CN114350624A - Method for preparing hepatitis A inactivated vaccine - Google Patents
Method for preparing hepatitis A inactivated vaccine Download PDFInfo
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- CN114350624A CN114350624A CN202111626701.2A CN202111626701A CN114350624A CN 114350624 A CN114350624 A CN 114350624A CN 202111626701 A CN202111626701 A CN 202111626701A CN 114350624 A CN114350624 A CN 114350624A
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- hepatitis
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 229940031551 inactivated vaccine Drugs 0.000 title abstract description 19
- 208000005252 hepatitis A Diseases 0.000 title abstract description 9
- 241000709721 Hepatovirus A Species 0.000 claims abstract description 37
- 239000000243 solution Substances 0.000 claims abstract description 33
- 238000001179 sorption measurement Methods 0.000 claims abstract description 26
- 210000001840 diploid cell Anatomy 0.000 claims abstract description 23
- 210000004072 lung Anatomy 0.000 claims abstract description 23
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 21
- 238000002156 mixing Methods 0.000 claims abstract description 21
- 239000011550 stock solution Substances 0.000 claims abstract description 18
- 229940038490 inactivated hepatitis a vaccine Drugs 0.000 claims abstract description 16
- 239000013592 cell lysate Substances 0.000 claims abstract description 15
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 11
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 10
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 10
- 239000002671 adjuvant Substances 0.000 claims abstract description 8
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 claims abstract description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 7
- 238000005119 centrifugation Methods 0.000 claims abstract description 6
- 239000002244 precipitate Substances 0.000 claims description 25
- 238000012258 culturing Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 3
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 3
- 244000309466 calf Species 0.000 claims description 3
- 229960003964 deoxycholic acid Drugs 0.000 claims description 3
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims description 3
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims 2
- 239000001963 growth medium Substances 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 7
- 238000000605 extraction Methods 0.000 abstract description 6
- 239000000725 suspension Substances 0.000 abstract description 4
- 239000013049 sediment Substances 0.000 abstract 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 229940124870 Hepatitis A virus vaccine Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for preparing inactivated hepatitis A vaccine, which comprises the steps of inoculating 0.01-0.04MOI hepatitis A virus to a human embryo lung diploid cell 2BS strain, placing the human embryo lung diploid cell 2BS strain and the hepatitis A virus at normal temperature for mixed adsorption for 50-100min, extracting culture solution after step 3 is completed, replacing and carrying out mixed adsorption again, putting the culture solution into a centrifuge for centrifugation and taking sediment after multiple times of mixed adsorption, adding cell lysate into the sediment and putting the cell lysate into a mixer for mixing for 50-100min, taking the sediment and adding polyethylene glycol for mixing for 50-100min again, taking out the sediment and adding phosphate buffer solution for suspension to prepare hepatitis A virus stock solution, carrying out ultrafiltration on the hepatitis A virus stock solution, carrying out adsorption by an aluminum hydroxide adjuvant to prepare the inactivated hepatitis A vaccine, and by adopting the preparation method, multiple times of mixed adsorption can be realized, so that the hepatitis A virus stock solution can be better extracted and prepared, thereby achieving the high-efficiency extraction and high-quality hepatitis A inactivated vaccine.
Description
Technical Field
The invention relates to preparation of an inactivated vaccine, in particular to a method for preparing an inactivated vaccine for hepatitis A, and belongs to the technical field of preparation of inactivated vaccines.
Background
Inactivated vaccines are those in which a virus or bacterium is cultured and then inactivated by heat or a chemical agent (usually formalin), and the inactivated vaccine may consist of the whole virus or bacterium or its split fragments.
The inactivated vaccine is also called as a killed vaccine, and is a vaccine prepared by killing complete pathogenic microorganisms cultured in large quantity by heating or physical and chemical methods such as formaldehyde and the like, so that the pathogens lose infectivity and toxicity and keep immunogenicity, and combining corresponding adjuvants.
In the prior art, the extraction degree and the extraction quality of the inactivated vaccine are low in the preparation process of the inactivated vaccine, so that the quality of the inactivated vaccine sold in the market is relatively low, and a method for preparing the hepatitis A inactivated vaccine is designed to solve the problems.
