CN102038945B - Method for industrially producing swine parvovirus vaccine by utilizing bioreactor - Google Patents
Method for industrially producing swine parvovirus vaccine by utilizing bioreactor Download PDFInfo
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- CN102038945B CN102038945B CN2010102821349A CN201010282134A CN102038945B CN 102038945 B CN102038945 B CN 102038945B CN 2010102821349 A CN2010102821349 A CN 2010102821349A CN 201010282134 A CN201010282134 A CN 201010282134A CN 102038945 B CN102038945 B CN 102038945B
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Abstract
The invention provides a method for industrially producing a swine parvovirus vaccine by utilizing a bioreactor, comprising the following steps of: (1) sterilizing a micro-carrier and the bioreactor, adding a cell growth solution, inoculating, preparing the vaccine and culturing with cells, inoculating a swine parvovirus after the cell on the micro-carrier forms a compact single layer, and continuously culturing to propagate the virus; (2) stopping culturing until the cytopathy reaches more than 80%, and harvesting a virus solution; (3) carrying out ultrafiltration concentration and virus inactivation on the harvested virus solution; and (4) purifying and inactivating the virus through a column chromatography method to prepare the vaccine. The invention has the advantages of favorable controllability of processing parameters, high cell density and virus titer, favorable vaccine safety, stable and reliable quality, high production efficiency, and the like.
Description
Technical field
The present invention relates to the veterinary biologics technical field, be specifically related to a kind of method of applying biological reaction vessel suitability for industrialized production pig parvoviral vaccine.
Background technology
(porcine parvovirus PPV) is one of principal disease of causing the sow breeding difficulty to porcine parvovirus.Farrowing sow mainly shows as miscarriage, stillborn fetus, mummy tire, weak son etc., and is the most serious with the first-born sow, causes enormous economic loss for the pig industry in the whole world.Control to pig parvoviral mainly is to put prevention first with immunity at present, because the single and high immunogenicity of PPV serotype makes vaccination become a kind of efficient ways that this disease of control infects.
Vaccine in China's extensive use is the pig parvoviral inactivated vaccine at present, and what the production of vaccine was adopted is traditional rolling bottle technology, and this technology has been used many decades in my vaccine industry, and its operating technology is simple relatively and ripe.But each rolling bottle all is cell culture units independently; The quality of every bottle of cell, viral yield and titre are all different; Cause the vaccine differences between batches big, and operation labor intensity is big, production efficiency is low; The shortcomings such as high endotoxin that recessive pollution causes, the more and more requirement of the current vaccine large-scale production of incompatibility.
Summary of the invention
The present invention provides a kind of method of applying biological reaction vessel suitability for industrialized production pig parvoviral vaccine in order to overcome the defective of the existing production method of pig parvoviral vaccine, and its advantage is that occupation area of equipment is little, production scale is big; Automatic controlling level is high, and production efficiency is good; Culture device is airtight, is difficult for polluting; By-product is few, and the side reaction of vaccine is little; The viral liquid of the producing height of tiring, vaccine quality is stable.
Technical scheme of the present invention is:
The method of applying biological reaction vessel suitability for industrialized production pig parvoviral vaccine; After it is characterized in that comprising the steps: that (1) is with microcarrier and bioreactor sterilization, add cell growth medium, the inoculation seedling is cultivated with cell; Treat that the cell on the microcarrier forms fine and close monolayer; The inoculation pig parvoviral, and continue to cultivate, make virus breeding; It is the cell responsive to the pig parvoviral strain that cell is used in described seedling, and cell is IBRS-2, PK-15 or ST cell; (2) treat that cytopathy reaches 80% when above, stop to cultivate, gather in the crops viral liquid; (3) the viral liquid to results carries out ultrafiltration and concentration and inactivation of virus; (4) process vaccine through the virus of column chromatography method purification deactivation.
