CN116656628A - Preparation method of poxvirus vector vaccine - Google Patents
Preparation method of poxvirus vector vaccine Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 229940126580 vector vaccine Drugs 0.000 title claims abstract description 16
- 241000700605 Viruses Species 0.000 claims abstract description 21
- 239000000725 suspension Substances 0.000 claims abstract description 15
- 210000003837 chick embryo Anatomy 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 13
- 210000002966 serum Anatomy 0.000 claims abstract description 13
- 239000006285 cell suspension Substances 0.000 claims abstract description 12
- 239000003623 enhancer Substances 0.000 claims abstract description 11
- 239000004017 serum-free culture medium Substances 0.000 claims abstract description 11
- 230000009385 viral infection Effects 0.000 claims abstract description 11
- 108010016626 Dipeptides Proteins 0.000 claims abstract description 10
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 10
- 102000008100 Human Serum Albumin Human genes 0.000 claims abstract description 9
- 108091006905 Human Serum Albumin Proteins 0.000 claims abstract description 9
- 238000003306 harvesting Methods 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims description 16
- 239000012679 serum free medium Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 7
- 108010019160 Pancreatin Proteins 0.000 claims description 6
- 229940055695 pancreatin Drugs 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 239000003223 protective agent Substances 0.000 claims description 5
- 239000011550 stock solution Substances 0.000 claims description 5
- 230000010412 perfusion Effects 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 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 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000010008 shearing Methods 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 241000700647 Variola virus Species 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims description 2
- 239000000835 fiber Substances 0.000 claims description 2
- 229920000728 polyester Polymers 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims 1
- 238000011081 inoculation Methods 0.000 abstract description 5
- 239000003795 chemical substances by application Substances 0.000 abstract description 4
- 230000005727 virus proliferation Effects 0.000 abstract description 3
- 230000001954 sterilising effect Effects 0.000 abstract description 2
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 18
- 230000000694 effects Effects 0.000 description 10
- 239000007788 liquid Substances 0.000 description 7
- 229960005486 vaccine Drugs 0.000 description 7
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- 230000007774 longterm Effects 0.000 description 1
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Abstract
The application provides a preparation method of a poxvirus vector vaccine, which comprises the following steps: s1: preparing primary chick embryo cell suspension; s2: culturing poxvirus; injecting a serum-free culture medium containing 0.1-0.5% of human serum albumin and 0.1-1.0 mmol/L glutamine dipeptide into a disposable reaction bag, adding a cell suspension, and uniformly mixing; adding a virus infection enhancer into a serum-free culture medium, adding a virus suspension, uniformly mixing, standing at 4 ℃, adding the virus suspension into a reaction bag, uniformly mixing and culturing; s3: and (5) harvesting poxviruses. The preparation method adopts a disposable reactor for culture, and the system takes the flaky carrier as the support of primary chick embryo cells, does not need sterilization, and has simple and convenient operation and high yield; the addition of the virus proliferation agent increases the infectivity of the virus and reduces the inoculation amount of the virus; the serum-free culture medium is adopted for culture, so that the product purity is high, and no serum residue exists.
Description
Technical Field
The application relates to the field of vaccines, in particular to a preparation method of a poxvirus vector vaccine.
Background
The poxvirus is used as vaccine for preventing smallpox, has good safety after long-term use in a large number of people in China, has the advantages of long insertion foreign gene, multiple inoculation paths, capability of inducing humoral immunity and cellular immune response, easiness in production and preparation and the like, can overcome certain defects of the traditional vaccine, can greatly improve the disease preventability, and has been widely applied to vaccine development. The vaccine prepared by taking the recombinant poxvirus as an expression vector has been subjected to a plurality of clinical tests, and the recombinant viral vector vaccine has the advantages of long insertion foreign gene, multiple inoculation paths, capability of inducing humoral immunity and cellular immune response, easiness in production and preparation and the like, can overcome certain defects of the traditional vaccine, can greatly improve the disease preventability, and has been widely applied to vaccine development.
