CN101062411A - Method for producing influenza vaccine in large-scale by using bioreactor - Google Patents
Method for producing influenza vaccine in large-scale by using bioreactor Download PDFInfo
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- CN101062411A CN101062411A CN 200610011773 CN200610011773A CN101062411A CN 101062411 A CN101062411 A CN 101062411A CN 200610011773 CN200610011773 CN 200610011773 CN 200610011773 A CN200610011773 A CN 200610011773A CN 101062411 A CN101062411 A CN 101062411A
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Abstract
The invention discloses a method to mass produce flu vaccine with bioreactor, which comprises the following steps: proceeding passage adaptation on Africa green monkey kidney cell (Vero cell) with influenza virus; intensifying intrinsic appeal of influenza virus for Vero cell; fitting for Vero cell quickly; breeding the Vero cell with cell density above 10. 7deka/ml in bioreactor; seeding the influenza virus into bioreactor; adjusting various controlling parameter in the reactor; making the virus high effective breed in the reactor; proceeding continuous perfusion; harvesting virus liquid; deactivating the virus; condensing; purifying; cracking; getting the flu vaccine. This invention possesses the advantages of high antigen quantity and stable quality.
Description
Technical field
The present invention relates to a kind of method of producing influenza vaccine in large-scale by using bioreactor.
Background technology
The influenza vaccines that produce that get the Green Light at present are still the inactivated vaccine that Embryo Gallus domesticus is originated, but still there are many deficiencies in it, as, usually cause the strain variation after the Embryo Gallus domesticus continuous passage; Might there be the pollution of the exogenous virus factor in Embryo Gallus domesticus; Produce the outburst that influenza vaccines can not be satisfied needs, especially bird flu under the case of emergency with Embryo Gallus domesticus, also can influence the regular supply of Embryo Gallus domesticus.Therefore, the method for countries in the world searching to the cell strain producing influenza vaccine in large-scale of influenza virus sensitivity urged by World Health Organization (WHO).China Patent No. is the method that 02112013.7 patent of invention description discloses a kind of large-scale continuous production of virus vaccine, this method comprises the following steps: a) at the Celligen Plus with the basket stirring system of fixed bed 8. in the bioreactor, with polyester slice Fibra-CelTMDisks is carrier, and cultured cell is produced virus; B) when cell grows to certain density, inoculate described virus, make described virus infected cell; C) virus is bred in a large number under appropriate condition; D) purified back gathers in the crops virus.This is a kind of method of utilizing cell culture technology to produce viral vaccine, but has cultured cells density high continuous culture and viral yield advantages of higher, has realized the purpose of suitability for industrialized production vaccine.Because in the cell culture technology, virus is adsorbed onto on the cell and breeds, above-mentioned mad dog, hemorrhagic fever, encephalitis b, children's's poliovirus is adsorbed on the cell easily, so said method is as the rabies vaccine that is suitable for that is proposed in the literary composition, hemorrhagic fever vaccine, Vaccinum Encephalitidis Epidemicae, the preparation of children's's poliomyelitis vaccine and Rotavirus Vaccine, and influenza virus to produce influenza vaccines without the method that proposed in the above-mentioned patent of the cell adapted employing of Vero be difficult, this just so far influenza vaccines still by the Embryo Gallus domesticus preparation and reason that can not producing influenza vaccine in large-scale by using bioreactor.
Summary of the invention
The objective of the invention is to propose a kind of method that adapts to the producing influenza vaccine in large-scale by using bioreactor of suitability for industrialized production.
The present invention takes following method:
With influenza then epidemic isolates on the Vero cell, adapt to and go down to posterity, be stored in below-70 ℃ stand-by through the viral seed culture of viruses of adaptation of virus; In bioreactor, add microcarrier, inoculation Vero cell, treat to inoculate when the Vero cell grows to certain density above-mentioned influenza seed culture of viruses, virus is bred under appropriate condition in a large number, with the viral liquid of results through deactivation, concentrate, purification and cracking, hemagglutinin content according to the unit price stock solution of purification is mixed with trivalent influenza vaccines semi-finished product.