Described bioreactor setup parameter is: pH 6.5～7.8,33～37 ℃ of temperature, dissolved oxygen 30～80%, mixing speed 30～100rpm.Consider the optimal condition of cell culture, preferred pH 6.9～7.3, cell culture phase temperature are set 37 ℃, 35 ℃ of Virus culture phase temperature settings, dissolved oxygen 50%, mixing speed 30～100rpm.
The inoculum concentration of said virus is 0.0001～0.1MOI.
Described microcarrier is a Cytodex series microcarrier, and the use density of microcarrier is 2～25g/L.
Described bioreactor is a stirring type bioreactor.
Described bioreactor volume is 14～150L.The training mode that seedling can adopt 14L～40L or 14L～40L～150L to amplify step by step with cell; Promptly get off through the cell dissociation of pancreatin with the 14L bioreactor culture; Be inoculated into the bioreactor of 40L as seed cell; Be inoculated into the 150L bioreactor with being used as seed cell under the cultured cells digestion in the reaction vessel of 40L again; So form the process of amplification culture step by step between the bioreactor, thereby avoided the simple defective that pollutes and increase labor intensity with rolling bottle cultivation seed cell easily; Or, directly use the bioreactor culture of 14L, 40L or 150L viral.
The cultivation of described bioreactor adopts batch formula, stream to add or the mode of perfusion cultures, dabbling speed according to the density of cell with 0.5～4 working volume every day.
The present invention adopts bioreactor microcarrier culture technique to carry out the High Density Cultivation of cell, produces the pig parvoviral vaccine, compares with traditional rolling bottle production technology, and automatic controlling level is high, and production can be monitored in real time; Save manpower, reduce cost; The production land used is few, and scale is easy to enlarge; The virus titer of producing is high, and differences between batches are little, constant product quality, and side reaction is little.
Description of drawings
Fig. 1 is a process chart of the present invention.
Fig. 2 a is the microcarrier cell picture of 4h behind the cell inoculation.
Fig. 2 b is the microcarrier cell picture that connects poison back cell state
Fig. 3 is a cell growth curve.
The specific embodiment
Below be concrete embodiment of the present invention, with further elaboration the present invention, but the scope that can not be construed as limiting the invention.
Embodiment 1
Bioreactor: U.S. 14L of NBS company and 40L bioreactor;
Microcarrier: Cytodex-1 (life sciences portion of medical treatment group of General Electric (U.S.A.));
Pig parvoviral: HW-1 strain;
Cell growth medium: contain the DMEM that volumetric concentration is 8% calf serum (Beijing Qingdatianyi Bioisystech Co., Ltd);
Virus is kept liquid: contain the DMEM that volumetric concentration is 1% calf serum (Beijing Qingdatianyi Bioisystech Co., Ltd);
Cell culture: respectively in the 14L bioreactor, add Cytodex1 according to the concentration of 10g/L, after the aquation, eluriate 2 times with the phosphate buffer PBS of pH7.2, the IBRS-2 cell is inoculated in sterilization, cultivates; The cultured method parameter is: pH7.2,37 ℃ of temperature, DO50%, mixing speed 30～100rpm; Every day, timing sampling observation of cell growing state carried out cell counting, measured the consumption of glucose, when the density of cell reaches 1.5 * 10
6During/ml, begin perfusion, dabbling speed,, was cultivated 4～5 days to keep the growth of cell with 0.5～4 working volume every day according to the consumption of the density of cell, glucose, and the density of cell reaches 7 * 10
6/ ml.
The amplification that microcarrier is cultivated: the density of treating 14L bioreactor cell reaches 7 * 10
6/ ml collects the microcarrier that covers with cell in the particular encapsulated container, uses to contain mass concentration and be twice in the PBS buffer solution for cleaning cell of the pH7.2 of 0.02%EDTA; The mass concentration that contains that adds 37 ℃ of preheatings is that 0.02%EDTA, mass concentration are the trypsinization liquid of 0.25% pancreatin; Digestion 8min discharges unnecessary pancreatin solution, adds cell growth medium; Stop the digestion of remaining pancreatin; Start the container stirring and make it fully disperse the cell that digests, will digest the bioreactor of dispersive cell suspension inoculation then, cultivate according to above-mentioned cultural method to 40L.
