CN102492659A - Production method for hepatitis A virus for vaccine production - Google Patents
Production method for hepatitis A virus for vaccine production Download PDFInfo
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- CN102492659A CN102492659A CN2011103590914A CN201110359091A CN102492659A CN 102492659 A CN102492659 A CN 102492659A CN 2011103590914 A CN2011103590914 A CN 2011103590914A CN 201110359091 A CN201110359091 A CN 201110359091A CN 102492659 A CN102492659 A CN 102492659A
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Abstract
The invention relates to the field of biotechnology, in particular to a production method for a hepatitis A virus for vaccine production. The production method comprises the following steps sequentially: reviving, passaging and amplifying a cell; vaccinating a hepatitis A virus seed; keeping culturing the cell infected with hepatitis A virus; detecting the reproduction condition of viral infection; stopping culturing, discarding the maintenance medium, and reaping the cultured infected cell; collecting cell suspension; and extracting and purifying the cell suspension. The production method has the advantages that the operation is simple; the production intensity is low; the high-density large-scale cultivation of stromal cells is realized; the yield and the quality stability of hepatitis A vaccine are improved; and pollution caused by manual operation in the conventional manufacturing technology is avoided.
Description
Technical field
The present invention relates to biological technical field, especially a kind of working method of HAV.
Background technology
Hepatitis A is called for short hepatitis A, is the transmissible disease that is caused by hepatitis A virus, and it is main that this disease is propagated with fecal oral route; HAV excretes with ight soil, can be through the daily life contact, through the propagation of mode per os such as water, food, and developed country is mainly through person to person's contact transmission; Often occur in developing countries and regions through polluted source, food transmission, the most outstanding example is the twice hepatitis A great outburst in Shanghai, all causes without the blood clam that boils because of edible; There is 20,000 people morbidity nineteen eighty-three when breaking out for the first time; Spring in 1988, the patient is nearly 300,000, causes heavy losses for national economy and people ' s health.
At present the domestic mode of production of mainly taking is the rolling bottle formula taked and the static low density training method cultivation of container virus antigen such as cell factory; Again through prepared hepatitis A vaccines such as purifying; It is low that this production technique exists production level, and processing parameter is stable inadequately, simultaneously complicated operation; Production intensity is big, is prone to take place shortcomings such as pollution.Simultaneously because traditional processing technology is less in batches, thereby cause the vaccine product differences between batches big, constant product quality property is good inadequately.
Summary of the invention
The objective of the invention is to solve the deficiency of prior art, the working method of a kind of production of vaccine with HAV is provided.
For achieving the above object, the present invention has taked following technical scheme:
A kind of production of vaccine may further comprise the steps with the working method of HAV successively:
Step 1: the recovery of cell and the amplification of going down to posterity:, and adopt microcarrier bioreactor culture cell with cell recovery;
Step 2: inoculation HAV seed: when cell concn is 10
6Individual/as when ml is above, to abandon nutrient solution, according to the mixed inoculation HAV seed of 0.001～0.01MOI;
Step 3: keep and cultivate the cell that infects HAV: keep the cell of cultivating the infection HAV with keeping liquid, every day, liquid was kept in replacing, kept and cultivated 14～28 days;
Step 4: detect virus infection and duplicate situation: since the 14th day, the virus antigen in the determination of immunofluorescence method cell was adopted in sampling every day, detected virus infection and duplicated situation;
Step 5: stop to cultivate: at virus infection, duplicate the peak period and stop cultivating, abandon and keep liquid, the cells infected that results are cultivated;
Step 6: collecting cell suspension: adopt sterile water for injection or hypotonic solution swelling ruptured cell, and be aided with stirring, cell is peeled off from microcarrier, screen cloth separates microcarrier, collecting cell suspension;
Step 7: with cell suspension extraction, purifying.
Further technical scheme is the cell behaviour diploid cell strain of said step 1, any in continuous cell line and the primary cultured cell.
Further technical scheme is that said human diploid cell strain is WI-38, MRC-5,2BS, any among the KMB17.
Further technical scheme is that said continuous cell line is the Vero cell.
Further technical scheme is, the microcarrier of said step 1 is that particulate state solid microsphere carrier, porousness are microsphere supported, in reticular fiber structure carrier and the sheet-like fiber structure carrier any.
Further technical scheme is that the hepatitis A vaccine production of said step 2 uses viral seed to be hepatitis A virus HM175 strain.
Further technical scheme is that it is that MEM keeps liquid that said step 3 is kept liquid.
