CN103505723A - Method for preparing freeze-dried rabies vaccine preparation - Google Patents
Method for preparing freeze-dried rabies vaccine preparation Download PDFInfo
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- CN103505723A CN103505723A CN201310430624.2A CN201310430624A CN103505723A CN 103505723 A CN103505723 A CN 103505723A CN 201310430624 A CN201310430624 A CN 201310430624A CN 103505723 A CN103505723 A CN 103505723A
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Abstract
The invention discloses a method for preparing a freeze-dried rabies vaccine preparation. The method comprises the following steps: in the preparing process, adding nano-scale aluminum hydroxide, preparing a phosphate buffer solution containing maltose, human albumin, vitamin C, cysteine and urea as a stabilizer, correcting and optimizing a normal freeze-drying process route and parameters, and completing production of the freeze-dried preparation through pre-freezing, primary drying, secondary drying and analytical drying. Compared with the conventional rabies vaccine, stabilizer components and protective agent components in the freeze-drying process are optimized, so that the method plays good roles of preventing deterioration and preventing loosing efficacy on the storage process of the rabies vaccine; meanwhile, the freeze-drying process route is optimized, and more reasonable freeze-drying parameters are set, so that the crystal structure of the freeze-dried preparation is stable, and the appearance conforms to the national standard.
Description
Technical field
The present invention relates to the medicine preparation of rabies vaccine, be specifically related to the lyophilized formulations preparation method of human rabies vaccine.
Background technology
Rabies have another name called hydrophobia, are by the Natur al foca infecting both domestic animals and human acute infectious disease due to rabies virus.Popular wide, case fatality rate is high, is almost 100%.Rabies cause serious threat to people's life health.Human rabies is conventionally passed to people's human body by sick beast in the mode of biting and is infected.Clinical manifestation is distinctive hydrophobia, probably sound, aversion to wind, frightened and restless, pharyngismus, the paralysis of carrying out property etc.
Existing rabies treatment means is generally, and after people is bitten, injects immediately rabies vaccine, makes it in human body, form potent antibodies and prevents morbidity.Rabies vaccine generally adopts administered intramuscular, conventionally has aqueous injection and injectable powder.The preparation technology of rabies vaccine is to its shelf-life and the tremendous influence of having tired.Rabies vaccine lyophilized formulations, before preparation, need to add stabilizing agent, protective agent and excipient; The component of above-mentioned additive and content have protective effect to the heat stability of rabies vaccine, and meanwhile, the selection of freeze-dry process route and lyophilizing parameter also has obvious impact to the character of vaccine.Rabies vaccine is not common drug, and the more general medicine of annual consumption is compared, and consumption seldom, is therefore badly in need of strengthening its stability, makes its shelf-life longer.
In order to solve above-mentioned deficiency of the prior art, the present invention proposes a kind of new solution.
Summary of the invention
The preparation method that the object of this invention is to provide a kind of lyophilized formulations of rabies vaccine; the present invention is by being optimized combination to the stabilizing agent of vaccinogen liquid and protective agent; improve freeze-dry process route and parameter, when making shelf-life of lyophilizing rabies vaccine longer, vaccine valence meets national standard.
For reaching above-mentioned purpose, the technical solution adopted in the present invention is: a kind of preparation method of rabies vaccine lyophilized formulations is provided, comprises following preparation process;
A, in purified vaccinogen liquid, add nanoscale aluminum hydroxide adjuvant, its quality proportioning is that in 100 parts of vaccinogen liquids, to add 10 parts ~ 15 parts, particle diameter be the aluminum hydroxide adjuvant of 50 nanometer ~ 90 nanometers, mixed liquor is placed in to 4 ℃ ~ 6 ℃ absorption after adding aluminum hydroxide adjuvant;
B, the configuration Ph phosphate buffer that is 7.2 ~ 7.8 adds maltose, 1% ~ 5% human albumin, 3% vitamin C and 5% cysteine and 2% the carbamide of phosphate buffer quality 2% ~ 5% in phosphate buffer;
C, the phosphate buffer in step B is added in the solution of steps A and mixes, then add 1% sodium bicarbonate solution, 1% dextran, 2% mannitol, 2% hydroxy methocel and 0.1% ~ 0.5% sodium selenite of mixed liquor gross mass; By mixed liquor lyophilizing;
D, aforementioned freeze-dry process comprise
Pre-freeze: the vaccinogen liquid of processing through step C is distributed in lyophilizing bottle, and lyophilizing bottle is placed in freeze dryer, makes its pre-freeze 4h in subzero 20 ℃, then pre-freeze 2h in subzero 35 ℃; Open vacuum machine simultaneously, make the vacuum of freeze dryer maintain 8pa ~ 12pa;
Primary drying: regulate baffle temperature, control baffle temperature and maintain subzero 30 ℃ ~ subzero 28 ℃, vacuum maintains 15Pa, insulation 10h;
Redrying: regulate baffle temperature, maintain between subzero 28 ℃ ~ subzero 25 ℃, vacuum maintains 15Pa, insulation 15h;
Parsing-desiccation: baffle temperature is warming up to 15 ℃, and vacuum is 15Pa, maintains 2h; Then baffle temperature is warming up to 30 ℃, vacuum is 10Pa, insulation 5h.
