freeze-drying process is calculated.
From the data in Table 2, the sugar types in the freeze-dried preparation can influence the appearance, moisture and potency stability of the preparation, the maltose has optimal freeze-drying protection effect on the vaccine under the same human serum albumin concentration, the total weight percentage of sugar substances is 3-10% when analyzed from the data of prescriptions 6, 7, 8, 9 and 10, the prescription appearance and moisture of the preparation containing 2-6% of maltose are good, the potency loss before and after freeze-drying is less than 10%, and the heat stability accords with the quality standard.
EXAMPLE 2 Effect of human serum albumin concentration on vaccine formulations
Mixing the inactivated virus purified solution with vaccine protectant according to the amount of 5 IU/dose, setting maltose concentration to 4% and 5%, and adjusting human serum albumin concentration to prepare vaccine semi-finished product, wherein the pH value of the semi-finished product is 7.5. Wherein the composition of the rabies vaccine composition is as shown in table 3.
The preparation of vaccine semi-finished products and the specific formulation composition are shown in Table 3.
TABLE 3 prescription information example 2
2) Lyophilization of semi-finished products
Lyophilization was performed by the following lyophilization protocol:
(1) Cleaning and sterilizing by a freeze dryer: the inner cavity of the freeze dryer is cleaned once by water for injection, then pure steam is introduced for sterilization for 30min at 121 ℃, and the freeze dryer is dried and cooled to room temperature.
(2) Split charging and box feeding: the prepared vaccine semi-finished product is respectively filled into 2mL neutral boron-silicon penicillin bottles, 1.0 mL/bottle, the bottle stopper is half-added, the penicillin bottles are placed on a plate layer of a freeze dryer, after the box feeding is finished, the box door is closed, and the freeze drying procedure is started.
(3) Pre-freezing: setting the temperature to be quickly reduced to minus 50 ℃ within 1h, pre-freezing for 4h at minus 50 ℃ and controlling the vacuum to be 0bar;
(4) Pre-vacuumizing: the vacuum pump was turned on and a pre-vacuum was applied at-45℃to 0.1mbar.
(5) Primary drying (sublimation drying): adjusting the temperature of the setting plate layer, controlling the temperature of the setting plate layer to minus 20 ℃, maintaining the vacuum degree to be 0.15mbar, and preserving the heat for 14 hours.
(6) And (5) analysis and drying: adjusting the temperature of the plate layer, quickly heating the temperature of the baffle plate to 5 ℃ within 1h, and maintaining the vacuum degree to 0.1mbar for 8h; then the temperature of the separator was raised to 25℃and the vacuum was 0mbar and incubated for 6h. And (5) reducing the vacuum compaction bottle stopper of the plate layer after the freeze-drying is finished, and taking out the rabies vaccine from the box, rolling the cover and sealing the cover.
3) Quality evaluation of rabies vaccine
Quality evaluation of rabies vaccine was as in example 1. The freeze-dried semi-finished product and the finished product detection result and specific test data are shown in table 4.
TABLE 4 test results of example 2
By combining with the prescription 1 of the example 1, the data show that the human serum albumin content is in the range of 2% -5%, the preparation has good appearance, short redissolution time and less potency loss before and after freeze-drying, and meets the quality standard requirement.
Example 3
Mixing the inactivated virus purified solution with vaccine protectant according to the amount of 4 IU/dose, setting maltose concentration to 5%, human serum albumin concentration to 3%, sucrose concentration to 2%, and preparing vaccine semi-finished product, wherein the pH value of semi-finished product is 7.5. Wherein the composition of the rabies vaccine composition is as follows.
The preparation of vaccine semi-finished products and the specific formulation composition are shown in Table 5.
TABLE 5 example 3 prescription information
2) Lyophilization of semi-finished products
Lyophilization was performed by the following lyophilization protocol:
(1) Cleaning and sterilizing by a freeze dryer: the inner cavity of the freeze dryer is cleaned once by water for injection, then pure steam is introduced for sterilization for 30min at 121 ℃, and the freeze dryer is dried and cooled to room temperature.
(2) Split charging and box feeding: the prepared vaccine semi-finished product is respectively filled into 2mL boron-silicon penicillin bottles, 1.0 mL/bottle, half-pressing bottle stoppers are arranged on a plate layer of a freeze dryer, after the box feeding is finished, the box door is closed, and the freeze drying procedure is started.