Disclosure of Invention
The invention mainly aims to provide a method for preparing inactivated hepatitis A virus vaccine, which comprises the steps of selecting a culture solution, culturing a human embryo lung diploid cell 2BS strain in the culture solution, inoculating 0.01-0.04MOI hepatitis A virus to the human embryo lung diploid cell 2BS strain, placing the human embryo lung diploid cell 2BS strain and the hepatitis A virus at normal temperature for mixed adsorption for 50-100min, extracting the culture solution after the step 3 is finished, replacing the culture solution for mixed adsorption again, placing the culture solution into a centrifuge for centrifugation after multiple mixed adsorption, taking out the precipitate, adding cell lysate into the precipitate, placing the cell lysate into the mixer for mixing for 50-100min, taking out the precipitate, adding polyethylene glycol for mixing for 50-100min again, taking out the precipitate, adding phosphate buffer solution for suspension to prepare hepatitis A virus stock solution, performing ultrafiltration on the hepatitis A virus stock solution, performing adsorption by using an aluminum hydroxide adjuvant, the preparation method can realize multiple times of mixed adsorption, so that the hepatitis A virus stock solution can be better extracted and prepared, and the high-efficiency extraction and high-quality hepatitis A inactivated vaccine is achieved.
The purpose of the invention can be achieved by adopting the following technical scheme:
a method for preparing a hepatitis A inactivated vaccine,
step 1: selecting a culture solution and culturing a human embryo lung diploid cell 2BS strain in the culture solution;
step 2: inoculating 0.01-0.04MOI hepatitis A virus to the human embryo lung diploid cell 2BS strain;
and step 3: placing human embryo lung diploid cell 2BS strain and hepatitis A virus at normal temperature, mixing and adsorbing for 50-100 min;
and 4, step 4: after the step 3 is finished, extracting the culture solution for replacement, mixing and adsorbing again;
and 5: putting the mixture into a centrifuge for centrifugation after multiple times of mixed adsorption to obtain precipitates;
step 6: adding cell lysate into the precipitate, and mixing in a mixer for 50-100 min;
and 7: adding polyethylene glycol into the precipitate, and mixing for 50-100 min;
and 8: taking out the precipitate, adding phosphate buffer solution, and suspending to prepare hepatitis A virus stock solution;
and step 9: and (3) carrying out ultrafiltration membrane ultrafiltration on the hepatitis A virus stock solution, and adsorbing by an aluminum hydroxide adjuvant to prepare the inactivated hepatitis A vaccine.
Preferably, the concentration of the human embryonic lung diploid cell 2BS strain in step 1 is 105-107/ml。
Preferably, the hepatitis A Virus selected in step 2 is at 0.02 MOI.
Preferably, in step 3, the normal temperature is 20-30 ℃, and the mixed adsorption time is 80 min.
Preferably, the culture solution used in step 4 is 199 medium or MEM medium containing 2-10% newborn calf serum, and the pH is adjusted to 7.3.
Preferably, 3-5 medium changes are performed in step 4.
Preferably, the mixer is run for 100min in step 6 and again for 100min in step 7.
Preferably, the cell lysate added in the step 6 acts for 20-30h at 2-10 ℃, and the cell lysate is 0.2-0.5% sodium deoxycholate solution containing 7-10% of disodium ethylene diamine tetraacetate by mass percentage.
Preferably, the molecular weight of the polyethylene glycol in step 7 is 6000.
The invention has the beneficial technical effects that:
the invention provides a method for preparing hepatitis A virus inactivated vaccine, which comprises the steps of selecting a culture solution, culturing a human embryo lung diploid cell 2BS strain in the culture solution, inoculating 0.01-0.04MOI hepatitis A virus to the human embryo lung diploid cell 2BS strain, placing the human embryo lung diploid cell 2BS strain and the hepatitis A virus at normal temperature for mixed adsorption for 50-100min, extracting the culture solution after the step 3 is completed, replacing, carrying out mixed adsorption again, placing the culture solution into a centrifuge after multiple times of mixed adsorption, centrifuging to obtain a precipitate, adding a cell lysate into the precipitate, placing the cell lysate into the mixer for mixing for 50-100min, taking the precipitate, adding polyethylene glycol for mixing again for 50-100min, taking out the precipitate, adding a phosphate buffer solution for suspension to prepare a hepatitis A virus stock solution, carrying out ultrafiltration on the hepatitis A virus stock solution by an ultrafiltration membrane, and carrying out adsorption by an aluminum hydroxide adjuvant to obtain the hepatitis A virus inactivated vaccine, by adopting the preparation method, multiple times of mixed adsorption can be realized, so that the hepatitis A virus stock solution can be better extracted and prepared, and the high-efficiency extraction and high-quality hepatitis A inactivated vaccine can be realized.