Wherein, in step C, also added 0.5% NaCl.
Wherein, vaccinogen liquid adds after aluminum hydroxide adjuvant absorption, uses ultrasound wave to disperse, and jitter time is 20 minutes.
Wherein, the gelatin that has also added phosphate buffered solution gross mass 5% in phosphate buffered solution.
In sum, the present invention has the following advantages:
The present invention compares with existing rabies vaccine; by the stabilizer component in freeze-drying process and protectant component; the storing process of rabies vaccine has been played to good anti-degeneration, inefficacy effect; optimized freeze-dry process route simultaneously; be provided with more rational lyophilizing parameter; make lyophilized formulations product crystal Stability Analysis of Structures, outward appearance meets national standard.
The specific embodiment
Embodiment 1
The preparation method of rabies vaccine lyophilized formulations, comprises following preparation process;
Configuration aluminum hydroxide adjuvant mixed liquor: add nanoscale aluminum hydroxide adjuvant in purified vaccinogen liquid, its quality proportioning is that in 100 parts of vaccinogen liquids, to add 15 parts, particle diameter be the aluminum hydroxide adjuvant of 90 nanometers, after adding aluminum hydroxide adjuvant, mixed liquor is placed in to 4 ℃ ~ 6 ℃ absorption, absorption refers to and allows vaccinogen liquid and aluminum hydroxide adjuvant mix, allows vaccinogen liquid be combined with aluminum hydroxide adjuvant.Vaccinogen liquid adds after aluminum hydroxide adjuvant absorption, uses ultrasound wave to disperse, and jitter time is 20 minutes.Above-mentioned nanometer aluminum hydroxide adjuvant can adopt chemical method preparation, also can adopt physical method preparation.
The configuration of phosphate buffer stabilizing agent: the configuration Ph phosphate buffer that is 7.2 ~ 7.8 adds maltose, 1% human albumin, 3% vitamin C and 5% cysteine and 2% the carbamide of phosphate buffer quality 2% in phosphate buffer; Then, the gelatin that adds phosphate buffered solution gross mass 5% in phosphate buffered solution.
Phosphate buffer stabilizing agent is added in aluminum hydroxide adjuvant mixed liquor and is mixed, and its mixed proportion is to add 20g phosphate buffer stabilizing agent in every 100g aluminum hydroxide adjuvant mixed liquor.When mixing, first phosphate buffer is warming up in water-bath to 80 ℃; Then add 1% sodium bicarbonate solution, 1% dextran, 2% mannitol, 2% hydroxy methocel, 0.1% sodium selenite of total mixed liquor quality and 0.5% NaCl; By mixed liquor lyophilizing.
Aforementioned freeze-dry process comprises
Pre-freeze: treated vaccinogen liquid is distributed in lyophilizing bottle, and lyophilizing bottle is placed in freeze dryer, makes its pre-freeze 4h in subzero 20 ℃, then pre-freeze 2h in subzero 35 ℃; Open vacuum machine simultaneously, make the vacuum of freeze dryer maintain 8pa;
Primary drying: regulate baffle temperature, control baffle temperature and maintain subzero 30 ℃, vacuum maintains 15Pa, insulation 10h;
Redrying: regulate baffle temperature, maintain between subzero 25 ℃, vacuum maintains 15Pa, insulation 15h;
Parsing-desiccation: baffle temperature is warming up to 15 ℃, and vacuum is 15Pa, maintains 2h; Then baffle temperature is warming up to 30 ℃, vacuum is 10Pa, insulation 5h.
Rabies vaccine lyophilized formulations product is tested, its inspection item comprise initially tire, tire after heat stabilization test, outward appearance etc.Its assay is as table 1.Meanwhile, the finished product in embodiment 1 is carried out to the evaluation of drug safety, carry out zoopery and check its safety.Vaccine is carried out respectively to the experiment of the experiment of vaccine intracerebral injection, anaphylaxis and injection site irritation test, and result shows, Novel presentation does not appear in mice, Continuous Observation 10 days, and experiment mice is all healthy.Wherein, by rabies vaccine intraperitoneal injection of mice, and it is observed 7 days.Mice was all good for and deposits in 7 days, and Normal-weight increases.
