CN1234412C - Method for large-scale continuous production of virus vaccine - Google Patents
Method for large-scale continuous production of virus vaccine Download PDFInfo
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- CN1234412C CN1234412C CNB021120137A CN02112013A CN1234412C CN 1234412 C CN1234412 C CN 1234412C CN B021120137 A CNB021120137 A CN B021120137A CN 02112013 A CN02112013 A CN 02112013A CN 1234412 C CN1234412 C CN 1234412C
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention provides a method for large-scale continuous production of virus vaccine, which comprises the following steps: a) in a Celligen Plus R bioreactor with a fixed bed basket type agitation system, polyester chips Fibra-cel<TM> Disks are used as carriers so as to culture cells and produce viruses; b) when the cells grow to certain density, the viruses are vaccinated, and thus, the cells are affected by the viruses; c) the viruses are greatly reproduced under the proper condition; d) after purified, the viruses are harvested. The method has the advantages that the density of the cultured cells is high, the cells can be continuously cultured, the virus yield is high, etc.
Description
Invention field
The present invention relates to bioengineering field.Specifically, the present invention relates to the method for large-scale continuous production of virus vaccine.
Technical field
The existing a lot of reports of applying biological reactor large-scale culture zooblast (as CHO, BHK, 293 cells, hybridoma, insect cell etc.).Producible gene engineering product, as protein excretion type product: erythropoietin (EPO) and histiotype plasminogen (t-PA) etc. all has report.
Yet,, so utilize method that bioreactor comes the large-scale production viral vaccine also seldom now because there are very big difference in protein excretion type product and viral vaccine on culture medium prescription, production technology.With the human rabies vaccine is example: rabies are a kind of disease of natural focus, are all susceptible people beast of all homoiothermic animals that caused by the rabies property suffered from diseases altogether, in case morbidity, 100% death.According to the statistics of World Health Organization (WHO), there are every year 30,000 people to die from rabies approximately.According to the ministry of Health of China statistics, the case fatality rate of China's calendar year 2001 rabies human is in first, Category B notifiable disease first place up to 95.88%.Develop good rabies vaccine and not only can prevent rabies, also can treat rabies.The domestic traditional rabies vaccine production method of China is that the primary cell hamster kidney cell is inoculated rabies virus through after rolling flask culture, forms through concentrating then.The rabies vaccine poor quality that this production method is produced, it is low to tire, and some vaccine need add adjuvant, systemic anaphylaxis can occur after part patient's vaccination, and immunity and therapeutic effect are not very good, have many sequela.Calendar year 2001 Chinese Government prohibited production and selling with hamster kidney cell through concentrating and the rabies vaccine of fill, only allow the temporary transient refining rabies vaccine of producing behind the purification of hamster kidney cell." the temporary transient permission " is meant that country will execute clean animal relevant laws and regulations, and all animals that are used for bio-pharmaceutical must be raised in meeting the Animal House of national standard.The production cost that with the hamster kidney cell is the rabies vaccine of raw material when the time comes will improve greatly.And country to allow the another kind of type rabies vaccine of production and selling be exactly to be host's rabies vaccine with passage cell Vero cell (from the African green monkey kidney cell cultivation).At present, domestic existing part unit produces the Vero cell rabies vaccine with Rotary Machine technology, and purified refining forming.But still be in the starting stage, and show as small scale, the cost height, quality differs bigger between the product batches, and the total quality instability.The at present domestic report of producing rabies vaccine with bioreactor of not seeing as yet.
Produce rabies vaccine with bioreactor at present abroad and mainly contain two kinds of processes.(1) in maxicell culture tank (tank volume rises between 1200 liters 800 usually), as host cell, (trade name: Cytodex-1) as the cell growing carrier, every liter of culture medium can only add 3-5 gram microcarrier with spherical microcarrier with the Vero cell; Batch formula is cultivated, and reaches virus inoculation behind certain cell density.The shortcoming of this method is conspicuous: every liter of added microcarrier of culture medium very little can not continuous culture, and is huge if pollute loss in the production process, thereby the product cost extra-high-speed, and the market price is corresponding higher.(2) rise in the bioreactor with a kind of special stirring paddle (trade name: Cell-Lift Impeller) at 7.5-30 that can continuous culture, with the Vero cell is host cell, spherical microcarrier (trade name: Cytodex-1) as the cell growing carrier, every liter of culture medium can add 25 gram microcarriers, continuous culture, results continuously.Though the method is a kind of before relatively improvement arranged, will join the cell settlement system on bioreactor, and microcarrier can only reuse twice at most, equipment investment is bigger, and consumables cost is higher in the production process.
