CN108434106B - Freeze-dried preparation of rabies vaccine - Google Patents
Freeze-dried preparation of rabies vaccine Download PDFInfo
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- CN108434106B CN108434106B CN201711457418.5A CN201711457418A CN108434106B CN 108434106 B CN108434106 B CN 108434106B CN 201711457418 A CN201711457418 A CN 201711457418A CN 108434106 B CN108434106 B CN 108434106B
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- 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
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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
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- 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
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- 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
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- 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 invention discloses a freeze-dried preparation of rabies vaccine, which contains rabies vaccine stock solution and a vaccine stabilizer, wherein the vaccine stabilizer consists of human serum albumin and sucrose. The invention has the beneficial effects that: (1) the auxiliary materials have simple components, do not contain components derived from animal sources, and have lower risk. (2) Effectively controlling the activity loss rate of the vaccine in the freeze-drying process. (3) After the preparation is prepared into a finished product, the product shows good stability through stability research.
Description
Technical Field
The invention belongs to the technical field of virus biology, and particularly relates to a freeze-dried preparation of rabies vaccine.
Background
Rabies is a highly fatal disease, and death is almost inevitable once the disease stage is reached after exposure to viruses. Vaccination of humans, dogs and other sensitive host animals with frequent human contact is the only effective means to control rabies incidence and prevalence.
Inactivated whole virus vaccine preparations are generally administered subcutaneously or intramuscularly, and the ability of immune cells, particularly Antigen Presenting Cells (APCs), abundant in these sites to recruit immune cells and immune-related cytokines to these sites in other sites is utilized to induce the body to rapidly generate protective immune responses. Therefore, this type of vaccine preparation is usually prepared by injection, including water injection and freeze-dried powder injection. In contrast, the freeze-dried powder injection has the advantages of longer effective period, more convenient storage and transportation, better consistency of various key quality indexes in the effective period and the like, and becomes the optimal preparation formulation of the inactivated whole virus vaccine product.
The vaccine adjuvant refers to an excipient and an additive used in the prescription preparation of a vaccine product, is an indispensable important component in the vaccine preparation, can endow the vaccine preparation with necessary physical, chemical, pharmacological and biological characteristics, and has positive key effects on the stability, the biological activity, the product quality guarantee, the development of new preparation forms and new varieties, the prevention and use requirements and the like of the vaccine preparation.
The related information of the adverse reactions after vaccination published by the World Health Organization (WHO) in 2000 provides that 1-64 people among 1 ten thousand vaccinees in the United states of America and vaccinated with Japanese encephalitis inactivated vaccine have anaphylactic reaction, severe systemic urticaria, facial angioedema or respiratory distress, and the reasons are related to the gelatin stabilizer in the product. Recent studies have shown that individuals with allergic reactions after monovalent measles, mumps and rubella vaccination have immunoglobulin E antibodies to gelatin stabilizers. Therefore, the adverse reactions of the vaccine after inoculation are caused by raw and auxiliary materials used in the production process in a certain proportion. Therefore, in the biological product, the raw and auxiliary materials are not considered as inactive substances any more, and the physical and chemical properties and safety of the raw and auxiliary materials for producing the biological product have direct influence on the effectiveness and safety of the biological product.
The human serum albumin is mainly used as an auxiliary material of an injection and as a protein stabilizer in a prescription. Can also be used as antifreeze in the freeze drying process to prevent other proteins from being adsorbed on the surface. Sucrose can be used as a stabilizer and lyoprotectant for biological products. In the process of preparing the freeze-dried rabies vaccine for human, in addition to the auxiliary materials, the components such as gelatin, trehalose, a surfactant and the like are required to be added so as to enhance the protection effect in the freeze-drying process and the preservation process and reduce the loss rate of the titer of the vaccine, but the introduced components can cause great influence on the safety of the product.
Therefore, it would be of great interest to develop a vaccine formulation that is simpler in composition, safer, stable and less toxic.
Disclosure of Invention
The invention aims to provide a vaccine stabilizer with simple components and good freeze-drying effect.
The invention also aims to provide a rabies vaccine preparation with simple auxiliary material components and good safety.
The technical scheme adopted by the invention is as follows:
a vaccine stabilizer comprises human serum albumin and sucrose.
Preferably, in the vaccine stabilizer, the mass ratio of human serum albumin to sucrose is as follows: (1-10):(2-20).
