CN113817688B - Chick embryo culture process of tetravalent influenza virus split vaccine and preparation method of tetravalent influenza virus split vaccine - Google Patents
Chick embryo culture process of tetravalent influenza virus split vaccine and preparation method of tetravalent influenza virus split vaccine Download PDFInfo
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- 210000003837 chick embryo Anatomy 0.000 title claims abstract description 35
- 241000712461 unidentified influenza virus Species 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 24
- 229960005486 vaccine Drugs 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 27
- 238000001816 cooling Methods 0.000 claims abstract description 24
- 241000700605 Viruses Species 0.000 claims abstract description 20
- 238000003306 harvesting Methods 0.000 claims abstract description 18
- 238000007710 freezing Methods 0.000 claims abstract description 12
- 230000008014 freezing Effects 0.000 claims abstract description 8
- 230000035755 proliferation Effects 0.000 claims abstract description 8
- 238000011081 inoculation Methods 0.000 claims abstract description 7
- 239000012530 fluid Substances 0.000 claims description 21
- 238000012258 culturing Methods 0.000 claims description 16
- 239000007853 buffer solution Substances 0.000 claims description 14
- 241000713196 Influenza B virus Species 0.000 claims description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- 241000712431 Influenza A virus Species 0.000 claims description 7
- 239000012297 crystallization seed Substances 0.000 claims description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 238000010276 construction Methods 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 abstract description 10
- 230000035931 haemagglutination Effects 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- DVKFVGVMPLXLKC-PUGXJXRHSA-N [(2s,3r,4s,5s,6r)-2-[(2s,3s,4s,5r)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] dihydrogen phosphate Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@]1(CO)[C@@]1(OP(O)(O)=O)[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DVKFVGVMPLXLKC-PUGXJXRHSA-N 0.000 description 2
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- 229940124873 Influenza virus vaccine Drugs 0.000 description 1
- 210000001643 allantois Anatomy 0.000 description 1
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- 239000000243 solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000005727 virus proliferation Effects 0.000 description 1
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Abstract
The embodiment of the invention provides a culture process of tetravalent influenza virus split vaccine chick embryos, a preparation method of tetravalent influenza virus split vaccine and related products. The culture process comprises the steps of warehouse building, inoculation, proliferation and embryo cooling, and is characterized by further comprising a quick freezing step before the embryo cooling step: freezing at-20+ -5deg.C for 0.5-1.5 hr. By implementing the invention, the process production time can be greatly shortened, and the hemagglutination titer and the virus titer of the harvest liquid can be improved; reducing the protein content and clarifying the feed liquid.
Description
Technical Field
The invention relates to the technical field of influenza vaccines, in particular to a culture process of tetravalent influenza virus split vaccine chick embryos, a tetravalent influenza virus split vaccine and a preparation method thereof.
Background
Tetravalent influenza virus split vaccines are basically prepared by chick embryo proliferation culture. And (3) proliferating and culturing viruses in chick embryos, harvesting chick embryo allantoic fluid, purifying the chick embryo allantoic fluid to obtain monovalent stock solution, and mixing to obtain the tetravalent influenza virus split vaccine. In the prior art, the culture process of tetravalent influenza virus chick embryos mainly comprises the steps of virus inoculation, culture, embryo cooling, allantoic fluid harvesting and the like. The prior culture process of tetravalent influenza virus chick embryo can be adopted to obtain monovalent stock solution of tetravalent influenza virus split vaccine which basically meets pharmacopoeia requirements. However, the time required is long, the blood coagulation titer and the virus titer of the harvest liquid are low, the protein content is high, and the feed liquid is not clear enough.
Disclosure of Invention
The invention aims to solve the technical problem of providing a culture process of chick embryo allantois of tetravalent influenza virus split vaccine, which can greatly shorten the production time and improve the hemagglutination titer and the virus titer of the harvest liquid; reducing the protein content and clarifying the feed liquid.
In order to solve the technical problems, the invention discloses the following technical scheme:
the chick embryo allantoic culture process of the tetravalent influenza virus split vaccine comprises the steps of stock building, inoculation, proliferation and embryo cooling, and the chick embryo allantoic culture process further comprises the following steps before the embryo cooling step:
quick-freezing: freezing at-20+ -5deg.C for 0.5-1.5 hr.
As some possible embodiments, the step of building a library includes:
diluting virus seeds to a proper concentration by using a buffer solution, inoculating 9-11-day-old SPF chick embryos for culture, and harvesting allantoic fluid; wherein the buffer solution is phosphate buffer solution with pH value of 8.0 and added with sucrose, and the sucrose content is 2-5% by mass.
As some possible embodiments, the step of building a library specifically includes:
and (3) establishing a master seed batch library: the original seeds are opened and diluted to 3.0-7.0lg EID by the buffer solution 50 After the concentration of the strain/ml, 9-11 days old SPF chick embryos are inoculated, and allantoic fluid is obtained after culturing influenza A viruses for 48-54 hours and influenza B viruses for 60-66 hours at the temperature of 33-35 ℃.