Therefore, still need the method for the lower large-scale continuous production of a kind of cost such as viral vaccines such as rabies vaccine in this area.
Summary of the invention
Purpose of the present invention just provides the method for the lower large-scale continuous production of virus vaccine of a kind of cost.
The present invention relates to a kind of method of large-scale continuous production of virus vaccine, it is characterized in that, this method comprises the following steps:
A) in having the Celligen Plus bioreactor of the basket stirring system of fixed bed, with polyester slice Fibra-Cel
TMDisks is a carrier, cultivates the cell that is used to produce virus;
B) when cell grows to certain density, inoculate described virus, make described virus infected cell;
C) make virus multiplication;
D) purification results virus.
In the method for the invention, used Celligen Plus bioreactor is available from New BrunswickScientific Co., the bioreactor of Inc. company.The basket culture systems of fixed bed (commodity are called the basket stirring system of fixed bed (Fibrous-Bed Basket System)) is housed in this bioreactor, this culture systems is a kind of stirring system that is used for cell culture, its characteristics are: the shearing force that forms when a) stirring is very low, especially almost nil in the shearing force that acts between the 50-120rpm on the cell; B) design has special gas return path, in the cultured cell process, need to feed all gases to bioreactor, air for example, oxygen, the bubble that is produced when carbon dioxide and nitrogen can be along specific loop turnover, and can as other culture systems, not allow cell be attached to bubble surface, when bubble rose to liquid level and breaks, cell was also dead thereupon; C) be unique cell culture system that uses the polyester slice carrier.
It is the bioreactor of 5.0 liters and 7.5 liters that the inventive method can adopt cumulative volume, but those skilled in the art can expect after the description of having read this description, and the bioreactor that volume is bigger also is applicable to the inventive method.
The carrier that adopts in the inventive method is the polyester slice carrier of being produced by Britain BIBBY STERILIN COMPANY (Fibra-Cel Disks).This carrier components is 50% polyester nonwoven fiber and 50% polypropylene foil.Through finding that this polyester slice carrier can provide the cell growing space in particular environment.
The cell (that is, host cell) that is used for producing viral vaccine in the present invention can be those zooblasts well-known to those skilled in the art, Chinese hamster ovary celI for example, bhk cell, 293 cells, Vero cell etc.
In a preferable embodiment of the present invention, should adopt the Vero cell as the virus production cell.Because the Vero cell is the wide spectrum host cell, behind the inoculation rabies virus, can produce rabies vaccine; The inoculation hemorrhagic fever virus can be produced hemorrhagic fever vaccine; In like manner, also can produce Vaccinum Encephalitidis Epidemicae, children's's poliomyelitis vaccine, viral vaccines such as Rotavirus Vaccine.Therefore estimate the present invention is widely used in the production of above-mentioned vaccine and other various vaccines.
Therefore, in a preferable embodiment of the present invention, described vaccine is selected from viral vaccines such as rabies vaccine, hemorrhagic fever vaccine, Vaccinum Encephalitidis Epidemicae, children's's poliomyelitis vaccine and Rotavirus Vaccine.Better vaccine is rabies vaccine, hemorrhagic fever vaccine and Vaccinum Encephalitidis Epidemicae.
In the method for the invention, at first be in above-mentioned bioreactor, in the presence of above-mentioned polyester slice,, and then inoculate the extremely certain density of the cell culture of producing viral usefulness.The time of virus inoculation is one of principal element that influences vaccine output.In the present invention, cell density can be 1.0 * 10 during virus inoculation
7-2.0 * 10
8Between/the milliliter.In preferable embodiment, the cell density during virus inoculation is 1.0-1.4 * 10
8/ milliliter.In a good especially embodiment, the cell density during virus inoculation is every milliliter 1.2 * 10
8About individual cell (just cell was cultivated in the reactor about 5-7 days).
In a preferable embodiment, behind virus inoculation, should under the stirring at low speed condition, cultivate, so that the viral infection host cell.Preferable, should under the mixing speed of 50-100rpm (better at 60-80rpm), cultivate.
It is can continuous culture that the inventive method is contrasted one of different characteristics of prior art.Virus is constantly bred in host cell (as the Vero cell) and is discharged in the culture supernatant in culture of continuous cultivation, is discharged to outside the reactor by peristaltic pump; Meanwhile, another peristaltic pump then adds fresh culture in the reactor, so that the nutrition that cell generates and virus multiplication is consumed to be provided.On the other hand, the amount (groundwater increment) of the culture medium that add every day can not be very few, generally need make the glucose content in the culture medium remain on 2g/L above (usually at the 2-3 grams per liter, more generally at the 2-2.5 grams per liter), so just can keep the virus of high yield.Simultaneously, if mixing speed is too low, then output will greatly reduce even cause all cells death.