Detailed Description
In order to make the technical solutions of the present invention more clear and definite for those skilled in the art, the present invention is further described in detail with reference to the following examples, but the embodiments of the present invention are not limited thereto.
This example provides a method for preparing inactivated hepatitis A vaccine,
step 1: selecting a culture solution and culturing a human embryo lung diploid cell 2BS strain in the culture solution;
step 2: inoculating 0.01-0.04MOI hepatitis A virus to the human embryo lung diploid cell 2BS strain;
and step 3: placing human embryo lung diploid cell 2BS strain and hepatitis A virus at normal temperature, mixing and adsorbing for 50-100 min;
and 4, step 4: after the step 3 is finished, extracting the culture solution for replacement, mixing and adsorbing again;
and 5: putting the mixture into a centrifuge for centrifugation after multiple times of mixed adsorption to obtain precipitates;
step 6: adding cell lysate into the precipitate, and mixing in a mixer for 50-100 min;
and 7: adding polyethylene glycol into the precipitate, and mixing for 50-100 min;
and 8: taking out the precipitate, adding phosphate buffer solution, and suspending to prepare hepatitis A virus stock solution;
and step 9: and (3) carrying out ultrafiltration membrane ultrafiltration on the hepatitis A virus stock solution, and adsorbing by an aluminum hydroxide adjuvant to prepare the inactivated hepatitis A vaccine.
Selecting a culture solution, culturing a human embryo lung diploid cell 2BS strain in the culture solution, inoculating 0.01-0.04MOI hepatitis A virus to the human embryo lung diploid cell 2BS strain, placing the human embryo lung diploid cell 2BS strain and the hepatitis A virus at normal temperature for mixed adsorption for 50-100min, after the step 3 is finished, extracting the culture solution for replacement, performing mixed adsorption again, putting the mixture into a centrifuge for centrifugation after multiple times of mixed adsorption, taking out a precipitate, adding a cell lysate into the precipitate, putting the cell lysate into a mixer for mixing for 50-100min, taking out the precipitate, adding polyethylene glycol into the precipitate, mixing for 50-100min again, taking out the precipitate, adding a phosphate buffer solution for suspension to prepare an hepatitis A virus stock solution, performing ultrafiltration membrane on the hepatitis A virus stock solution, performing adsorption by an aluminum hydroxide adjuvant to prepare the inactivated hepatitis A vaccine, and by adopting the preparation method, the multiple times of mixed adsorption can be realized, so that the hepatitis A virus stock solution can be better extracted and prepared, thereby achieving the high-efficiency extraction and high-quality hepatitis A inactivated vaccine.
In this example, the concentration of the human embryonic lung diploid cell 2BS strain was set to 10 in step 15-107/ml。
In this example, the hepatitis A Virus selected in step 2 was 0.02 MOI.
In this example, in step 3, the normal temperature is 20 to 30 ℃ and the mixed adsorption is 80 min.
In this example, the culture solution used in step 4 was 2-10% newborn calf serum 199 medium or MEM medium, and the pH was adjusted to 7.3.
In this example, 3 to 5 medium changes were performed in step 4.
In this example, the mixer was run for 100min in step 6, and remixing was also 100min in step 7.
In this embodiment, the cell lysis solution added in step 6 is reacted at 2-10 ℃ for 20-30h, and the cell lysis solution is 0.2-0.5% sodium deoxycholate solution containing 7-10% disodium ethylenediaminetetraacetate by mass.
In this example, the molecular weight of the polyethylene glycol was 6000 in step 7.
The above description is only for the purpose of illustrating the present invention and is not intended to limit the scope of the present invention, and any person skilled in the art can substitute or change the technical solution of the present invention and its conception within the scope of the present invention.
