CN104436184A - Cell production method for influenza virus vaccine - Google Patents
Cell production method for influenza virus vaccine Download PDFInfo
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- CN104436184A CN104436184A CN201410684948.3A CN201410684948A CN104436184A CN 104436184 A CN104436184 A CN 104436184A CN 201410684948 A CN201410684948 A CN 201410684948A CN 104436184 A CN104436184 A CN 104436184A
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Abstract
The invention discloses a cell production method for an influenza virus vaccine. The cell production method comprises the following steps: preparing of virus-seeds, microcarrier suspension culture of a Vero cell in a bioreactor, breeding of virus liquid for preparing the vaccine, and inactivation and purification of the virus liquid. According to the invention, chick embryo tissue is replaced with cells for culturing and preparing the influenza virus vaccine, so that the problems of chick embryo pollution and exogenous virus pollution to the chick embryo are solved; through the strict control over the raw materials and the culture conditions, that the produced vaccine is pure can be ensured and the safety of the vaccine is ensured; the bioreactor is used for producing the vaccine, so that advantages of high degree of automation, labor saving and simple and stable production processes are achieved; the production cost can be reduced greatly, the raw material supply limitation does not exist, and the production cycle is short; by using the method for producing the vaccine, less environmental pollution and high easiness in treatment are achieved, and the problems of a great amount of generated waste, high difficulty in treatment, biosafety and public health of the conventional chick embryo production method are solved.
Description
Technical field
The present invention relates to influenza virus vaccine field, particularly, relate to a kind of cells produce method of influenza virus vaccine.
Background technology
traditional method preparing influenza virus vaccine uses Embryo Gallus domesticus to prepare, and domestic use chick embryo culture vaccine has had the history of more than 50 year.The influenza vaccines of current use are the influenza trivalent split vaccine of deactivation prepared of Embryo Gallus domesticus mainly, comprises influenza A virus strain, H3N2 and influenza B virus strain and large influenza vaccines.The main source of sick Seedling strain is deciding with the surface antigen of influenza virus epidemic strain for next year influenza vaccines production of being recommended according to the Changing Pattern of annual Global influenza virus by WHO, the popular street strain recommend WHO and Embryo Gallus domesticus high yield strain double infection produce reprovision or anti-hereditism's reprovision, obtain new vaccine strain, Embryo Gallus domesticus has the characteristic of high yield, but Embryo Gallus domesticus has the possibility of potential pollution, and the allergy that potential Embryo Gallus domesticus albumen causes, and cultivation cycle is long, be not easy to control output.Be unfavorable for for tackling large-scale flu outbreak; This traditional handicraft labor intensity is large, and length consuming time, efficiency are low, and production cost is high; Easily by environmental pollution; Difference between Embryo Gallus domesticus different batches is large; Be difficult to expanding production; There is quality and potential safety hazard in Embryo Gallus domesticus self is caused production vaccine by antibacterial or other virus contamination; The useless embryo quantity of producing after vaccine is many, and harmless treatment difficulty is large, relates to bio-safety and public health problem.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of cells produce method of influenza virus vaccine, there is the problem of potential safety hazard with the vaccine that the preparation method production cycle overcoming existing influenza virus vaccine is long, prepared.
The present invention's adopted technical scheme that solves the problem is: the cells produce method of influenza virus vaccine, comprises the following steps:
1) preparation of poison is planted
A) the going down to posterity and cultivation of cell: by Vero cell through pancreatin cell dispersal liquid had digestive transfer culture, the culture medium of serum-free medium continues Cultivation of Vero and makes it to form cell monolayer;
B) inoculation, the breeding of poison is planted: with cell maintenance medium, H1 or H3 provide source WHO or Type B epidemic isolates, dilution 10
-4be seeded in well-grown above-mentioned cell monolayer doubly, add the Type IX pancreatin of 1-3ug/L simultaneously, continue to cultivate, during pathological changes above to cell 50--100%, gather in the crops virus liquid; Again using this virus liquid as kind of a poison, cell carries out cell adaptation continuous passage 6 generation as seeding;
2) microcarrier suspension culture of Vero cell in bioreactor: get well-grown Vero cell, is prepared as cell suspension through the digestion of pancreatin cell dispersal liquid, is seeded in bioreactor after cell counting;
3) breeding of seedling virus liquid: when the Vero cell on the microcarrier in bioreactor covers with substantially, and above-mentioned kind poison is seeded on Vero cell after reaching 1 × more than 106/ml by cell counts, the Type IX pancreatin of 1-3ug/L is added in culture fluid after planting poison inoculation, it is 0.0001-0.1MOI that kind poison connects poison amount, get microcarrier in reactor at regular intervals, by microscope observing cell pathological changes situation, and the HA detecting supernatant tires and TCID50, cells on microcarriers to be seen substantially all comes off, and DO value is obvious ascendant trend, stopped reaction device stirs, after microcarrier all sinks to reactor bottom, results supernatant is virus liquid, described for needing cleaning before carrier use, sterilizing,
4) deactivation of virus liquid and purification: concentrate after above-mentioned virus liquid deactivation, again with little DNA and the albumen of removing cell derived with molecular sieve, use ion exchange column to remove DNA and other cell component with negative charge of cell derived, virus liquid carries out the concentrated virus liquid obtaining purification again.