The poxvirus vector vaccine is usually prepared from a culture medium containing serum, so that the risk of serum residue exists, the harvested stock solution contains more cell fragments, and the residual quantity of bovine serum is more, so that the purity of the product is not high; the virus liquid is obtained after the culture of cell rotating bottles or cell factories and freeze thawing and filtering, and the process has the advantages of more manual operation, easy pollution, high labor intensity and more occupied area; the culture efficiency of the process product is low, the MOI (the number of viruses infected by each cell) adopted is high, and the consumption of viruses is high. Therefore, there is a need for a method for preparing poxvirus vector vaccines that is easy to operate, less prone to contamination, high in yield and high in purity.
Disclosure of Invention
Aiming at the defects in the prior art, the application provides a preparation method of a poxvirus vector vaccine.
The application provides a preparation method of a poxvirus vector vaccine, which comprises the following steps:
s1: preparing primary chick embryo cell suspension;
s2: culturing poxvirus;
injecting a serum-free culture medium containing 0.1-0.5% of human serum albumin and 0.1-1.0 mmol/L glutamine dipeptide into a disposable reaction bag, placing the disposable reaction bag into a swinging bed reactor for use, adding cell suspension, and uniformly mixing; adding a virus infection enhancer into a serum-free culture medium, adding a virus suspension, uniformly mixing, standing at 4 ℃, adding the virus suspension into a reaction bag, and uniformly mixing; after culturing for 18 hours, carrying out perfusion culture until the time is 68-82 hours, and stopping the culture; the human serum albumin and glutamine dipeptide function to provide nutrition for cells and improve cell activity.
S3: harvesting poxviruses;
after the culture is terminated, the supernatant is discarded, PBS is used for cleaning, a protective agent is added to the solution to be frozen at the temperature of minus 80 ℃, the solution is dissolved at room temperature, the solution is collected, and the supernatant is obtained after centrifugation, thus obtaining the poxvirus stock solution.
Further, the specific steps of the step S1 are as follows:
taking chick embryos which develop for 9-11 days old, removing heads, shearing, digesting with pancreatin, adding an equal volume of culture medium containing serum to neutralize pancreatin, centrifuging to remove supernatant, adding the culture medium to blow dispersed cells, and collecting suspension to obtain primary chick embryo cell suspension.
Further, the disposable reaction bag in the step S2 is provided with 30 g/L of sheet-shaped carrier.
Further, in the step S2, a serum-free medium containing human serum albumin and glutamine dipeptide is injected into a disposable reaction bag, and the ratio of the serum-free medium to the glutamine dipeptide is 1-5 multiplied by 10 6 Cell suspensions were added to each cell/mL.
In the step S2, the virus infection enhancer is added into the serum-free culture medium, and the virus suspension is added according to the MOI of 0.001-0.01.
Further, after the standing at 4 ℃ in the step S2, the virus suspension is added into a reaction bag, and the culture conditions are 37+/-1 ℃, 7 degrees, 70+/-20% dissolved oxygen and 7.6+/-0.1 pH.
Further, the protective agent in the step S3 is sucrose with a mass-volume ratio of 1-5%.
The application also provides the poxvirus vector vaccine prepared by the preparation method.
The application also provides a preparation method or application of the poxvirus vector vaccine in preparation of poxvirus vector vaccine products.
In conclusion, compared with the prior art, the application achieves the following technical effects:
(1) The preparation method adopts a disposable reactor for culture, and the system uses a flaky carrier as a support of primary chick embryo cells to culture poxviruses. The process has the advantages that: a is a disposable reaction bag, sterilization is not needed, and the operation is simple. The b yield is high. One 2.5L working volume reaction bag can replace 5 3L roller bottles.
(2) The preparation method of the application adds the virus proliferation agent, increases the infectivity of the virus and reduces the inoculation amount of the virus.
(3) The preparation method adopts a serum-free culture medium for culture, and has high product purity and no serum residue. Adding 0.1-0.5% human serum albumin and 0.1-1.0 mmol/L glutamine dipeptide.