Virus breeding: when the density of 40L bioreactor cell reaches 8 * 10
6/ ml is replaced with virus and keeps liquid, according to 0.01MOI inoculation pig parvoviral; Meet poison back 6h and begin perfusion cultures,, treat that cytopathy reaches 80% when above, gathers in the crops viral liquid, mensuration viral level 8.0logTCID simultaneously to keep the necessary nutrient substance of virus breeding
50/ ml,, the viral liquid of results is placed 2～8 ℃, and be kept at-20 ℃.
Virus concentrates, purification, join Seedling: with the viral liquid of above-mentioned results with concentrated 10 times of the ultrafilter membrane bag of 100,000 molecular cut offs; By inactivator and viral concentrated solution volume ratio is that 1: 2000 adding mass concentration is after 37% formalin inactivator carries out deactivation, obtains purified vaccine through gel chromatography column Sepharose 4FF column chromatography again; Add oily adjuvant in antigen and 1: 1 ratio of oily adjuvant, emulsifying is mixed with inactivated vaccine.The Span-80 that is formulated as 94% (V/V) white oil, 6% (V/V) of oil adjuvant, 2% (g/V) aluminium stearate.
Embodiment 2
Bioreactor: U.S. 14L of NBS company and 40L bioreactor;
Microcarrier: Cytodex-1 (life sciences portion of medical treatment group of General Electric (U.S.A.));
Pig parvoviral: HW-1 strain;
Cell growth medium: contain the DMEM that volumetric concentration is 8% calf serum (Beijing Qingdatianyi Bioisystech Co., Ltd);
Virus is kept liquid: contain the DMEM that volumetric concentration is 1% calf serum (Beijing Qingdatianyi Bioisystech Co., Ltd);
Cell culture: respectively in the 14L bioreactor, add Cytodex-1 according to the concentration of 10g/L, after the aquation, eluriate 2 times with the phosphate buffer PBS of pH7.2, the PK-15 cell is inoculated in sterilization, cultivates; The cultured method parameter is: pH7.2,37 ℃ of temperature, DO50%, mixing speed 30～100rpm; Every day, timing sampling observation of cell growing state carried out cell counting, measured the consumption of glucose, when the density of cell reaches 1.5 * 10
6During/ml, begin perfusion, dabbling speed,, was cultivated 4 days to keep the generation of cell with 0.5～4 working volume every day according to the consumption of the density of cell, glucose, and the density of cell reaches 7 * 10
6/ ml.
The amplification that microcarrier is cultivated: the density of treating 14L bioreactor cell reaches 7 * 10
6/ ml collects the microcarrier that covers with cell in the particular encapsulated container, uses to contain mass concentration and be twice in the PBS buffer solution for cleaning cell of the pH7.2 of 0.02%EDTA; The mass concentration that contains that adds 37 ℃ of preheatings is that 0.02%EDTA, mass concentration are the trypsinization liquid of 0.25% pancreatin; Digestion 10min discharges unnecessary pancreatin solution, adds cell growth medium; Stop the digestion of remaining pancreatin; Start the container stirring and make it fully disperse the cell that digests, will digest the bioreactor of dispersive cell suspension inoculation then, cultivate according to above-mentioned cultural method to 40L.
Virus breeding: when the density of 40L bioreactor cell reaches 8 * 10
6/ ml is replaced with virus and keeps liquid, according to 0.01MOI inoculation pig parvoviral; Meet poison back 6h and begin perfusion cultures,, treat that cytopathy reaches 80% when above, gathers in the crops viral liquid, mensuration viral level 7.8logTCID simultaneously to keep the necessary nutrient substance of virus breeding
50/ ml places 2～8 ℃ with the viral liquid of gathering in the crops, and is kept at-20 ℃.