Further, microcarrier is plastic plus microcarrier.Select plastic plus microcarrier as the carrier of Growth of Cells, this carrier does not meet the requirement of bio-safety containing zoogenous albumen.
Further, pancreatin is Thermo scientific HyQTase-pancreatin.Thermo scientific HyQTase-pancreatin is the pancreatin of plant origin, avoids animal-based protein.
Further, the concentrated employing 750K Da MWCO doughnut of virus liquid.750K Da MWCO doughnut efficiently can be spent virus liquid fast and be concentrated, and can avoid introducing undesirable protein sources or impurity virus.
A kind of influenza virus vaccine, described influenza virus vaccine is prepared from by the cells produce method of influenza virus vaccine.
An application for influenza virus vaccine, is applied to influenza prevention by influenza virus vaccine.
To sum up, the invention has the beneficial effects as follows:
1, the problem that the present invention cell line replaces chicken embryo tissue to cultivate manufacturing bird flu virus can solve Embryo Gallus domesticus self and pollute by exogenous virus, by the strict control of raw material and condition of culture, ensures that the vaccine produced is pure, guarantees the safety of vaccine.
2, the present invention can reduce production cost in a large number, can not be limited by raw material supply, and with short production cycle, and each production cycle only needs 5-7 days, and more than 15 days that compare existing cultivation in the chick embryo shorten greatly.
3, applying biological reactor of the present invention carries out production of vaccine, and have automaticity high, employment is few, production technology simple and stable, and the large occupied ground of easy to operate, output is little, is easy to rapid expansion production scale, and quality is easy to realize equalization stable.
4, apply this method produce vaccine, low in the pollution of the environment and be easy to process.And the refuses such as a large amount of useless embryos that existing Embryo Gallus domesticus working system can produce, and intractability is large, relates to bio-safety and public health problem.
Accompanying drawing explanation
Fig. 1 is the process chart of cell legal system for influenza vaccines.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, to the detailed description further of invention do, but embodiments of the present invention are not limited thereto.
Embodiment:
As shown in Figure 1, the cells produce method of influenza virus vaccine, is characterized in that, comprise the following steps:
1) preparation of poison is planted
A) the going down to posterity and cultivation of cell: by Vero cell through Thermo scientific HyQTase-pancreatin cell dispersal liquid had digestive transfer culture, continues to cultivate with HycloneSFM4MegaVir or suitable serum-free medium cell growth medium and forms cell monolayer;
B) inoculation, the breeding of poison is planted: with cell maintenance medium, popular then H1, the H3 that provide source WHO and Type B epidemic isolates, dilution 10
-4be seeded in well-grown above-mentioned cell monolayer, add the Type IX pancreatin of 3ug/L simultaneously, continue to cultivate, during pathological changes above to cell 50--100%, gather in the crops virus liquid; Again using this virus liquid as kind of a poison, cell carries out cell adaptation continuous passage 6 generation as seeding;
2) microcarrier suspension culture of Vero cell in bioreactor: get well-grown Vero cell, is prepared as cell suspension, after cell counting in reaction of inoculation device through Thermo scientific HyQTase-pancreatin cell dispersal liquid digestion;
3) breeding of seedling virus liquid: the Vero cell on the microcarrier in bioreactor covers with substantially, and above-mentioned kind poison is seeded on Vero cell after reaching 1 × more than 106/ml by cell counts, the Type IX pancreatin of 3ug/L is added in culture fluid after planting poison inoculation, it is 0.0001-0.1MOI that kind poison connects poison amount, get microcarrier in reactor at regular intervals, by microscope observing cell pathological changes situation, and the HA detecting supernatant tires and TCID50, cells on microcarriers to be seen substantially all comes off, and DO value is obvious ascendant trend, stopped reaction device stirs, after microcarrier all sinks to reactor bottom, results supernatant is virus liquid, described for needing cleaning before carrier use, sterilizing,
4) deactivation of virus liquid and purification: first above-mentioned virus liquid carries out deactivation with B-propiolactone, with 750K Da MWCO doughnut, results are also concentrated, with molecular sieve as the little DNA of sepharose 4 fast flow removal cell derived and albumen, use ion exchange column such as Q sepharose XL virus licensed to remove DNA and other cell component with negative charge of cell derived, with 750K Da MWCO doughnut, the concentrated virus liquid obtaining purification is carried out to virus liquid.
Described microcarrier is plastic plus microcarrier.
As mentioned above, the present invention can be realized preferably.