Detailed Description
In order that those skilled in the art will better understand the present application, a technical solution of the embodiments of the present application will be clearly and completely described below, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application.
The application adopts a disposable reaction bagCulturing poxvirus vector vaccine, wherein the reactor is provided with 30 g/L flaky carrier which is made of polyester fiber and takes the shape of book pages, and the specific surface area of the carrier is 1200 cm 2 And/g. The process adopts primary chick embryo cells as cells, adopts a serum-free culture medium containing 0.1% -0.5 human serum albumin and 0.1% -1.0 mmol/L glutamine dipeptide as a culture medium, and adopts the MOI of 0.001-0.01, and according to the inoculation amount, poxviruses are firstly mixed with 81 mL virus proliferation agent and then added into a reaction bag for culture. Culture parameters: culturing at 37+ -1deg.C with dissolved oxygen of 70+ -20% and pH of 7.6+ -0.1 for 48 hr, and changing liquid to continue culturing; parameters after 48 hours: culturing at 34+/-1 ℃ with dissolved oxygen 70+/-20% and pH value 7.4+/-0.1 for 68-84 hours, discarding the supernatant, washing with PBS, adding hypotonic solution for cell lysis, and obtaining the virus liquid as stock solution.
1. The raw materials and sources used are as follows:
(1) Serum-free medium: purchased from Shanghai source culture Biotech Co., ltd., virusPro cube CEF, cat: H5E5LJ, lot number: F220805.
(2) Low serum medium: the serum-free medium is a serum-free medium containing 2% bovine serum, and the source of the serum-free medium is shown in (1).
(3) Viral value-added agent: envirus, yingen Biol, cat: 30001; hiTransGP may also be used.
2. The testing method comprises the following steps:
titer: the method is carried out according to the virus titration blood cell adsorption method in the biological product procedure (1979 edition) of the tissue culture smallpox vaccine manufacturing and detecting procedure.
EXAMPLE 1 preparation method of poxvirus vector vaccine of the present application
The method comprises the following specific steps:
(1) Preparation of Primary chick embryo cells
Taking chick embryos which develop for 9-11 days old, removing heads, shearing, digesting with 0.25% pancreatin, adding an equal volume of a culture medium containing 2% serum to neutralize pancreatin, centrifuging to remove supernatant, adding a small amount of culture medium to blow dispersed cells, and collecting suspension to obtain primary chick embryo cell suspension.
(2) Poxvirus culture
Human serum albumin with 0.1-0.5%Serum-free medium 2.0L containing protein and 0.1-1.0 mmol/L glutamine dipeptide is injected into a disposable reaction bag according to 1-5×10 6 The cells/mL are added to the cell suspension and mixed well. Adding 60-100 mL of virus infection enhancer into a serum-free culture medium, adding virus suspension according to the MOI of 0.001-0.01, uniformly mixing, standing at 4 ℃ for 15 minutes, adding the virus suspension into a reaction bag, uniformly mixing, and culturing under the condition: after culturing at 37.+ -. 1 ℃ and 7 degrees, dissolved oxygen at 70%.+ -. 20% and pH at 7.6.+ -. 0.1 for 18 hours, perfusion culture was performed at 3.5 mL/min, and the perfusion volume was 3.5L. Culturing for 68-82 hours, and stopping culturing.
(3) Poxvirus harvesting
After the culture is stopped, the supernatant is discarded, after the culture is washed for 1 time by PBS, 400-600 mL of protective agent (1-5% m/v sucrose) is added to the culture solution to be frozen at the temperature of minus 80 ℃, the culture solution is dissolved at room temperature, the solution is collected, and the supernatant is obtained by centrifugation at 2500 rpm, thus obtaining the poxvirus stock solution.
Comparative example 1 comparison of Effect of Using Disposable reaction bag and bottle
The same batch of cells was cultured and harvested according to Table 1 below, and as can be seen from the results, the yield of 1 2.5L working volume reaction bags was equivalent to 11 roller bottles.