A rabies vaccine lyophilized preparation, the formulation of the preparation comprises rabies vaccine antigen and vaccine stabilizer, the vaccine stabilizer is as above.
Preferably, the concentration of each component in the rabies vaccine lyophilized preparation is as follows according to the mass percentage before lyophilization:
further preferably, in the rabies vaccine freeze-dried preparation, the concentration of human serum albumin is 1-3%.
Further preferably, in the rabies vaccine freeze-dried preparation, the concentration of sucrose is 3-6%.
Further preferably, the buffer solution is a phosphate buffer solution with the pH value of 7.2-8.0.
The invention has the beneficial effects that:
(1) the components in the stabilizer are simple, no animal-derived component is used, complex high-risk components such as gelatin, dextran, trehalose and surfactant are not needed, and the safety is higher.
(2) Effectively controlling the activity loss rate of the vaccine in the freeze-drying process.
(3) The preparation of the invention has good stability.
Detailed Description
The present invention will be further illustrated by the following examples, but is not limited thereto.
Example 1
1.1 preparing a vaccine semi-finished product according to the following formula, and detecting the freeze-drying effect of the vaccine semi-finished product:
human serum albumin 1.5 (w/w)%;
sucrose 5.0 (w/w)%;
50mL of rabies virus antigen stock solution;
supplementing phosphate buffer solution with pH of 7.2-8.0 to 100 mL.
1.2 preparation method of rabies vaccine
Preparing a semi-finished product from the purified rabies virus antigen stock solution according to the formula;
subpackaging the semi-finished product into 2ml tube bottles, each tube contains 0.5ml, and freeze-drying with a small freeze dryer.
1.3 vaccine assay methods
And (3) measuring the titer: measured according to the 3503 method of the three general rules of the chapter of the book of Chinese pharmacopoeia 2015 edition;
moisture content: measured according to 0832 method of the three ministry of communications in 2015 pharmacopoeia of China;
abnormal toxicity test: measured according to 1141 method of the three ministry of communications of the 2015 version of Chinese pharmacopoeia;
thermal stability test: the vaccine is placed in a thermostat with the temperature of 37 +/-1 ℃ and stored for 0 week, 4 weeks, 5 weeks and 6 weeks, and the potency is measured by adopting an NIH mouse in-vivo immunization method after the storage;
long-term stability test: the vaccine is stored in a medicine storage box at 4 +/-2 ℃, taken out at 0 month, 3 months, 6 months and 9 months, and the potency is determined by adopting an NIH mouse in-vivo immunization method.
1.4 results of the study
1.4.1 loss ratio of titer before and after lyophilization of rabies vaccine
And (4) performing titer determination on the vaccine semi-finished product before freeze-drying and the finished product preparation after freeze-drying, and calculating the loss rate of the vaccine activity in the freeze-drying process.
TABLE 1 comparison of vaccine titer loss rates before and after lyophilization
The freeze-drying titer loss rate of the formula is within an acceptable range.
1.4.2 assay results after lyophilization
The lyophilized preparation is subjected to the detection of titer, moisture and abnormal toxicity test items.
Table 2 quality index assay results of lyophilized formulations
1.4.3 accelerated stability Studies of lyophilized formulations
The lyophilized preparation was stored at 37. + -. 1 ℃ and the samples were taken out for different times according to Table 3 for titer determination.
The data in table 3 show that the vaccine titer is not less than 2.5 IU/dose after 4 weeks of accelerated thermal stability examination, and meets the requirements of the three parts (2015 edition) of the Chinese pharmacopoeia on rabies vaccine for freeze-dried human.
Table 3 accelerated stability study results of lyophilized formulations
1.4.4 Long-term stability Studies of lyophilized formulations
The lyophilized preparation was stored at 4 + -2 deg.C, and samples were taken out after different times of storage according to Table 4 for titer determination.
Table 4 long-term stability study results of lyophilized formulations
As can be seen from the data in the table, the lyophilized formulation has better long-term stability.
Example 2
2.1 Effect of different sucrose dosages on vaccine formulations
The concentration of human serum albumin in the vaccine semi-finished product is set to be 1.5%, the concentration of sucrose in the vaccine semi-finished product is adjusted, and the influence of the concentration of sucrose on the freeze-drying effect of the vaccine is observed. Specific comparison results are shown in table 5:
TABLE 5 Effect of different sucrose concentrations on the Freeze drying Effect
The data in table 5 show that, within the sucrose concentration range of 1-10%, the appearance, the reconstitution time and the moisture content all meet the requirements of the three parts (2015 edition) of the Chinese pharmacopoeia on rabies vaccine for freeze-dried human, but within the sucrose concentration range of 3-6%, the appearance and the reconstitution time are optimal.