Embodiment 2
The preparation method of rabies vaccine lyophilized formulations, comprises following preparation process;
Configuration aluminum hydroxide adjuvant mixed liquor: add nanoscale aluminum hydroxide adjuvant in purified vaccinogen liquid, its quality proportioning is that in 100 parts of vaccinogen liquids, to add 10 parts, particle diameter be the aluminum hydroxide adjuvant of 50 nanometers, after adding aluminum hydroxide adjuvant, mixed liquor is placed in to 4 ℃ ~ 6 ℃ absorption, absorption refers to and allows vaccinogen liquid and aluminum hydroxide adjuvant mix, allows vaccinogen liquid be combined with aluminum hydroxide adjuvant.Vaccinogen liquid adds after aluminum hydroxide adjuvant absorption, uses ultrasound wave to disperse, and jitter time is 20 minutes.Above-mentioned nanometer aluminum hydroxide adjuvant can adopt chemical method preparation, also can adopt physical method preparation.
The configuration of phosphate buffer stabilizing agent: the configuration Ph phosphate buffer that is 7.2 ~ 7.8 adds maltose, 5% human albumin, 3% vitamin C and 5% cysteine and 2% the carbamide of phosphate buffer quality 5% in phosphate buffer; Then, the gelatin that adds phosphate buffered solution gross mass 5% in phosphate buffered solution.
Phosphate buffer stabilizing agent is added in aluminum hydroxide adjuvant mixed liquor and is mixed, and its mixed proportion is to add 20g phosphate buffer stabilizing agent in every 100g aluminum hydroxide adjuvant mixed liquor.When mixing, first phosphate buffer is warming up in water-bath to 80 ℃; Then add 1% sodium bicarbonate solution, 1% dextran, 2% mannitol, 2% hydroxy methocel, 0.5% sodium selenite of total mixed liquor quality and 0.5% NaCl; By mixed liquor lyophilizing.
Aforementioned freeze-dry process comprises
Pre-freeze: treated vaccinogen liquid is distributed in lyophilizing bottle, and lyophilizing bottle is placed in freeze dryer, makes its pre-freeze 4h in subzero 20 ℃, then pre-freeze 2h in subzero 35 ℃; Open vacuum machine simultaneously, make the vacuum of freeze dryer maintain 8pa;
Primary drying: regulate baffle temperature, control baffle temperature and maintain subzero 28 ℃, vacuum maintains 15Pa, insulation 10h;
Redrying: regulate baffle temperature, maintain between subzero 25 ℃, vacuum maintains 15Pa, insulation 15h;
Parsing-desiccation: baffle temperature is warming up to 15 ℃, and vacuum is 15Pa, maintains 2h; Then baffle temperature is warming up to 30 ℃, vacuum is 10Pa, insulation 5h.
Rabies vaccine lyophilized formulations product is tested, its inspection item comprise tire, heat stability, outward appearance etc.Its assay is as table 1.Meanwhile, the finished product in embodiment 2 is carried out to the evaluation of drug safety, carry out zoopery and check its safety.Vaccine is carried out respectively to the experiment of the experiment of vaccine intracerebral injection, anaphylaxis and injection site irritation test, and result shows, Novel presentation does not appear in mice, Continuous Observation 10 days, and experiment mice is all healthy.Wherein, by rabies vaccine intraperitoneal injection of mice, and it is observed 7 days.Mice was all good for and deposits in 7 days, and Normal-weight increases.The detection of vaccine detects according to the summation approach of wanting in pharmacopeia.In embodiment 1 and embodiment 2, the heat stability experiment of vaccine is adopted and accelerates experiment, the vaccine of embodiment 1 and embodiment 2 is placed in respectively at 38 ℃, detect its effectively tiring after 7 days, after 14 days, after 28 days.According to current edition " Products in China rules ", the test specimen of lyophilizing is carried out to titration respectively, it is tired and is not less than 2.5IU/dose dosage is qualified.
Table 1 heat stability experimental result
As can be seen from the above table, the vaccine in embodiment 1 and embodiment 2 is after heat stability accelerated test, and it is tired and still can be greater than national standard, so the present invention is with respect to existing rabies vaccine, and its stability strengthens.
