CN102526720A - Preparation method of influenza virus vaccine - Google Patents
Preparation method of influenza virus vaccine Download PDFInfo
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- CN102526720A CN102526720A CN2012100076872A CN201210007687A CN102526720A CN 102526720 A CN102526720 A CN 102526720A CN 2012100076872 A CN2012100076872 A CN 2012100076872A CN 201210007687 A CN201210007687 A CN 201210007687A CN 102526720 A CN102526720 A CN 102526720A
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Abstract
The invention discloses a preparation method of influenza virus vaccine and belongs to the technical field of vaccine preparation. The method comprises the steps that: firstly, mammal cells are subjected to monocloning, a monoclonal cell bank is built, then, monoclonal cell strains adapted by influenza viruses are sieved, next, the virus adaptability cell strain virus culture condition is optimized, finally, virus adaptability cell strains are cultured in a bioreactor, influenza viruses are inoculated when the cells grow to a certain density, the viruses are cultured through referring to the optimized virus culture condition, the viruses are subjected to concentration, purification, inactivation and cracking after being harvested, the hemagglutinin content is measured, and the influenza virus vaccine is prepared. The method disclosed by the invention has the advantages that the virus yield is greatly improved, and safety, validity and stability are realized.
Description
Technical field
The invention belongs to the vaccine production technical field, be specifically related to a kind of method for preparing of influenza virus vaccine.
Background technology
Influenza infection causes acute respiratory infectious disease-influenza, causes serious harm for human health and social life.Influenza virus type is many, variation is fast, and when new subtype virus occurred, crowd's specific immunity often was difficult to prove effective.
Inoculation epidemic strain vaccine is that flu-prevention is popular, most economical, the effective means of protection Susceptible population.The influenza vaccines that produce that get the Green Light at present mainly are the inactivated vaccines in Embryo Gallus domesticus source, and there are many deficiencies in this vaccine, and are high like production cost, the cycle is long; Anaphylaxis; The Embryo Gallus domesticus continuous passage can cause the virus antigenicity variation, and also there is the pollution of possible exogenous factor etc. in Embryo Gallus domesticus, if particularly bird flu outburst; Chicken crowd mortality, Embryo Gallus domesticus supply and output can become bottleneck.Therefore, the preparation of the influenza virus vaccine of various countries' development mammalian cell cultivation is actively recommended by World Health Organization (WHO).
At present, multiple mammalian cell is applied to the production of some viral vaccines, and generally acknowledge in the Vero cell whole world, the mdck cell U.S., Europe approval.And mdck cell is acknowledged as the only cell line of cultivation influenza virus; This cell is more responsive to influenza virus than other cells known today; Short time can obtain the virus of higher titre; The stability that keeps influenza antigen simultaneously, but mdck cell is not all-round sensitivity for the influenza vaccines Strain that annual WHO issues, and make the virus titer of producing still have the uncertain bottleneck that has become production of vaccine.It is different (Govorkova EA, Murti G, Meignier B with quantity that the type of the influenza virus receptor of bibliographical information mammalian cell of the same race surface existence is arranged; Taisne C DE, Webster RG.African Green Monkey Kidney (Vero) Cells Provide an AlternativeHost Cell System for Influenza A and B Viruses.J Virol, 1996; 70 (8) 5519-5524.), therefore, set up the cell monoclonal storehouse; The responsive monoclonal strain of screening virus; Obtain viral adaptability cell monoclonal, improve viral yield, can be the suitability for industrialized production influenza vaccines and lay the foundation.
Summary of the invention
The object of the present invention is to provide a kind of method for preparing of influenza virus vaccine, improve influenza virus output, make it be applicable to suitability for industrialized production.