The adaptation of above-mentioned influenza pandemic strain on the Vero cell gone down to posterity and taked following measure: place the cultivation vessel to go down to posterity with EDTA or trypsinization cell Vero, after cell grows up to monolayer, outwell growth-promoting media, use Hank ' s liquid or PBS that cell surface is cleaned 1～2 time, inoculation influenza seed culture of viruses, add and to contain certain density trypticly cultivate after keeping liquid, with cell freeze thawing 1～2 time, continue to go down to posterity with quadrat method before the results by above-mentioned.
The bioreactor that is adopted is the basket stirred reactor of fixed bed, and carrier is a chip carrier, and its composition is a polyester fiber.
The bioreactor that is adopted is the suspension culture reactor, and microcarrier is a dextran polymer.
The present invention adopts the influenza seed culture of viruses is carried out adaptation of virus on African green monkey kidney cell (Vero cell), under the effect of the decomposition agent of debita spissitudo, influenza virus strengthens Vero cell infection ability, and very fast adaptation Vero cell, will be inoculated at bioreactor at the cell adapted influenza seed culture of viruses of Vero then and breed to 10
7In the Vero cell of individual/above cell density of ml, the various control parameters of conditioned reaction device, as mixing speed, pH value, D0 value, perfusion rate etc., make virus colleges and universities' breeding in the bioreactor of high-cell density, and continuous perfusion, gather in the crops viral liquid, carry out then virus deactivation, concentrate, steps such as purification, cracking make influenza vaccines.
Producing influenza vaccine in large-scale by using bioreactor of the present invention, solved adopt in the past Embryo Gallus domesticus produce the existing exogenous virus of influenza vaccines pollute be difficult to control, pay that reaction is big, cost is high, production scale is difficult for the shortcoming that enlarges, it has safe and effective, antigen amount height, steady quality, be easy to advantage such as expansion scale.
The specific embodiment
Embodiment 1
The preparation method of influenza vaccines is as follows:
Influenza vaccines preparation in, what at first will solve is that influenza virus adapts to the Vero cell, this is the key of this preparation method, take following steps: Vero cell of recovery from the seed cell storehouse, the cell adding is had in the cell culture vessel of growth-promoting media, growth-promoting media is MEM or M199+8% Ox blood serum PH7.0～7.2, after 37 ℃ of cultivations grew up to monolayer in 48 hours, outwell growth-promoting media, use Hank ' s liquid that cell surface is cleaned 1～2 time, Ox blood serum remaining in the growth-promoting media is cleaned (, can influence duplicating of influenza virus), then inoculated the influenza seed culture of viruses owing to contain the non-specific inhibin of influenza virus in the Ox blood serum, put 33～35 ℃ of absorption 1-2 hour, add then and keep liquid, keep liquid and take serum-free medium (containing 1～5 μ g/ml trypsin), adding tryptic purpose is in order to improve the replication capacity of influenza virus in cell, cultivate three days results through 33-35 ℃, with cell freeze thawing 1-2 time, cell rupture virus discharges, and can improve the virus titer of results liquid before the results.In the same way the influenza seed culture of viruses is continued to go down to posterity, withhold through 6～8 and obtain, the viral hemoagglutination titre is stabilized to 1: 320, seed culture of viruses is stored in below-70 ℃ stand-by.The virus that obtains by said method has adapted to the Vero cell.
The used capital equipment of industrialization continuous production influenza vaccines is available from 14 liters of bioreactors of U.S. NBS company.The stirring system that this bioreactor adopts is the basket stirring system of fixed bed (FibrousTM-Bed Basket system), and tank body is the high temperature resistant pressure glass of a jacket type tank body.10 liters of working volumes.
In the tank body of 14 liters of bioreactors, add 400 gram chip carriers, chip carrier is produced by U.S. NBS company, its composition is a polyester slice, in above-mentioned tank body, add 10 liters of phosphate buffer PBS (pH7.3) simultaneously, sterilized 1 hour for 121 ℃, after the sterilization, emptying PBS, add Vero cell growth medium (DMEM+8% calf serum), 37 ℃ of preheatings 30 minutes.Inoculate 27 bottles of (3L cell bottle) cells in tank body, adjust the retort parameter, mixing speed is 60-90rpm, dissolved oxygen 50-80%, pH7.0-7.2, press glucose content whole growth-promoting media in the drain tank after the continuous perfusion 5-6 of 0.8-1.8g/L days, and after using Hank ' s liquid that tank body and carrier are washed 3 times, add 10L and keep liquid (containing 1～5 μ g/ml trypsin), inoculate above-mentioned influenza seed culture of viruses simultaneously, conditioned reaction jar parameter (34 ℃ of temperature, mixing speed 60-90rpm, dissolved oxygen 50-80% pH7.4-7.8), carries out cultivation and the results of influenza virus at 0.8-1.8g/L continuous irrigation stream by glucose content after 24 hours.