Claims (4)
1. the preparation method of rabies vaccine lyophilized formulations, comprises following preparation process;
A, in purified vaccinogen liquid, add nanoscale aluminum hydroxide adjuvant, its quality proportioning is that in 100 parts of vaccinogen liquids, to add 10 parts ~ 15 parts, particle diameter be the aluminum hydroxide adjuvant of 50 nanometer ~ 90 nanometers, mixed liquor is placed in to 4 ℃ ~ 6 ℃ absorption after adding aluminum hydroxide adjuvant;
B, the configuration Ph phosphate buffer that is 7.2 ~ 7.8 adds maltose, 1% ~ 5% human albumin, 3% vitamin C and 5% cysteine and 2% the carbamide of phosphate buffer quality 2% ~ 5% in phosphate buffer;
C, the phosphate buffer in step B is added in the solution of steps A and mixes, then add 1% sodium bicarbonate solution, 1% dextran, 2% mannitol, 2% hydroxy methocel and 0.1% ~ 0.5% sodium selenite of mixed liquor gross mass; By mixed liquor lyophilizing;
D, aforementioned freeze-dry process comprise
Pre-freeze: the vaccinogen liquid of processing through step C is distributed in lyophilizing bottle, and lyophilizing bottle is placed in freeze dryer, makes its pre-freeze 4h in subzero 20 ℃, then pre-freeze 2h in subzero 35 ℃; Open vacuum machine simultaneously, make the vacuum of freeze dryer maintain 8pa ~ 12pa;
Primary drying: regulate baffle temperature, control baffle temperature and maintain subzero 30 ℃ ~ subzero 28 ℃, vacuum maintains 15Pa, insulation 10h;
Redrying: regulate baffle temperature, maintain between subzero 28 ℃ ~ subzero 25 ℃, vacuum maintains 15Pa, insulation 15h;
Parsing-desiccation: baffle temperature is warming up to 15 ℃, and vacuum is 15Pa, maintains 2h; Then baffle temperature is warming up to 30 ℃, vacuum is 10Pa, insulation 5h.
2. the preparation method of rabies vaccine lyophilized formulations as claimed in claim 1, is characterized in that: in step C, added 0.5% NaCl.
3. the preparation method of rabies vaccine lyophilized formulations as claimed in claim 1, is characterized in that: vaccinogen liquid adds after aluminum hydroxide adjuvant absorption, and use ultrasound wave to disperse, jitter time is 20 minutes.
4. the preparation method of rabies vaccine lyophilized formulations as claimed in claim 1, is characterized in that: the gelatin that has added phosphate buffered solution gross mass 5% in phosphate buffered solution.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103721252A (en) * | 2014-01-08 | 2014-04-16 | 天津津斯特疫苗有限责任公司 | Gelatin-free rubella vaccine freeze-dried formula and process |
| CN103736089A (en) * | 2014-01-17 | 2014-04-23 | 成都康华生物制品有限公司 | Human diploid cell cultured rabies vaccine and preparation method thereof |
| CN104353068A (en) * | 2014-11-18 | 2015-02-18 | 成都康华生物制品有限公司 | Large-scale production method of rabies vaccines by using human diploid cells |
| CN114931557A (en) * | 2022-03-18 | 2022-08-23 | 辽宁成大生物股份有限公司 | Freeze-drying protective agent, vaccine freeze-drying preparation and preparation method thereof |
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| CN1824302A (en) * | 2005-12-29 | 2006-08-30 | 中国人民解放军第三军医大学第三附属医院 | Preparation method of rabies vaccine for human and its use |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103721252A (en) * | 2014-01-08 | 2014-04-16 | 天津津斯特疫苗有限责任公司 | Gelatin-free rubella vaccine freeze-dried formula and process |
| CN103736089A (en) * | 2014-01-17 | 2014-04-23 | 成都康华生物制品有限公司 | Human diploid cell cultured rabies vaccine and preparation method thereof |
| WO2015106483A1 (en) * | 2014-01-17 | 2015-07-23 | 成都康华生物制品有限公司 | Rabies vaccine cultured from human diploid cells and method for preparing same |
| CN103736089B (en) * | 2014-01-17 | 2016-04-20 | 成都康华生物制品有限公司 | Rabies vaccine that human diploid cell is cultivated and preparation method thereof |
| CN104353068A (en) * | 2014-11-18 | 2015-02-18 | 成都康华生物制品有限公司 | Large-scale production method of rabies vaccines by using human diploid cells |
| CN114931557A (en) * | 2022-03-18 | 2022-08-23 | 辽宁成大生物股份有限公司 | Freeze-drying protective agent, vaccine freeze-drying preparation and preparation method thereof |
| CN114931557B (en) * | 2022-03-18 | 2023-07-07 | 辽宁成大生物股份有限公司 | Freeze-drying protective agent, vaccine freeze-drying preparation and preparation method thereof |
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| CN103505723B (en) | 2014-12-31 |
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Address after: 610100 No. 182 Beijing Road, national economic and Technological Development Zone, Chengdu, Sichuan Patentee after: Chengdu Hong Wah biological products Limited by Share Ltd Address before: 610100 No. 182 Beijing Road, national economic and Technological Development Zone, Chengdu, Sichuan Patentee before: Chengdu Kanghua Biological Products Co., Ltd. |