Virus concentrates, purification, join Seedling: with the viral liquid of above-mentioned results with concentrated 50 times of the ultrafilter membrane bag of 100,000 molecular cut offs; By inactivator and viral concentrated solution volume ratio is after 1: 2000 adding 37% formalin inactivator carries out deactivation, to obtain purified vaccine through gel chromatography column Sepharose 4FF column chromatography again; Add oily adjuvant in antigen and 1: 1 ratio of oily adjuvant, emulsifying is mixed with inactivated vaccine, the Span-80 that is formulated as 94% (V/V) white oil, 6% (V/V) of oily adjuvant, 2% (g/V) aluminium stearate.
Embodiment 3
Bioreactor: U.S. 14L of NBS company and 40L bioreactor;
Microcarrier: Cytodex-1 (life sciences portion of medical treatment group of General Electric (U.S.A.));
Pig parvoviral: HW-1 strain;
Cell growth medium: contain the DMEM/F12 that volumetric concentration is 8% calf serum;
Virus is kept liquid: contain the DMEM/F12 that volumetric concentration is 1% calf serum;
Cell culture: respectively in the 14L bioreactor, add Cytodex1 according to the concentration of 10g/L, after the aquation, eluriate 2 times with the phosphate buffer PBS of pH7.2, the ST cell is inoculated in sterilization, cultivates; The cultured method parameter is: pH7.2,37 ℃ of temperature, DO50%, mixing speed 30～100rpm; Every day, timing sampling observation of cell growing state carried out cell counting, measured the consumption of glucose, when the density of cell reaches 1～1.5 * 10
6During/ml, begin perfusion, dabbling speed with 0.5～4 working volume every day, is cultivated 4d according to the consumption of the density of cell, glucose, and the density of cell reaches 7 * 10
6/ ml.
The amplification that microcarrier is cultivated: the density of treating 14L bioreactor cell reaches 7 * 10
6/ ml collects the microcarrier that covers with cell in the particular encapsulated container, uses to contain mass concentration and be twice in the PBS buffer solution for cleaning cell of the pH7.2 of 0.02%EDTA; The mass concentration that contains that adds 37 ℃ of preheatings is that 0.02%EDTA, mass concentration are the trypsinization liquid of 0.25% pancreatin; Digestion 10min discharges unnecessary pancreatin solution, adds cell growth medium; Stop the digestion of remaining pancreatin; Start the container stirring and make it fully disperse the cell that digests, will digest the bioreactor of dispersive cell suspension inoculation then, cultivate according to above-mentioned cultural method to 40L.
Virus breeding: when the density of 40L bioreactor cell reaches 8 * 10
6/ ml is replaced with virus and keeps liquid, according to 0.01MOI inoculation pig parvoviral; Meet poison back 6h and begin perfusion cultures,, treat that cytopathy reaches 80% when above, gathers in the crops viral liquid, mensuration viral level 7.8logTCID simultaneously to keep the necessary nutrient substance of virus breeding
50/ ml places 2～8 ℃ with the viral liquid of gathering in the crops, and is kept at-20 ℃.
Virus concentrates, purification, join Seedling: with the viral liquid of above-mentioned results with concentrated 30 times of the ultrafilter membrane bag of 100,000 molecular cut offs; By inactivator and viral concentrated solution volume ratio is that 1: 2000 adding mass concentration is after 37% formalin inactivator carries out deactivation, obtains purified vaccine through gel chromatography column Sepharose 4FF column chromatography again; Add oily adjuvant in antigen and 1: 1 ratio of oily adjuvant, emulsifying is mixed with inactivated vaccine.The Span-80 that is formulated as 94% (V/V) white oil, 6% (V/V) of oil adjuvant, 2% (g/V) aluminium stearate.