As some possible embodiments, the step of building a library further includes:
batch construction of working seeds: diluting the main seed lot to 3.0-7.0lg EID by the buffer solution 50 After the concentration of the strain/ml, 9-11 days old SPF chick embryos are inoculated, and allantoic fluid is obtained after culturing influenza A viruses for 48-54 hours and influenza B viruses for 60-66 hours at the temperature of 33-35 ℃.
As some possible embodiments, in the stock building step, the quick freezing step and the embryo cooling step are performed once before each time allantoic fluid is harvested.
As some possible embodiments, the inoculating step specifically includes:
diluting the virus seed of the working seed lot with buffer solution to 5.0-7.0lg EID 50 /ml。
As some possible embodiments, the proliferation step specifically includes:
according to the inoculum size of 0.2+/-0.05 ml per embryo, culturing for 48-72 hours at the culture temperature of 34+/-1 ℃ and the humidity of 65+/-10%.
As some possible embodiments, the cooling step specifically includes:
and (3) harvesting allantoic fluid after cooling the embryo at 2-8 ℃ for 5-10 hours.
Correspondingly, the invention also discloses a preparation method of the tetravalent influenza virus split vaccine, and the chick embryo allantoic culture process of the tetravalent influenza virus split vaccine is adopted.
Correspondingly, the invention also discloses a tetravalent influenza virus vaccine which is prepared by the method.
The beneficial effects of the invention are as follows:
according to the embodiment of the invention, the quick-freezing step is implemented before the embryo cooling step of the tetravalent influenza virus chick embryo culture process, namely, the chick embryo is frozen for 0.5-1.5 hours at the temperature of minus 20+/-5 ℃, so that the production time is greatly shortened, the hemagglutination titer and the virus titer of the harvest liquid are improved, the impurity protein content is reduced, and the feed liquid is clearer.
Detailed Description
For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the following detailed description of the present invention is given in conjunction with specific examples, which are given by way of illustration of the present invention only and are not to be construed as limiting the present invention.
The invention provides an embodiment of a tetravalent influenza virus chick embryo culture process, which comprises the steps of library establishment, inoculation, proliferation and embryo cooling, and the chick embryo culture process further comprises the following steps before the embryo cooling step:
quick-freezing: freezing at-20+ -5deg.C for 0.5-1.5 hr.
As some possible embodiments, the step of building a library includes:
diluting virus seeds to a proper concentration by using a buffer solution, inoculating 9-11-day-old SPF chick embryos for culture, and harvesting allantoic fluid; wherein the buffer solution is phosphate buffer solution with pH value=8.0 and added with sucrose, and the sucrose content is 2-5% by mass.
As some possible embodiments, the step of building a library specifically includes:
and (3) establishing a master seed batch library: the original seeds are opened and diluted to 3.0-7.0lg EID by the buffer solution 50 After the concentration of the strain/ml, 9-11 days old SPF chick embryos are inoculated, and allantoic fluid is obtained after culturing influenza A viruses for 48-54 hours and influenza B viruses for 60-66 hours at the temperature of 33-35 ℃.
As some possible embodiments, the step of building a library further includes:
batch construction of working seeds: principal seed warpThe buffer is diluted to 3.0-7.0lg EID 50 After the concentration of the strain/ml, 9-11 days old SPF chick embryos are inoculated, and allantoic fluid is obtained after culturing influenza A viruses for 48-54 hours and influenza B viruses for 60-66 hours at the temperature of 33-35 ℃.
As some possible embodiments, in the stock building step, the quick freezing step and the embryo cooling step are performed once before each time allantoic fluid is harvested.
As some possible embodiments, the inoculating step specifically includes:
the virus seeds of the working seed lot are taken, and the virus seeds of the working seed lot are diluted into 5.0 to 7.0lgEID by buffer solution 50 /ml。
As some possible embodiments, the proliferation step specifically includes:
according to the inoculum size of 0.2+/-0.05 ml per embryo, culturing for 48-72 hours at the culture temperature of 34+/-1 ℃ and the humidity of 65+/-10%.
As some possible embodiments, the cooling step specifically includes: and (3) harvesting allantoic fluid after cooling the embryo at 2-8 ℃ for 5-10 hours.
The chick embryo allantoic culture process of the tetravalent influenza virus split vaccine chick embryo allantoic culture process provided by the embodiment of the invention can be basically the same as the prior art.
The tetravalent influenza virus split vaccine provided by the embodiment of the invention can be prepared by the preparation method described in the previous embodiment.
In order to further explain the technical means and effects adopted by the embodiment to achieve the preset aim of the invention, the specific steps of the chick embryo allantoic culture process of the tetravalent influenza virus split vaccine of the embodiment are described in detail by example data as follows.