A kind of method for preparing of influenza virus vaccine, carry out according to following steps:
(1) foundation in mammalian cell monoclonal storehouse
Mammalian cell is cultivated in culture bottle, treated that cell grows to degree of converging 70-90%, with trypsinization that contains EDTA and counting; Get 100 cell dilutions to the 20ml cell culture fluid, mixing is inoculated in 96 orifice plates; Every hole 200 μ l put in 37 ℃ of cell culture incubators and cultivate, and treat that cell grows to 40-60% degree of converging; Be transferred to after the digestion and continue in 24 orifice plates to cultivate; Each strain monoclonal cell transfer to a hole digests behind the 85-95% degree of converging to be grown to, is transferred in two 24 holes and continues to cultivate, and 85-95% degree of converging to be grown to carries out cell frozen;
(2) screening of viral high-adaptability cell monoclonal strain
Get monoclonal cell and be seeded to 24 orifice plates, overnight incubation is inhaled and is abandoned culture fluid, and PBS washes 2-6 time, adds influenza virus and TPCK-Trypsin, measures the blood clotting titre in the cells and supernatant behind 37 ℃ of cultivation 48-90h, and the blood clotting titre is greater than 2
8Be the strain of viral high-adaptability cell monoclonal;
(3) optimize the Virus culture condition
Get viral high-adaptability cell monoclonal strain shop to 24 orifice plates; Overnight incubation; Optimize temperature, the harvest time of Virus culture, amount and the MOI of infestation index of interpolation TPCK-Trypsin respectively, obtain virus optimum growh parameter in the strain of viral high-adaptability cell monoclonal;
(4) in bioreactor, cultivate the strain of viral high-adaptability cell monoclonal; Collect culture fluid after cultivating 48-90h; The culture fluid of collecting is concentrated through the ultrafilter membrane bag, SDGC purification, ultra from desaccharide, deactivation, is mixed with unit price or multivalence influenza vaccines behind the mensuration hemagglutinin content.
Said mammalian cell is MDCK MDCK, African green monkey kidney cell Vero, PER.C6 cell or human diploid cell.
Said influenza virus is influenza A virus, Influenza B virus or the reprovision Strain with influenza virus phenotype.
The mode of said bioreactor culture virus high-adaptability cell monoclonal strain is microcarrier suspension culture or carrier-free suspension culture.
Microcarrier in the said microcarrier suspension culture is a dextran microcarrier, and consumption is 10-20g/L.
Beneficial effect of the present invention: the present invention is through setting up cell monoclonal storehouse, screening viral high-adaptability cell monoclonal strain, using extensive bioreactor and carry out the mass cell cultured method and produce influenza virus vaccine; Overcome traditional Embryo Gallus domesticus and produce that the influenza vaccines production cost is high, the cycle is long; Anaphylaxis, Embryo Gallus domesticus continuous passage can cause shortcomings such as virus antigenicity variation; Improve viral yield greatly, safety, effective, stable.
Description of drawings
Fig. 1 is the blood clotting titre detection that the influenza A H1N1 influenza virus vaccine strain is cultivated in the strain of MDCK monoclonal cell.
Fig. 2 is the microphotograph that the viral adaptability cell monoclonal strain of large-scale culture is grown on microcarrier cytodex 3.
Fig. 3 is the electromicroscopic photograph behind the influenza virus purification of cell culture.
Fig. 4 is a SDS-PAGE electrophoretogram behind the influenza virus purification of cell culture;
Among the figure, the H1N1 influenza virus in M-albumen marker, 1-cell source, the H1N1 influenza virus in 2-Embryo Gallus domesticus source.
The specific embodiment
Below in conjunction with accompanying drawing and specific embodiment the present invention is further specified.
To screen the strain of influenza A H1N1 influenza virus vaccine strain (A/reassortant/NYMC X-179A) high-adaptability mdck cell monoclonal, to use this monoclonal cell strain production unit price influenza A H1N1 influenza virus vaccine is example.
(1) foundation in mdck cell monoclonal storehouse:
Will be from the mdck cell (p=53) of the biological article collecting center of Unite States Standard (ATCC) introduction at 75cm
2Cultivate in the culture bottle, treat that cell grows to degree of converging 80%,, get 100 cell dilutions to the 20ml cell culture fluid with trypsinization that contains EDTA and counting; Mixing is inoculated in 96 orifice plates, every hole 200 μ l; Put in 37 ℃ of cell culture incubators and cultivate, the next day, observe, and changes liquid in good time; Treat that cell grows to 50% degree of converging, be transferred to after the digestion and continue in 24 orifice plates to cultivate each strain monoclonal cell transfer to a hole; Wait to grow to digestion behind 90% degree of converging, be transferred in two 24 holes and continue to cultivate, wait to grow to 90% degree of converging and get an amount of cell and carry out frozenly, set up mdck cell monoclonal storehouse.