Claims (9)
1. A method for preparing inactivated hepatitis A vaccine is characterized in that: the method comprises the following steps:
step 1: selecting a culture solution and culturing a human embryo lung diploid cell 2BS strain in the culture solution;
step 2: inoculating 0.01-0.04MOI hepatitis A virus to the human embryo lung diploid cell 2BS strain;
and step 3: placing human embryo lung diploid cell 2BS strain and hepatitis A virus at normal temperature, mixing and adsorbing for 50-100 min;
and 4, step 4: after the step 3 is finished, extracting the culture solution for replacement, mixing and adsorbing again;
and 5: putting the mixture into a centrifuge for centrifugation after multiple times of mixed adsorption to obtain precipitates;
step 6: adding cell lysate into the precipitate, and mixing in a mixer for 50-100 min;
and 7: adding polyethylene glycol into the precipitate, and mixing for 50-100 min;
and 8: taking out the precipitate, adding phosphate buffer solution, and suspending to prepare hepatitis A virus stock solution;
and step 9: and (3) carrying out ultrafiltration membrane ultrafiltration on the hepatitis A virus stock solution, and adsorbing by an aluminum hydroxide adjuvant to prepare the inactivated hepatitis A vaccine.
2. The method for preparing inactivated hepatitis A vaccine according to claim 1, wherein: in step 1, the concentration of the human embryo lung diploid cell 2BS strain is up to 105-107/ml。
3. The method for preparing inactivated hepatitis A vaccine according to claim 1, wherein: the hepatitis A Virus chosen in step 2 was 0.02 MOI.
4. The method for preparing inactivated hepatitis A vaccine according to claim 1, wherein: in step 3, the normal temperature is 20-30 ℃, and the mixing and adsorption time is 80 min.
5. The method for preparing inactivated hepatitis A vaccine according to claim 1, wherein: the culture solution used in step 4 was 199 medium or MEM medium containing 2-10% newborn calf serum, and the pH was adjusted to 7.3.
6. The method for preparing inactivated hepatitis A vaccine according to claim 1, wherein: the culture medium was changed 3 to 5 times in step 4.
7. The method for preparing inactivated hepatitis A vaccine according to claim 1, wherein: the mixer was run for 100min in step 6 and again for 100min in step 7.
8. The method for preparing inactivated hepatitis A vaccine according to claim 1, wherein: and (3) acting the cell lysate added in the step (6) for 20-30h at the temperature of 2-10 ℃, wherein the cell lysate is 0.2-0.5% of sodium deoxycholate solution containing 7-10% of disodium ethylene diamine tetraacetate in percentage by mass.
9. The method for preparing inactivated hepatitis A vaccine according to claim 1, wherein: in step 7, the molecular weight of the polyethylene glycol is 6000.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111626701.2A CN114350624A (en) | 2021-12-28 | 2021-12-28 | Method for preparing hepatitis A inactivated vaccine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111626701.2A CN114350624A (en) | 2021-12-28 | 2021-12-28 | Method for preparing hepatitis A inactivated vaccine |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816786A (en) * | 2010-04-30 | 2010-09-01 | 长春生物制品研究所 | Inactivated hepatitis A vaccine and preparation method thereof |
| CN102492659A (en) * | 2011-11-14 | 2012-06-13 | 成都康华生物制品有限公司 | Production method for hepatitis A virus for vaccine production |
| CN102988975A (en) * | 2012-11-30 | 2013-03-27 | 深圳康泰生物制品股份有限公司 | Combined hepatitis A and B vaccine and preparation method thereof |
| CN110669739A (en) * | 2019-09-30 | 2020-01-10 | 长春生物制品研究所有限责任公司 | Preparation method of novel hepatitis A virus antigen |
-
2021
- 2021-12-28 CN CN202111626701.2A patent/CN114350624A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816786A (en) * | 2010-04-30 | 2010-09-01 | 长春生物制品研究所 | Inactivated hepatitis A vaccine and preparation method thereof |
| CN102492659A (en) * | 2011-11-14 | 2012-06-13 | 成都康华生物制品有限公司 | Production method for hepatitis A virus for vaccine production |
| CN102988975A (en) * | 2012-11-30 | 2013-03-27 | 深圳康泰生物制品股份有限公司 | Combined hepatitis A and B vaccine and preparation method thereof |
| CN110669739A (en) * | 2019-09-30 | 2020-01-10 | 长春生物制品研究所有限责任公司 | Preparation method of novel hepatitis A virus antigen |
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