The viral liquid of results is added 1/4000 formaldehyde, and 20-22 ℃ of deactivation 96 hours concentrates 30-50 doubly with 100,000 molecular weight ultrafilter membranes then, and usefulness sucrose density gradient centrifugation method purification carries out cracking with 5%Triton * 100.
According to the hemagglutinin content of the unit price stock solution of purification, by final 30 μ g/ agent. hypotype is mixed with the influenza vaccines semi-finished product.
Embodiment 2
Bioreactor adopts the suspension culture reactor, and carrier is a dextran polymer, and operating procedure is basic identical with embodiment 1.
Claims (4)
1, a kind of method of producing influenza vaccine in large-scale by using bioreactor is characterized in that: with influenza then epidemic isolates on the Vero cell, adapt to and go down to posterity, be stored in below-70 ℃ stand-by through the viral seed culture of viruses of adaptation of virus; In bioreactor, add microcarrier, inoculation Vero cell, treat to inoculate when the Vero cell grows to certain density above-mentioned influenza seed culture of viruses, virus is bred under appropriate condition in a large number, with the viral liquid of results through deactivation, concentrate, purification and cracking, hemagglutinin content according to the unit price stock solution of purification is mixed with trivalent influenza vaccines semi-finished product.
2, the method for producing influenza vaccine in large-scale by using bioreactor according to claim 1, it is characterized in that: the adaptation of above-mentioned influenza pandemic strain on the Vero cell gone down to posterity and taked following measure: place the cultivation vessel to go down to posterity with EDTA or trypsinization cell Vero, after cell grows up to monolayer, outwell growth-promoting media, use Hank ' s liquid or PBS that cell surface is cleaned 1～2 time, inoculation influenza seed culture of viruses, add and keep the cultivation of liquid (containing certain density trypsin) back, with cell freeze thawing 1～2 time, continue to go down to posterity with quadrat method before the results by above-mentioned.
3, the method for producing influenza vaccine in large-scale by using bioreactor according to claim 1 is characterized in that: bioreactor is the basket stirred reactor of fixed bed, and carrier is a chip carrier, and its composition is a polyester fiber.
4, the method for producing influenza vaccine in large-scale by using bioreactor according to claim 1 is characterized in that: bioreactor is the suspension culture reactor, and microcarrier is a dextran polymer.
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127525A (en) * | 2010-12-27 | 2011-07-20 | 吉林亚泰生物药业股份有限公司 | Adaption method of influenza virus vaccine strains on Vero cells |
| CN102154220A (en) * | 2010-12-30 | 2011-08-17 | 浙江易邦生物技术有限公司 | Method and equipment for ultrahigh-density and large-scale production of porcine reproductive and respiratory syndrome virus (PRRSV) |
| CN102205116A (en) * | 2010-03-29 | 2011-10-05 | 杭州安普生物工程有限公司 | Method for producing vaccine by virtue of culturing animal cells |
| CN102268411A (en) * | 2011-08-15 | 2011-12-07 | 江苏省农业科学院 | Method for IBDV (Infectious Bursal Disease Virus) serum-free microcarrier suspension culture proliferation |
| CN102327609A (en) * | 2010-08-27 | 2012-01-25 | 丽珠集团疫苗工程股份有限公司 | Production method of encephalitis B vaccine |
| CN102453699A (en) * | 2010-10-18 | 2012-05-16 | 北京清大天一科技有限公司 | Method for suspension culture of sensitive cells and method for producing blue ear disease vaccine by using sensitive cells |