(3) Pre-freezing: setting the temperature to be quickly reduced to minus 50 ℃ within 1h, pre-freezing for 4h at minus 50 ℃ and controlling the vacuum to be 0bar;
(4) Pre-vacuumizing: the vacuum pump was turned on and a pre-vacuum was applied at-45℃to 0.1mbar.
(5) Primary drying (sublimation drying): adjusting the temperature of the setting plate layer, controlling the temperature of the setting plate layer to-15 ℃, maintaining the vacuum degree to be 0.15mbar, and preserving the heat for 14h.
(6) And (5) analysis and drying: adjusting the temperature of the plate layer, quickly heating the temperature of the baffle plate to 5 ℃ within 1h, and maintaining the vacuum degree to 0.1mbar for 8h; then the temperature of the separator was raised to 25℃and the vacuum was 0mbar and incubated for 6h. And (5) reducing the vacuum compaction bottle stopper of the plate layer after the freeze-drying is finished, and taking out the rabies vaccine from the box, rolling the cover and sealing the cover.
3) Quality evaluation of rabies vaccine
Quality evaluation of rabies vaccine was as in example 1. The freeze-dried semi-finished product and the finished product detection result and specific test data are shown in Table 6.
TABLE 6 test results of example 3
The test results show that: the existing freeze-drying prescription is used, the drying temperature of-15 ℃ is set as the drying temperature of primary drying (sublimation drying), and after freeze-drying is finished, the quality of the sample meets the standard requirement.
Example 4 influence of rabies virus dose on formulation
1) Preparation of vaccine semi-finished products
The vaccine semi-finished product was formulated in the same way as in example 1, the pH of the semi-finished product being 7.5. The composition of the rabies vaccine composition is shown in table 7.
TABLE 7 prescription information example 4
2) Lyophilization of semi-finished products
Lyophilization was performed by the following lyophilization protocol:
(1) Cleaning and sterilizing by a freeze dryer: the inner cavity of the freeze dryer is cleaned once by water for injection, then pure steam is introduced for sterilization for 30min at 121 ℃, and the freeze dryer is dried and cooled to room temperature.
(2) Split charging and box feeding: the prepared vaccine semi-finished product is respectively filled into 2mL neutral boron-silicon penicillin bottles, 1.0 mL/bottle, the bottle stopper is half-added, the penicillin bottles are placed on a plate layer of a freeze dryer, after the box feeding is finished, the box door is closed, and the freeze drying procedure is started.
(3) Pre-freezing: setting the temperature to be quickly reduced to minus 50 ℃ within 1h, pre-freezing for 4h at minus 50 ℃ and controlling the vacuum to be 0bar;
(4) Pre-vacuumizing: the vacuum pump was turned on and a pre-vacuum was applied at-45℃to 0.1mbar.
(5) Primary drying (sublimation drying): adjusting the temperature of the setting plate layer, controlling the temperature of the setting plate layer to 15 ℃ below zero, maintaining the vacuum degree to 0.2mbar, and preserving the heat for 14h.
(6) And (5) analysis and drying: adjusting the temperature of the plate layer, quickly heating the temperature of the baffle plate to 5 ℃ within 1h, and maintaining the vacuum degree to 0.1mbar for 8h; then the temperature of the separator was raised to 25℃and the vacuum was 0mbar and incubated for 6h. And (5) reducing the vacuum compaction bottle stopper of the plate layer after the freeze-drying is finished, and taking out the rabies vaccine from the box, rolling the cover and sealing the cover.
3) Quality evaluation of rabies vaccine
Quality evaluation of rabies vaccine was as in example 1. The freeze-dried semi-finished product and the finished product detection result and specific test data are shown in Table 8.
TABLE 8 test results of example 4
Test data show that when the antigen content is above 3.0IU/mL, the potency result of the preparation sample accords with the pharmacopoeia standard.
EXAMPLE 5 Effect of Primary drying (sublimation drying) temperature on formulation
Mixing the inactivated virus purified solution with vaccine protectant according to the amount of 4 IU/dose, setting maltose concentration to 5%, sucrose concentration to 2%, and human serum albumin concentration to 3%, and preparing vaccine semi-finished product with pH value of 7.5, wherein rabies vaccine composition has the following composition.