The inventive method can be divided into three phases: 1) host cell growth stage, 2) the viral infection stage and 3) virus multiplication and results stage.Used culture medium composition, condition of culture (as temperature, pH, dissolved oxygen) etc. are not difficult to be determined according to used host cell type by those of ordinary skills in this three phases.Yet, it should be noted that the temperature in virus multiplication stage and temperature and the pH that pH should be lower than preceding two stages.For example, for produce rabies vaccine with the Vero cell for, be preferably 7.2-7.5 (preferable is 7.3) in the host cell growth stage and the pH in viral infection stage, temperature should be about 37 ℃; And produce the stage in virus, and pH should be 7.0-7.2 (preferable be 7.1), temperature should be between 32-35 ℃ (preferable is 34 ℃).
Behind certain hour behind the viral infection, just can begin to gather in the crops continuously the virus in the culture supernatant.Virus titer can be measured with conventional means (as the injected in mice method).
Compared with prior art, the method for large-scale continuous production of virus vaccine of the present invention has cultured cells density height, can gather in the crops continuously and the advantage of output height (average titer height).It is reported, produce in the method for rabies vaccine utilizing the Rotary Machine Cultivation of Vero, 300 milliliters of supernatant of the each results of each rolling bottle, each cycle every bottle of results 2-3 time, harvest cycle is 9 days, pollution rate 3%, putting the frame rate is 90%, is used to produce malicious Rotary Machine and approximately can only accounts for 60% of whole Rotary Machine quantity.And the present invention is by using 150 gram polyester supports, be equivalent to 150 internal surface areas and be 1200 square centimeters rolling bottle, but continuous culture can be gathered in the crops 7000 milliliters of supernatants every day, and the continuous culture time be 20 days, harvest time, pollution rate was zero.Cell density in the prior art during virus inoculation only is 1 * 10
6Cells/ml, and the cell density during the inventive method virus inoculation can reach 1 * 10
8Cells/ml.Comprehensive comparable factorial analysis contrast, the Rotary Machine method has improved 100 times in the production ratio prior art of the inventive method.
Specific embodiments
Providing following embodiment purpose is to illustrate the present invention, limits the scope of the invention but can not be construed to.
The large-scale production of embodiment 1 rabies vaccine
In the present embodiment, the capital equipment of employing is 5.0 liters of bioreactors (trade names: CELLIGEN PLUS
, available from U.S. NEW BRUNSWICK SCIENTIFIC CO., INC.).The tank body of this bioreactor is a jacket type high temperature high voltage resistant glass tank body, 5 liters of cumulative volumes, 3.5 liters of working volumes.The stirring system that this bioreactor adopts is the basket stirring system (Fibrous of fixed bed
TM-Bed Basket System).
Used culture medium is M199 culture medium or DMEM culture medium in the present embodiment.Used other reagent comprises hyclone, pancreatin, human serum albumin, FeSO
4, AlCl
3, ZnSO
4Be analytical reagent, and filter membrane, filter element, silica gel tube etc. are the cell culture common used material.
1 Vero cell of recovery from the seed cell storehouse (the wide spectrum host cell of being cultivated by African green monkey kidney cell that goes down to posterity is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute) went down to posterity, and used 175cm in 48 hours
2Square vase increases 30 bottles, makes total cellular score reach 1-2 * 10
9Cell.
In the bioreactor tank body, add 150 gram polyester slice (Fibra-Cel
TMDisks available from NEWBRUNSWICK SCIENTIFIC CO., INC) as carrier, and adds 3.5 liters of phosphate buffer PBS (pH7.4), sterilizes 30 minutes for 121 ℃.
After the sterilization, the emptying phosphate buffer.Adding Vero cell growth medium (M199+5%FBS (hyclone)), and the inoculation seed cell, is that 40-60%, mixing speed are to cultivate under the 90-120rpm at 37 ℃, pH7.2-7.4, dissolved oxygen.Maximum perfusion rate is that 8-10 rises culture medium/sky (making the glucose content in the culture medium remain on the 1-2 grams per liter).