Claims (6)
1. the cells produce method of influenza virus vaccine, is characterized in that, comprise the following steps:
1) preparation of poison is planted
A) the going down to posterity and cultivation of cell: by Vero cell through pancreatin cell dispersal liquid had digestive transfer culture, the culture medium of serum-free medium continues Cultivation of Vero and makes it to form cell monolayer;
B) inoculation, the breeding of poison is planted: with cell maintenance medium, H1 or H3 provide source WHO or Type B epidemic isolates, dilution 10
-4be seeded in well-grown above-mentioned cell monolayer doubly, add the Type IX pancreatin of 1-3ug/L simultaneously, continue to cultivate, during pathological changes above to cell 50--100%, gather in the crops virus liquid; Again using this virus liquid as kind of a poison, cell carries out cell adaptation continuous passage 6 generation as seeding;
2) microcarrier suspension culture of Vero cell in bioreactor: get well-grown Vero cell, is prepared as cell suspension through the digestion of pancreatin cell dispersal liquid, is seeded in bioreactor after cell counting;
3) breeding of seedling virus liquid: when the Vero cell on the microcarrier in bioreactor covers with substantially, and above-mentioned kind poison is seeded on Vero cell after reaching 1 × more than 106/ml by cell counts, the Type IX pancreatin of 1-3ug/L is added in culture fluid after planting poison inoculation, it is 0.0001-0.1MOI that kind poison connects poison amount, get microcarrier in reactor at regular intervals, by microscope observing cell pathological changes situation, and the HA detecting supernatant tires and TCID50, cells on microcarriers to be seen substantially all comes off, and DO value is obvious ascendant trend, stopped reaction device stirs, after microcarrier all sinks to reactor bottom, results supernatant is virus liquid, described for needing cleaning before carrier use, sterilizing,
4) deactivation of virus liquid and purification: concentrate after above-mentioned virus liquid deactivation, again with little DNA and the albumen of removing cell derived with molecular sieve, use ion exchange column to remove DNA and other cell component with negative charge of cell derived, virus liquid carries out the concentrated virus liquid obtaining purification again.
2. the cells produce method of influenza virus vaccine according to claim 1, is characterized in that, described microcarrier is plastic plus microcarrier.
3. the cells produce method of influenza virus vaccine according to claim 1 and 2, is characterized in that, described pancreatin is Thermo scientific HyQTase-pancreatin.
4. the cells produce method of influenza virus vaccine according to claim 1 and 2, is characterized in that, the concentrated employing 750K Da MWCO doughnut of described virus liquid.
5. an influenza virus vaccine, is characterized in that, described influenza virus vaccine is prepared from by the cells produce method of influenza virus vaccine according to claim 1.
6. an application for influenza virus vaccine, is characterized in that, influenza virus vaccine as claimed in claim 2 is applied to influenza prevention.
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| CN201410684948.3A CN104436184A (en) | 2014-11-25 | 2014-11-25 | Cell production method for influenza virus vaccine |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108570445A (en) * | 2017-11-09 | 2018-09-25 | 甘肃健顺生物科技有限公司 | PK15 cells tame suspension process and second order virus production technique |
| CN108570454A (en) * | 2017-11-09 | 2018-09-25 | 甘肃健顺生物科技有限公司 | MDBK tames suspension process and second order virus production technique |
| CN108570444A (en) * | 2017-11-09 | 2018-09-25 | 甘肃健顺生物科技有限公司 | ST cells tame suspension process and second order virus production technique |
| WO2019090565A1 (en) * | 2017-11-09 | 2019-05-16 | 甘肃健顺生物科技有限公司 | Method for acclimating and suspending vero and second-order production process for virus |
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| CN102078605A (en) * | 2010-12-27 | 2011-06-01 | 吉林亚泰生物药业股份有限公司 | Method for preparing Vero cell influenza virus vaccine |
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Patent Citations (3)
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| CN102078605A (en) * | 2010-12-27 | 2011-06-01 | 吉林亚泰生物药业股份有限公司 | Method for preparing Vero cell influenza virus vaccine |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108570445A (en) * | 2017-11-09 | 2018-09-25 | 甘肃健顺生物科技有限公司 | PK15 cells tame suspension process and second order virus production technique |
| CN108570454A (en) * | 2017-11-09 | 2018-09-25 | 甘肃健顺生物科技有限公司 | MDBK tames suspension process and second order virus production technique |
| CN108570444A (en) * | 2017-11-09 | 2018-09-25 | 甘肃健顺生物科技有限公司 | ST cells tame suspension process and second order virus production technique |
| WO2019090565A1 (en) * | 2017-11-09 | 2019-05-16 | 甘肃健顺生物科技有限公司 | Method for acclimating and suspending vero and second-order production process for virus |
| US11629338B2 (en) | 2017-11-09 | 2023-04-18 | Jianshun Biosciences Co., Ltd. | Method for acclimating and suspending Vero and second order production process for virus |
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