(2) the viral high-adaptability cell monoclonal of screening strain
The monoclonal cell of getting equal number is seeded to 24 orifice plates; Overnight incubation; Culture fluid is abandoned in suction, and PBS washes 3 times, adds equivalent influenza virus (10 μ l) and 1 μ g/ml TPCK-Trypsin with the cell maintenance medium dilution; Measure the blood clotting titre in the cells and supernatant behind 37 ℃ of cultivation 72h, the blood clotting titre is greater than 2
8Be influenza virus adaptability cell strain M77 (Fig. 1).
(3) optimize the Virus culture condition
High cell strain (M77) shop that adapts to of influenza virus of acquisition of getting equal number is to 24 orifice plates, and overnight incubation is optimized temperature, the harvest time of Virus culture, amount and infestation index (MOI=0.001,0.005,0.01 of interpolation TPCK-Trypsin respectively; 0.025,0.1,0.25,0.5; 1,1.5,2,3; 5,7), obtain influenza virus optimum growh parameter in adapted strain, thereby obtain the susceptible poison of more multithread.The influenza virus optimal culture conditions is: cultivation temperature is 37 ℃, adds 1 μ g/ml TPCK-Trypsin, and the MOI of infestation index is between 0.001 to 0.005, and viral harvest time is 72h.
(4) the large-scale culture cell is to cultivate influenza virus and vaccine production
The capital equipment that uses is the U.S. 7.5L of a NBS company bioreactor, and working volume is 4L.In the bioreactor tank body, add 40g cytodex 3 type microcarriers (GE Healthcare); Add 2L phosphate buffer PBS simultaneously; 8 pounds of high pressure 40min, emptying PBS adds 10% hyclone DMEM cell culture fluid; Adjustment bioreactor rotating speed, temperature and ventilation, overnight incubation in advance.The cell inoculation of 4 10 confluent monolayer cells factories of digestion is gone in the jar, and cultivation temperature is made as 37 ℃, and speed of agitator is made as 65rpm; Dissolved oxygen is made as 40%; PH is made as 7.4, cultivates, and 8h measures glucose content in the culture fluid at interval; Culture fluid in the drain tank when glucose content＜1g/L renews bright culture fluid and continues to cultivate.Treat that cell covers with microcarrier (Fig. 2), all culture fluid in the drain tank uses Hank ' s liquid that tank body and microcarrier are washed 3 times, adds the cell maintenance medium 4L that contains 1 μ g/ml TPCK-Trypsin, inoculates influenza virus (MOI=0.001-0.005 of infestation index) simultaneously.8h measures and keeps the liquid concentration of glucose at interval, adds glucose when being lower than 1g/L to 4g/L, collects virus-culturing fluid behind the 72h, contains high concentration influenza virus (Fig. 3) in the culture fluid.The virus-culturing fluid of results is concentrated through 300kD molecular weight ultrafilter membrane bag, SDGC purification (electrophoresis detection behind the purification is as shown in Figure 4), ultra from desaccharide, formalin-inactivated, Triton X-100 cracking; The single expansion measured hemagglutinin content, is mixed with influenza vaccines according to 15 μ g HA (hemagglutinin)/agent.