Claims (2)
1. the method for applying biological reaction vessel suitability for industrialized production pig parvoviral vaccine; After it is characterized in that comprising the steps: that (1) is with microcarrier and bioreactor sterilization, add cell growth medium, the inoculation seedling is cultivated with cell; Treat that the cell on the microcarrier forms fine and close monolayer; The inoculation pig parvoviral, and continue to cultivate, make virus breeding; It is the cell responsive to the pig parvoviral strain that cell is used in described seedling, and cell is IBRS-2, PK-15 or ST cell; (2) treat that cytopathy reaches 80% when above, stop to cultivate, gather in the crops viral liquid; (3) the viral liquid to results carries out ultrafiltration and concentration and inactivation of virus; (4) process vaccine through the virus of column chromatography method purification deactivation.
2. the method for applying biological reaction vessel suitability for industrialized production pig parvoviral vaccine according to claim 1, it is characterized in that: described bioreactor setup parameter is: pH 6.5～7.8,33～37 ℃ of temperature, dissolved oxygen 30～80% and mixing speed 30～100rpm.
3, the method for applying biological reaction vessel suitability for industrialized production pig parvoviral vaccine according to claim 1 is characterized in that: the inoculum concentration of virus is 0.0001～0.1MOI.
4, according to the method for the described applying biological reaction vessel of one of claim 1～3 suitability for industrialized production pig parvoviral vaccine, it is characterized in that: described bioreactor volume is 14～150L; Or, seedling with cell through 14L～40L or 14L～40L～150L step by step after the amplification culture, in 40L or 150L bioreactor culture rabies virus; Or, directly use the bioreactor culture of 14L, 40L or 150L viral.
5, according to the method for the described applying biological reaction vessel of one of claim 1～3 suitability for industrialized production pig parvoviral vaccine, it is characterized in that: described microcarrier is a Cytodex series microcarrier, and the use density of microcarrier is 2～25g/L.
6, according to the method for the described applying biological reaction vessel of one of claim 1～3 suitability for industrialized production pig parvoviral vaccine, it is characterized in that: described bioreactor is a stirring type bioreactor.
7, according to the method for the described applying biological reaction vessel of one of claim 1～3 suitability for industrialized production pig parvoviral vaccine; It is characterized in that: formula is criticized in the cultivation employing of described bioreactor, stream adds or the mode of perfusion cultures, and dabbling speed is 0.5～4 working volume every day according to the density of cell.
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| CN102690791A (en) * | 2011-10-25 | 2012-09-26 | 哈药集团生物疫苗有限公司 | Method for generating porcine pseudorabies virus by culturing ST cell in microcarrier of bioreactor |
| CN102988972B (en) * | 2012-12-14 | 2014-05-28 | 山东滨州沃华生物工程有限公司 | Method for producing porcine parvovirus inactivated vaccine by using torrent bioreactor |
| CN103157106B (en) * | 2012-12-27 | 2014-07-23 | 瑞普（保定）生物药业有限公司 | Method for preparing porcine parvovirus inactivated vaccines |
| CN103877572A (en) * | 2014-03-21 | 2014-06-25 | 吉林正业生物制品股份有限公司 | Method for preparing porcine parvovirus disease inactivated vaccine |
| CN105749270A (en) * | 2016-03-03 | 2016-07-13 | 深圳康泰生物制品股份有限公司 | Rotavirus vaccine and preparation method thereof |
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| CN1970080A (en) * | 2006-12-04 | 2007-05-30 | 上海乔南生泰科学仪器有限公司 | Method for producing virus vaccine by using suspended Vero cell |
| CN101745106A (en) * | 2008-12-09 | 2010-06-23 | 上海佳牧生物制品有限公司 | Porcine parvnvirus living vaccine and preparation method thereof |
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