Compared with prior art, the present invention has following beneficial effect:
(1) the present invention adopts microcarrier bioreactor culture cell, has realized that the high-density large-scale of stroma cell is cultivated, and has improved the output and the quality stability of hepatitis A vaccine.
(2) the present invention has avoided in the conventional production process because the pollution that manual operation brings.
(3) high expression level of the online detection of the present invention viral cultures that guaranteed to be gathered in the crops has guaranteed the quality of production.
(4) the present invention is simple to operate, and production intensity is little.
Embodiment
Embodiment 1
A kind of production of vaccine may further comprise the steps with the working method of HAV successively:
Step 1: the recovery of cell and the amplification of going down to posterity:, and adopt particulate state solid microsphere carrier organism reactor drum culturing cell with cell recovery;
Step 2: inoculation HAV seed: when cell concn is 10
6Individual/as during ml, to abandon nutrient solution, according to the mixed inoculation hepatitis A virus HM175 strain of 0.001MOI;
Step 3: keep and cultivate the cell that infects hepatitis A virus HM175 strain: keep liquid with MEM and keep the cell of cultivating infection hepatitis A virus HM175 strain, change MEM and keep liquid every day, keeps and cultivated 14 days;
Step 4: detect virus infection and duplicate situation: since the 14th day, the virus antigen in the determination of immunofluorescence method cell was adopted in sampling every day, detected virus infection and duplicated situation;
Step 5: stop to cultivate: at virus infection, duplicate the peak period and stop cultivating, abandon MEM and keep liquid, the cells infected that results are cultivated;
Step 6: collecting cell suspension: adopt sterile water for injection swelling ruptured cell, and be aided with stirring, cell is peeled off screen cloth separating particles shape solid microsphere carrier, collecting cell suspension from particulate state solid microsphere carrier;
Step 7: with cell suspension extraction, purifying.
Further technical scheme is said cell behaviour diploid cell strain WI-38.
Embodiment 2
A kind of production of vaccine may further comprise the steps with the working method of HAV successively:
Step 1: the recovery of cell and the amplification of going down to posterity:, and adopt the microsphere supported bioreactor culture cell of porousness with cell recovery;
Step 2: inoculation HAV seed: when cell concn is 10
7Individual/as during ml, to abandon nutrient solution, according to the mixed inoculation hepatitis A virus HM175 strain of 0.01MOI;
Step 3: keep and cultivate the cell that infects hepatitis A virus HM175 strain: keep liquid with MEM and keep the cell of cultivating infection hepatitis A virus HM175 strain, change MEM and keep liquid every day, keeps and cultivated 28 days;
Step 4: detect virus infection and duplicate situation: since the 14th day, the virus antigen in the determination of immunofluorescence method cell was adopted in sampling every day, detected virus infection and duplicated situation;
Step 5: stop to cultivate: at virus infection, duplicate the peak period and stop cultivating, abandon MEM and keep liquid, the cells infected that results are cultivated;
Step 6: collecting cell suspension: adopt sterile water for injection swelling ruptured cell, and be aided with stirring, cell is peeled off from porousness is microsphere supported, it is microsphere supported that screen cloth separates porousness, the collecting cell suspension;
Step 7: with cell suspension extraction, purifying.
Further technical scheme is said cell behaviour diploid cell strain KMB17.
Embodiment 3
A kind of production of vaccine may further comprise the steps with the working method of HAV successively:
Step 1: the recovery of cell and the amplification of going down to posterity:, and adopt particulate state solid microsphere carrier organism reactor drum culturing cell with cell recovery;
Step 2: inoculation HAV seed: when cell concn is 10
6Individual/as during ml, to abandon nutrient solution, according to the mixed inoculation hepatitis A virus HM175 strain of 0.001MOI;
Step 3: keep and cultivate the cell that infects hepatitis A virus HM175 strain: keep liquid with MEM and keep the cell of cultivating infection hepatitis A virus HM175 strain, change MEM and keep liquid every day, keeps and cultivated 14 days;
Step 4: detect virus infection and duplicate situation: since the 14th day, the virus antigen in the determination of immunofluorescence method cell was adopted in sampling every day, detected virus infection and duplicated situation;
Step 5: stop to cultivate: at virus infection, duplicate the peak period and stop cultivating, abandon MEM and keep liquid, the cells infected that results are cultivated;
Step 6: collecting cell suspension: adopt sterile water for injection swelling ruptured cell, and be aided with stirring, cell is peeled off screen cloth separating particles shape solid microsphere carrier, collecting cell suspension from particulate state solid microsphere carrier;
Step 7: with cell suspension extraction, purifying.