Claims (5)
1. the method for preparing of an influenza virus vaccine is characterized in that, carries out according to following steps:
(1) foundation in mammalian cell monoclonal storehouse
Mammalian cell is cultivated in culture bottle, treated that cell grows to degree of converging 70-90%, with trypsinization that contains EDTA and counting; Get 100 cell dilutions to the 20ml cell culture fluid, mixing is inoculated in 96 orifice plates; Every hole 200 μ l put in 37 ℃ of cell culture incubators and cultivate, and treat that cell grows to 40-60% degree of converging; Be transferred to after the digestion and continue in 24 orifice plates to cultivate; Each strain monoclonal cell transfer to a hole digests behind the 85-95% degree of converging to be grown to, is transferred in two 24 holes and continues to cultivate, and 85-95% degree of converging to be grown to carries out cell frozen;
(2) screening of viral high-adaptability cell monoclonal strain
Get monoclonal cell and be seeded to 24 orifice plates, overnight incubation is inhaled and is abandoned culture fluid, and PBS washes 2-6 time, adds influenza virus and TPCK-Trypsin, measures the blood clotting titre in the cells and supernatant behind 37 ℃ of cultivation 48-90h, and the blood clotting titre is greater than 2
8Be the strain of viral high-adaptability cell monoclonal;
(3) optimize the Virus culture condition
Get viral high-adaptability cell monoclonal strain shop to 24 orifice plates; Overnight incubation; Optimize temperature, the harvest time of Virus culture, amount and the MOI of infestation index of interpolation TPCK-Trypsin respectively, obtain virus optimum growh parameter in the strain of viral high-adaptability cell monoclonal;
(4) in bioreactor, cultivate the strain of viral high-adaptability cell monoclonal; Collect culture fluid after cultivating 48-90h; The culture fluid of collecting is concentrated through the ultrafilter membrane bag, SDGC purification, ultra from desaccharide, deactivation, is mixed with unit price or multivalence influenza vaccines behind the mensuration hemagglutinin content.
2. according to the method for preparing of the said a kind of influenza virus vaccine of claim 1, it is characterized in that said mammalian cell is MDCK MDCK, African green monkey kidney cell Vero, PER.C6 cell or human diploid cell.
3. according to the method for preparing of the said a kind of influenza virus vaccine of claim 1, it is characterized in that said influenza virus is influenza A virus, Influenza B virus or the reprovision Strain with influenza virus phenotype.
4. according to the method for preparing of the said a kind of influenza virus vaccine of claim 1, it is characterized in that the mode of said bioreactor culture virus high-adaptability cell monoclonal strain is microcarrier suspension culture or carrier-free suspension culture.
5. according to the method for preparing of the said a kind of influenza virus vaccine of claim 4, it is characterized in that the microcarrier in the said microcarrier suspension culture is a dextran microcarrier, consumption is 10-20g/L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103160457A (en) * | 2013-04-11 | 2013-06-19 | 山东信得动物疫苗有限公司 | Large-scaled cell-culturing proliferating method and method for using proliferated cell in virus production |
| CN104046588A (en) * | 2014-06-20 | 2014-09-17 | 马忠仁 | Micro-carrier culture method of bioreactor of MDCK (Madin Darby Canine Kidney) cells and application thereof |
| CN106754722A (en) * | 2016-11-10 | 2017-05-31 | 中国食品药品检定研究院 | Vero cell line of stabilization expression bovine trypsinogen and application thereof |
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| CN101189326A (en) * | 2004-12-23 | 2008-05-28 | 米迪缪尼疫苗股份有限公司 | Non-tumorigenic MDCK cell line for propagating viruses |
| CN101627113A (en) * | 2006-09-15 | 2010-01-13 | 米迪缪尼有限公司 | Mdck cell lines supporting viral growth to high titers and bioreactor process using the same |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1433472A (en) * | 1999-11-26 | 2003-07-30 | 克鲁塞尔荷兰公司 | Production of vaccines |
| CN101094915A (en) * | 2004-05-20 | 2007-12-26 | 益得生物医学公司 | Process for the production of an influenza vaccine |
| CN101189326A (en) * | 2004-12-23 | 2008-05-28 | 米迪缪尼疫苗股份有限公司 | Non-tumorigenic MDCK cell line for propagating viruses |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103160457A (en) * | 2013-04-11 | 2013-06-19 | 山东信得动物疫苗有限公司 | Large-scaled cell-culturing proliferating method and method for using proliferated cell in virus production |
| CN103160457B (en) * | 2013-04-11 | 2014-10-01 | 山东信得动物疫苗有限公司 | Large-scaled cell-culturing proliferating method and method for using proliferated cell in virus production |
| CN104046588A (en) * | 2014-06-20 | 2014-09-17 | 马忠仁 | Micro-carrier culture method of bioreactor of MDCK (Madin Darby Canine Kidney) cells and application thereof |
| CN106754722A (en) * | 2016-11-10 | 2017-05-31 | 中国食品药品检定研究院 | Vero cell line of stabilization expression bovine trypsinogen and application thereof |
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