Preparation of vaccine semi-finished product the specific formulation composition was the same as formulation 17 in example 3.
2) Lyophilization of semi-finished products
Lyophilization was performed by the following lyophilization protocol:
(1) Cleaning and sterilizing by a freeze dryer: the inner cavity of the freeze dryer is cleaned once by water for injection, then pure steam is introduced for sterilization for 30min at 121 ℃, and the freeze dryer is dried and cooled to room temperature.
(2) Split charging and box feeding: the prepared vaccine semi-finished product is respectively filled into 2mL neutral boron-silicon penicillin bottles, 1.0 mL/bottle, the bottle stopper is half-added, the penicillin bottles are placed on a plate layer of a freeze dryer, after the box feeding is finished, the box door is closed, and the freeze drying procedure is started.
(3) Pre-freezing: setting the temperature to be quickly reduced to minus 50 ℃ within 1h, pre-freezing for 4h at minus 50 ℃ and controlling the vacuum to be 0bar;
(4) Pre-vacuumizing: the vacuum pump was turned on and a pre-vacuum was applied at-45℃to 0.1mbar.
(5) Primary drying (sublimation drying): adjusting the temperature of the setting plate layer, controlling the temperature of the setting plate layer to minus 10 ℃, maintaining the vacuum degree to be 0.15mbar, and preserving the heat for 14h.
(6) And (5) analysis and drying: adjusting the temperature of the plate layer, quickly heating the temperature of the baffle plate to 5 ℃ within 1h, and maintaining the vacuum degree to 0.1mbar for 8h; then the temperature of the separator was raised to 25℃and the vacuum was 0mbar and incubated for 6h. And (5) reducing the vacuum compaction bottle stopper of the plate layer after the freeze-drying is finished, and taking out the rabies vaccine from the box, rolling the cover and sealing the cover.
3) Quality evaluation of rabies vaccine
Quality evaluation of rabies vaccine was as in example 1. The freeze-dried semi-finished product and the finished product detection result and specific test data are shown in Table 9.
TABLE 9 test results of example 5
Analysis of the results shows that: when the temperature of freeze-drying (sublimation drying) is-10 ℃, the appearance of the preparation is qualified, the redissolution time data are all qualified, the appearance has the bottle climbing phenomenon, the antigen content and the potency loss before and after freeze-drying are large, and the potency data are far lower than the quality standard when the thermal stability is 28 days. By combining the experimental data of example 1, example 3, example 4, and thus setting the lyophilization temperature to-30 ℃ to-15 ℃ as the primary drying temperature (sublimation drying), a lyophilized vaccine product meeting the quality standard can be prepared.
EXAMPLE 6 Effect of maltose amount on formulation
Mixing the inactivated virus purified solution with vaccine protectant according to the amount of 4 IU/dose, and preparing vaccine semi-finished product according to prescription amount, wherein the final pH value is 7.5, wherein the rabies vaccine composition has the following composition.
The preparation of vaccine semi-finished products and the specific formulation composition are shown in Table 10.
TABLE 10 example 6 prescription information
2) Lyophilization of semi-finished products
Lyophilization was performed by the following lyophilization protocol:
(1) Cleaning and sterilizing by a freeze dryer: the inner cavity of the freeze dryer is cleaned once by water for injection, then pure steam is introduced for sterilization for 30min at 121 ℃, and the freeze dryer is dried and cooled to room temperature.
(2) Split charging and box feeding: the prepared vaccine semi-finished product is respectively filled into 2mL neutral boron-silicon penicillin bottles, 1.0 mL/bottle, the bottle stopper is half-added, the penicillin bottles are placed on a plate layer of a freeze dryer, after the box feeding is finished, the box door is closed, and the freeze drying procedure is started.
(3) Pre-freezing: setting the temperature to be quickly reduced to minus 50 ℃ within 1h, pre-freezing for 4h at minus 50 ℃ and controlling the vacuum to be 0bar;
(4) Pre-vacuumizing: the vacuum pump was turned on and a pre-vacuum was applied at-45℃to 0.1mbar.
(5) Primary drying (sublimation drying): adjusting the temperature of the setting plate layer, controlling the temperature of the setting plate layer to-15 ℃, maintaining the vacuum degree to be 0.15mbar, and preserving the heat for 14h.