| CN102526720A (en) * | 2012-01-11 | 2012-07-04 | 中国人民解放军军事医学科学院微生物流行病研究所 | Preparation method of influenza virus vaccine |
| CN102653728A (en) * | 2011-03-04 | 2012-09-05 | 北京清大天一科技有限公司 | Method for gradually culturing animal cells in scale-up manner by using bioreactor and micro-carrier |
| CN103773741A (en) * | 2012-10-18 | 2014-05-07 | 辽宁成大生物股份有限公司 | Method for preparing influenza vaccine by using cage type ventilating and stirring bioreactor |
| CN104027800A (en) * | 2014-06-19 | 2014-09-10 | 山东亦度生物技术有限公司 | Method for preparing rabies vaccines for human use |
| CN104436185A (en) * | 2014-11-25 | 2015-03-25 | 成都威尔诺生物科技有限公司 | Method for preparing influenza vaccine |
| CN104436184A (en) * | 2014-11-25 | 2015-03-25 | 成都威尔诺生物科技有限公司 | Cell production method for influenza virus vaccine |
| CN110106138A (en) * | 2019-05-17 | 2019-08-09 | 北京生物制品研究所有限责任公司 | A method of carrying out basket reactor inner cell digestion |
| CN112795530A (en) * | 2021-02-18 | 2021-05-14 | 武汉科前生物股份有限公司 | Method for culturing canine distemper virus and canine parvovirus and application |
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2006
- 2006-04-24 CN CN 200610011773 patent/CN101062411A/en active Pending
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102205116A (en) * | 2010-03-29 | 2011-10-05 | 杭州安普生物工程有限公司 | Method for producing vaccine by virtue of culturing animal cells |
| CN102327609A (en) * | 2010-08-27 | 2012-01-25 | 丽珠集团疫苗工程股份有限公司 | Production method of encephalitis B vaccine |
| CN102453699A (en) * | 2010-10-18 | 2012-05-16 | 北京清大天一科技有限公司 | Method for suspension culture of sensitive cells and method for producing blue ear disease vaccine by using sensitive cells |
| CN102127525B (en) * | 2010-12-27 | 2013-06-05 | 吉林亚泰生物药业股份有限公司 | Adaption method of influenza virus vaccine strains on Vero cells |
| CN102127525A (en) * | 2010-12-27 | 2011-07-20 | 吉林亚泰生物药业股份有限公司 | Adaption method of influenza virus vaccine strains on Vero cells |
| CN102154220A (en) * | 2010-12-30 | 2011-08-17 | 浙江易邦生物技术有限公司 | Method and equipment for ultrahigh-density and large-scale production of porcine reproductive and respiratory syndrome virus (PRRSV) |
| CN102653728A (en) * | 2011-03-04 | 2012-09-05 | 北京清大天一科技有限公司 | Method for gradually culturing animal cells in scale-up manner by using bioreactor and micro-carrier |
| CN102268411A (en) * | 2011-08-15 | 2011-12-07 | 江苏省农业科学院 | Method for IBDV (Infectious Bursal Disease Virus) serum-free microcarrier suspension culture proliferation |
| CN102526720A (en) * | 2012-01-11 | 2012-07-04 | 中国人民解放军军事医学科学院微生物流行病研究所 | Preparation method of influenza virus vaccine |
| CN103773741A (en) * | 2012-10-18 | 2014-05-07 | 辽宁成大生物股份有限公司 | Method for preparing influenza vaccine by using cage type ventilating and stirring bioreactor |
| CN103773741B (en) * | 2012-10-18 | 2016-07-06 | 辽宁成大生物股份有限公司 | The method that cage air agitation bioreactor prepares influenza vaccines |
| CN104027800A (en) * | 2014-06-19 | 2014-09-10 | 山东亦度生物技术有限公司 | Method for preparing rabies vaccines for human use |
| CN104436185A (en) * | 2014-11-25 | 2015-03-25 | 成都威尔诺生物科技有限公司 | Method for preparing influenza vaccine |
| CN104436184A (en) * | 2014-11-25 | 2015-03-25 | 成都威尔诺生物科技有限公司 | Cell production method for influenza virus vaccine |
| CN110106138A (en) * | 2019-05-17 | 2019-08-09 | 北京生物制品研究所有限责任公司 | A method of carrying out basket reactor inner cell digestion |
| CN112795530A (en) * | 2021-02-18 | 2021-05-14 | 武汉科前生物股份有限公司 | Method for culturing canine distemper virus and canine parvovirus and application |
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