Example 1
And (3) establishing a master seed batch library: the original seeds were opened and diluted to 3.0-7.0lg EID with phosphate buffer having ph=8.0 and added with 2-5% sucrose (mass ratio) 50 Inoculating 9-11 day old SPF chick embryo at concentration of/ml, culturing at 33-35deg.C, and culturing for type ACulturing the influenza virus for 48-54 hours, and culturing the influenza B virus for 60-66 hours; rapidly cooling at-20+ -5deg.C for 0.5-1.5 hr, transferring to cold embryo at 2-8deg.C for 5-10 hr, and collecting allantoic fluid.
Batch construction of working seeds: the main seed batch is diluted to 3.0-7.0lg EID by pH 8.0+2% sucrose phosphate buffer 50 Inoculating to 9-11 day old SPF chick embryo, culturing at 33-35deg.C, culturing for 48-54 hr for influenza A virus, and culturing for 60-66 hr for influenza B virus; rapidly cooling at-20+/-5 ℃ for 0.5-1.5 hours, transferring to a cold embryo at 2-8 ℃ for 5-10 hours, and harvesting allantoic fluid.
Virus inoculation: taking working seed virus seed, diluting the working seed virus seed to 5.0-7.0lg EID with phosphate buffer solution with pH value of 8.0 and 2-5% sucrose (mass ratio) 50 /ml。
Virus proliferation: the inoculation amount is 0.2+/-0.05 ml per embryo; the culture temperature is 34+/-1 ℃, the culture time is 48-72 hours, and the humidity is 65+/-10%.
Virus harvesting: rapidly cooling at-20+/-5 ℃ for 0.5-1.5, transferring to a cold embryo at 2-8 ℃ for 5-10 hours, and harvesting allantoic fluid.
The results of comparing various indexes with the prior art are shown in the following tables 1, 2 and 3.
TABLE 1 working seed lot hemagglutination titres and viral titres
TABLE 3 Haemophilus titre of harvest (log 2 hemagglutination titre)
According to the embodiment of the invention, the virus is diluted by the optimized virus diluent pH 8.0+2% sucrose phosphate buffer solution and inoculated, the influenza A virus is cultured for 48-54 hours in the optimized culture time, the influenza B virus is cultured for 60-66 hours, and the seed batch with higher hemagglutination titer and higher virus titer is achieved by the optimized embryo cooling mode (rapid cooling is firstly carried out for 0.5-1.5 hours and then cooling is carried out for 5-10 hours at 2-8 ℃), and the inoculum size is 0.2+/-0.05 ml per embryo during production inoculation; the culture temperature is 34+/-1 ℃, the culture time is 48-72 h, and the humidity is 65+/-10%, so that a virus harvest liquid with higher virus content is obtained, the harvest liquid is clearer, the content of foreign proteins is lower, the yield and the safety of the chick embryo influenza vaccine are improved, the conventional process embryo cooling time is shortened to 5-10h, and the working efficiency is improved.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the invention, and all such modifications are to be considered as falling within the scope of the invention.
Claims (2)
1. The chick embryo allantoic culture process of the tetravalent influenza virus split vaccine comprises the steps of library establishment, inoculation, proliferation and embryo cooling, and is characterized in that the library establishment step comprises the following steps:
diluting virus seeds to the concentration of 3.0-7.0 g EID50/ml by using a buffer solution, inoculating 9-11 days old SPF chick embryo for culture, and harvesting allantoic fluid; wherein the buffer solution is phosphate buffer solution with pH value=8.0 and added with sucrose, and the sucrose content is 2-5% by mass;
before the cold embryo step, the method further comprises the following steps:
quick-freezing: freezing at-20+ -5deg.C for 0.5-1.5 hr;
wherein, the step of building the library specifically comprises the following steps:
and (3) establishing a master seed batch library: the original seeds are opened and diluted to 3.0-7.0lg EID by the buffer solution 50 Inoculating 9-11 day old SPF chick embryo, culturing influenza A virus for 48-54 hr and influenza B virus for 60-66 hr at 33-35deg.C, and collecting allantoic fluid;
batch construction of working seeds: diluting the main seed lot to 3.0-7.0lg EID by the buffer solution 50 Inoculating 9-11 day old SPF chick embryo after/ml concentration, and performing para-type A under 33-35deg.CCulturing influenza virus for 48-54 hr, culturing influenza B virus for 60-66 hr, and collecting allantoic fluid;
the inoculation step specifically comprises the following steps:
diluting the virus seed of the working seed lot with buffer solution to 5.0-7.0lg EID 50 /ml；
The proliferation step specifically comprises the following steps:
culturing for 48-72 hours at 34+ -1deg.C and 65+ -10% humidity according to 0.2+ -0.05 ml inoculum size per embryo;
the cold embryo step specifically comprises the following steps:
harvesting allantoic fluid after cooling the embryo at 2-8deg.C for 5-10 hours;
in the warehouse building step, the quick freezing step and the embryo cooling step are carried out once before allantoic fluid is harvested.
2. A method for preparing a tetravalent influenza virus split vaccine, which is characterized in that the tetravalent influenza virus split vaccine chick embryo culture process of claim 1 is adopted.
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