(6) And (5) analysis and drying: adjusting the temperature of the plate layer, quickly heating the temperature of the baffle plate to 5 ℃ within 1h, and maintaining the vacuum degree to 0.1mbar for 8h; then the temperature of the separator was raised to 25℃and the vacuum was 0mbar and incubated for 6h. And (5) reducing the vacuum compaction bottle stopper of the plate layer after the freeze-drying is finished, and taking out the rabies vaccine from the box, rolling the cover and sealing the cover.
3) Quality evaluation of rabies vaccine
Quality evaluation of rabies vaccine was as in example 1. The freeze-dried semi-finished product and the finished product detection result and specific test data are shown in Table 11.
TABLE 11 test results of example 6
The test result further shows that under the same human serum albumin concentration, the total weight percentage of the saccharide is 3% -10%, the preparation prescription containing 2% -6% of maltose has good appearance and water, the potency loss before and after freeze-drying is less than 10%, and the heat stability is good.
The data of examples 1-6 above demonstrate that the present disclosure provides vaccine stabilizers that effectively control the rate of vaccine activity loss during lyophilization and a suitable lyophilization process.
The technical solution of the present disclosure is not limited to the above specific embodiments, and all technical modifications made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims (10)

1. A rabies vaccine composition for freeze-drying comprises inactivated rabies virus stock solution, saccharide and high molecular material freeze-drying protective agent.
2. The rabies vaccine composition for lyophilization according to claim 1, wherein the saccharide substance comprises at least one of glucose, lactose, sucrose, galactose, trehalose, maltose, mannitol, sorbitol or dextran; preferably, the carbohydrate substance comprises at least one of sucrose, trehalose, dextran or maltose.
3. The rabies vaccine composition for lyophilization according to claim 1 or 2, wherein the content of the saccharide is 3% -10.0% based on the total weight of the rabies vaccine composition.
4. The rabies vaccine composition for lyophilization according to any one of claims 1-3, wherein the saccharide comprises maltose or a combination of maltose and a second saccharide,
preferably, the second carbohydrate substance comprises at least one of glucose, lactose, sucrose, galactose, trehalose, mannitol, sorbitol or dextran,
preferably, the maltose is present in an amount of 1% -6% based on the total weight of the rabies vaccine composition.
5. The rabies vaccine composition according to any one of claims 1-4, wherein the polymeric material lyoprotectant comprises at least one of collagen, hyaluronic acid, sodium hyaluronate, whey protein, human serum albumin, preferably the polymeric material protectant comprises human serum albumin.
6. The rabies vaccine composition according to any one of claims 1-5, wherein the polymeric material lyoprotectant is present in an amount of 2.0% -5.0% based on the total weight of the rabies vaccine composition.
7. The lyophilized rabies vaccine composition according to any one of claims 1-6, further comprising a pH buffer solution and a solvent,
preferably, the pH buffer solution comprises at least one of histidine solution, glycine solution, tris solution, citric acid buffer solution, phosphate buffer solution, acetate buffer solution, preferably, the pH buffer solution comprises phosphate buffer solution;
preferably, the solvent is water for injection.
8. The lyophilized rabies vaccine composition according to any one of claims 1-7, wherein the inactivated rabies virus stock solution is an inactivated rabies virus human diploid cell stock solution,
preferably, the antigen content of the inactivated rabies virus human diploid cell stock solution is not less than 3.0IU/mL,
preferably, the titer of the inactivated rabies virus human diploid stock solution is not less than 4.0IU/mL,
preferably, the antigen content and potency loss of the inactivated rabies virus stock solution before and after lyophilization is no more than 10%; and/or
The rabies vaccine composition has a pH of 7.2-8.0.
9. Use of a rabies vaccine composition according to any one of claims 1-8 in the preparation of a rabies vaccine lyophilized formulation.
10. A process for preparing a lyophilized formulation of rabies vaccine, which comprises lyophilizing a vaccine semi-finished product comprising the rabies vaccine composition of any one of claims 1 to 8,
preferably, the freeze-drying comprises sublimation drying at a temperature of-30 to-15 ℃.
CN202311058374.4A 2023-08-21 2023-08-21 Rabies vaccine composition for freeze-drying and preparation method and application thereof Pending CN117018177A (en)

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