2.2 Effect of different human serum Albumin dosages on vaccine formulations
Setting the sucrose concentration in the vaccine semi-finished product to be 5%, adjusting the human serum albumin concentration in the vaccine semi-finished product, and observing the influence of the human serum albumin concentration on the vaccine. Specific comparison results are shown in table 6:
TABLE 6 Effect of different human serum albumin concentrations on the lyophilization Effect
The data in table 6 show that the freeze-dried preparation has good appearance within the concentration range of 0.5-5% of human serum albumin, and the freeze-dried form is poor under the concentration of 0.3% of human serum albumin.
Example 3 Effect of different combinations of vaccine stabilizers on vaccine formulations
The above experiments show that: the vaccine stabilizer effectively controls the activity loss rate of the vaccine in the freeze-drying process.
Claims (2)
1. The rabies vaccine freeze-dried preparation is characterized in that the preparation formula comprises rabies vaccine antigen and a vaccine stabilizer, the vaccine stabilizer consists of human serum albumin and sucrose, and the concentration of each component in 100mL of the preparation formula is as follows according to the mass percentage before freeze-drying:
0.5-5% of human serum albumin,
3 to 6 percent of cane sugar,
the volume of the rabies vaccine antigen is 50mL,
the buffer solution is the balance of phosphate buffer solution with the pH value of 7.2-8.0.
2. The lyophilized rabies vaccine formulation according to claim 1, wherein the concentration of human serum albumin is 1-3%.
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| CN2017102985571 | 2017-04-25 | ||
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| CN108434106B true CN108434106B (en) | 2021-07-30 |
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| 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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| MX2008014391A (en) * | 2006-05-12 | 2009-03-06 | Bharat Biotech Int Ltd | A composition useful as a vaccine. |
| CN101095950B (en) * | 2006-06-30 | 2011-01-26 | 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 |
| FR2944292B1 (en) * | 2009-04-08 | 2013-08-23 | Sanofi Pasteur | PROCESS FOR PURIFYING RABIC VIRUS |
| CN102399755B (en) * | 2011-09-21 | 2013-03-20 | 上海创宏生物科技有限公司 | Pig pseudorabies virus natural low-virulent C strain and heatproof preservation method thereof |
| CN102671194B (en) * | 2012-05-07 | 2013-08-14 | 成都康华生物制品有限公司 | Human vaccine for preventing hydrophobia and tetanus |
| CN103146655B (en) * | 2013-03-27 | 2015-05-13 | 广州银河阳光生物制品有限公司 | Seed selection of high-immunogenicity rabies virus fixed strain and application thereof in vaccine development |
| CN103690943A (en) * | 2013-12-12 | 2014-04-02 | 大连汉信生物制药有限公司 | Freeze-dried rabies vaccine for human use and preparation method thereof |
| CN105012963B (en) * | 2015-08-19 | 2018-10-23 | 肇庆大华农生物药品有限公司 | A kind of recombinant pseudorabies virus vaccine heat-resisting lyophilized protecting agent and preparation method thereof |
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| CN1966076A (en) * | 2006-11-14 | 2007-05-23 | 广州市嘉合生物技术有限公司 | Mass-production method of hydrophobic vaccine |
| CN102512686A (en) * | 2010-12-21 | 2012-06-27 | 成都生物制品研究所有限责任公司 | Vaccine protectant, hydrophobia vaccine and preparation method thereof |
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Effective date of registration: 20181221 Address after: Room 305, Block G, Guangzhou International Business Incubator, No. 3 Lanyue Road, Science City, Luogang District, Guangzhou City, Guangdong Province Applicant after: Guangzhou Rui Beisi Pharmaceutical Co. Ltd. Address before: Room 306, 307 and 308, Block G, No. 3 Lanyue Road, Science City, Guangzhou High-tech Industrial Development Zone, Guangdong Province Applicant before: Guangzhou Rui Beisi Pharmaceutical Co. Ltd. Applicant before: Guangzhou Galaxy Sun Biological Products Co., Ltd. |
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