TABLE 1 cultivation Effect of different incubators
Comparative example 2 Effect of adding Virus infection enhancer
Taking the same batch of cells, respectively adding two reaction bags according to the same cell density for culture, wherein the reaction bag 1 is added with a virus infection enhancer, and the reaction bag 2 is not added with the virus infection enhancer, and comparing the effects of the two. The details are shown in Table 2.
TABLE 2 Effect of viral infection enhancers
Comparative example 3 Effect of Using serum-free Medium
The same batch of cells are respectively inoculated into two reaction bags, the reaction bag 1 adopts serum-free culture, the reaction bag 2 adopts low-serum culture, the culture effects of the two are equivalent, but the harvest liquid of the serum-free culture has no serum residue, and the serum residue of the harvest liquid of the low-serum culture is 20-40 ng/mL. See in particular table 3.
TABLE 3 Effect of serum-free culture
As can be seen from the above examples, the preparation method of the present application can greatly improve the yield by using a disposable reaction bag; after the addition of the virus infection enhancer, the titer of the virus is higher and the yield is higher. The harvest liquid of the serum-free culture has no serum residue, and the serum residue of the harvest liquid of the low serum culture is 20-40 ng/mL, so that the purity and the safety of the product can be improved.
The foregoing description of the preferred embodiments of the application is not intended to limit the application to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the application are intended to be included within the scope of the application.
Claims (10)
1. A method for preparing a poxvirus vector vaccine comprising the steps of:
s1: preparing primary chick embryo cell suspension;
s2: culturing poxvirus;
injecting a serum-free culture medium containing 0.1-0.5% of human serum albumin and 0.1-1.0 mmol/L glutamine dipeptide into a disposable reaction bag, adding a cell suspension, and uniformly mixing; adding a virus infection enhancer into a serum-free culture medium, adding a virus suspension, uniformly mixing, standing at 4 ℃, adding the virus suspension into a reaction bag, and uniformly mixing; after culturing for 18 hours, carrying out perfusion culture until the time is 68-82 hours, and stopping the culture;
s3: harvesting poxviruses;
after the culture is terminated, the supernatant is discarded, PBS is used for cleaning, a protective agent is added to the solution to be frozen at the temperature of minus 80 ℃, the solution is dissolved at room temperature, the solution is collected, and the supernatant is obtained after centrifugation, thus obtaining the poxvirus stock solution.
2. The preparation method according to claim 1, wherein the specific steps of step S1 are as follows:
taking chick embryos which develop for 9-11 days old, removing heads, shearing, digesting with pancreatin, adding an equal volume of culture medium containing serum to neutralize pancreatin, centrifuging to remove supernatant, adding the culture medium to blow dispersed cells, and collecting suspension to obtain primary chick embryo cell suspension.
3. The method according to claim 1, wherein the disposable reaction bag in step S2 is loaded with 30 g/L of the sheet-like carrier.
4. The method according to claim 3, wherein the sheet-like carrier is a polyester fiber, has a book-like shape, and has a specific surface area of 1200 cm 2 /g。
5. The method according to claim 1, wherein the serum-free medium containing human serum albumin and glutamine dipeptide is filled into a disposable reaction bag in step S2, and the ratio is 1-5×10 6 Cell suspensions were added to each cell/mL.
6. The method according to claim 1, wherein the virus infection enhancer is added to the serum-free medium in the step S2, and the virus suspension is added at a MOI of 0.001-0.01.
7. The method according to claim 1, wherein the virus suspension is added to the reaction bag after the step S2 is performed at a temperature of 4 ℃, and the culture conditions are 37+ -1 ℃, 7 °, 70% + -20% dissolved oxygen, and 7.6+ -0.1.
8. The preparation method according to claim 1, wherein the protective agent in the step S3 is 1-5% of sucrose by mass/volume.
9. The poxvirus vector vaccine prepared by the preparation method of any one of claims 1 to 8.
10. Use of the preparation method of any one of claims 1 to 8 or the poxvirus vector vaccine of claim 9 in the preparation of a product for the prevention of smallpox.
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