After cultured cell 5-7 days, growth medium is all extracted out, (M199 contains 1% (W/V) human serum albumin, 1.5 grams per liter glucoses, 0.5 mg/litre FeSO to change virus production phase culture medium
4, 0.3 mg/litre AlCl
3, 0.195 mg/litre ZnSO
4, pH7.3).Infection multiplicity (MOI) inoculation aG rabies virus strain (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute) with 2-10.At 37 ℃, pH7.2-7.4, dissolved oxygen is that 40-60%, mixing speed are to cultivate 6-8 hour under the 60-80rpm, makes virus infected cell.
Then, be that 40-60%, mixing speed are to cultivate under the 120rpm at 32-35 ℃, pH7.0-7.2, dissolved oxygen, and recover the rate of flooding before the virus inoculation, make virus propagation in a large number in cell., begin to gather in the crops the supernatant that contains virus, and be used for purification after 36 hours at viral infection.
Supernatant harvest yield be 5-8 rise culture medium/sky (make glucose content in the culture medium remain on 2 grams per liters or more than), so can keep continuous harvest time more than 20 days.Total yield is 140 liters of viral supernatants.Record virus titer with the injected in mice method and can reach 9LgLD
50/ milliliter (logarithm of half lethal dose).
The production of embodiment 2 hemorrhagic fever virus vaccines
Adopt step substantially the same manner as Example 1 to carry out present embodiment, different is that the employing volume is 7.5 liters a bioreactor, and gives Vero cell inoculation hemorrhagic fever virus vaccine.The pH in virus production stage is 7.0, and temperature is 33 ℃.It is 250 liters of viral supernatants that result's results obtain.Record virus titer at 8LgLD with direct fluorescent antibody technique
50About/milliliter.
Though above according to some concrete preferable embodiments the present invention has been made detailed description, should think that the present invention is not limited to these specific embodiments.Those skilled in the art are reading behind the foregoing description and can do any change to the present invention under the condition that does not break away from the claims scope.
Claims (7)
1. the method for a large-scale continuous production of virus vaccine is characterized in that, this method comprises the following steps:
A) in having the Celligen Plus bioreactor of the basket stirring system of fixed bed, with polyester slice Fibra-Cel
TMDisks is a carrier, cultivates the cell that is used to produce virus;
B) when cell grows to certain density, inoculate described virus, make described virus infected cell;
C) make virus multiplication;
D) purification results virus.
2. method according to claim 1 is characterized in that, described viral vaccine is selected from rabies vaccine, hemorrhagic fever vaccine, Vaccinum Encephalitidis Epidemicae, children's's poliomyelitis vaccine and Rotavirus Vaccine.
3. method according to claim 1 is characterized in that, the cell density during described virus inoculation is 1.0 * 10
7-2.0 * 10
8/ milliliter.
4. method according to claim 1 is characterized in that, in step b), making viral low whipping speed is that 50-100rpm infects described cell down.
5. method according to claim 1 is characterized in that, the described cell that is used to produce virus is selected from Chinese hamster ovary celI, bhk cell, 293 cells and Vero cell.
6. method according to claim 5 is characterized in that, the described cell that is used to produce virus is the Vero cell.
7. method according to claim 1 is characterized in that, keeps in the culture medium glucose content at the 2-2.5 grams per liter during virus multiplication.
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| CNB021120137A CN1234412C (en) | 2002-06-10 | 2002-06-10 | Method for large-scale continuous production of virus vaccine |
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|---|---|---|---|
| CNB021120137A CN1234412C (en) | 2002-06-10 | 2002-06-10 | Method for large-scale continuous production of virus vaccine |
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| CN1234412C true CN1234412C (en) | 2006-01-04 |
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| CN102140477A (en) * | 2010-02-01 | 2011-08-03 | 上海比昂生物医药科技有限公司 | Method for producing viral vectors |
| 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 |
| CN102154220B (en) * | 2010-12-30 | 2012-12-26 | 浙江诺倍威生物技术有限公司 | Method and equipment for ultrahigh-density and large-scale production of porcine reproductive and respiratory syndrome virus (PRRSV) |
| 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 |
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| CN106047821B (en) * | 2016-05-17 | 2019-07-09 | 丽珠集团疫苗工程股份有限公司 | A method of utilizing bioreactor large-scale production Rotavirus Vaccine |
| CN109055321A (en) * | 2018-08-27 | 2018-12-21 | 武汉博沃生物科技有限公司 | A kind of cultural method of rotavirus |
| CN111793611A (en) * | 2020-07-14 | 2020-10-20 | 安徽安科生物工程（集团）股份有限公司 | Process optimization method for culturing oncolytic adenovirus by bioreactor tank flow |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101928693A (en) * | 2010-06-25 | 2010-12-29 | 辽宁安迪生物技术有限公司 | Method for culturing primary cells |
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