Further technical scheme is said cell behaviour diploid cell strain KMB17.
Embodiment 4
A kind of production of vaccine may further comprise the steps with the working method of HAV successively:
Step 1: the recovery of cell and the amplification of going down to posterity:, and adopt reticular fiber structure carrier organism reactor drum culturing cell with cell recovery;
Step 2: inoculation HAV seed: when cell concn is 10
6Individual/as during ml, to abandon nutrient solution, according to the mixed inoculation hepatitis A virus HM175 strain of 0.001MOI;
Step 3: keep and cultivate the cell that infects hepatitis A virus HM175 strain: keep liquid with MEM and keep the cell of cultivating infection hepatitis A virus HM175 strain, change MEM and keep liquid every day, keeps and cultivated 14 days;
Step 4: detect virus infection and duplicate situation: since the 14th day, the virus antigen in the determination of immunofluorescence method cell was adopted in sampling every day, detected virus infection and duplicated situation;
Step 5: stop to cultivate: at virus infection, duplicate the peak period and stop cultivating, abandon MEM and keep liquid, the cells infected that results are cultivated;
Step 6: collecting cell suspension: adopt hypotonic solution swelling ruptured cell, and be aided with stirring, cell is peeled off from the reticular fiber structure carrier, screen cloth separates reticular fiber structure carrier, collecting cell suspension;
Step 7: with cell suspension extraction, purifying.
Further technical scheme is that said cell is a Vero clone.
Embodiment 5
A kind of production of vaccine may further comprise the steps with the working method of HAV successively:
Step 1: the recovery of cell and the amplification of going down to posterity:, and adopt sheet-like fiber structure carrier bioreactor culture cell with cell recovery;
Step 2: inoculation HAV seed: when cell concn is 10
8Individual/as during ml, to abandon nutrient solution, according to the mixed inoculation hepatitis A virus HM175 strain of 0.01MOI;
Step 3: keep and cultivate the cell that infects hepatitis A virus HM175 strain: keep liquid with MEM and keep the cell of cultivating infection hepatitis A virus HM175 strain, change MEM and keep liquid every day, keeps and cultivated 28 days;
Step 4: detect virus infection and duplicate situation: since the 14th day, the virus antigen in the determination of immunofluorescence method cell was adopted in sampling every day, detected virus infection and duplicated situation;
Step 5: stop to cultivate: at virus infection, duplicate the peak period and stop cultivating, abandon MEM and keep liquid, the cells infected that results are cultivated;
Step 6: collecting cell suspension: adopt hypotonic solution swelling ruptured cell, and be aided with stirring, cell is peeled off screen cloth isolating plate-like fibrous texture carrier, collecting cell suspension from the sheet-like fiber structure carrier;
Step 7: with cell suspension extraction, purifying.
Further technical scheme is that said cell is a primary cultured cell.
Claims (7)
1. a production of vaccine is characterized in that: may further comprise the steps successively with the working method of HAV:
Step 1: the recovery of cell and the amplification of going down to posterity:, and adopt microcarrier bioreactor culture cell with cell recovery;
Step 2: inoculation HAV seed: when cell concn is 10
6Individual/as when ml is above, to abandon nutrient solution, according to the mixed inoculation HAV seed of 0.001～0.01MOI;
Step 3: keep and cultivate the cell that infects HAV: keep the cell of cultivating the infection HAV with keeping liquid, every day, liquid was kept in replacing, kept and cultivated 14～28 days;
Step 4: detect virus infection and duplicate situation: since the 14th day, the virus antigen in the determination of immunofluorescence method cell was adopted in sampling every day, detected virus infection and duplicated situation;
Step 5: stop to cultivate: at virus infection, duplicate the peak period and stop cultivating, abandon and keep liquid, the cells infected that results are cultivated;
Step 6: collecting cell suspension: adopt sterile water for injection or hypotonic solution swelling ruptured cell, and be aided with stirring, cell is peeled off from microcarrier, screen cloth separates microcarrier, collecting cell suspension;
Step 7: with cell suspension extraction, purifying.
2. a kind of production of vaccine according to claim 1 is characterized in that with the working method of HAV: the cell behaviour diploid cell strain of said step 1, any in continuous cell line and the primary cultured cell.
3. a kind of production of vaccine according to claim 2 is characterized in that with the working method of HAV: said human diploid cell strain is WI-38, MRC-5,2BS, any among the KMB17.
4. a kind of production of vaccine according to claim 2 is characterized in that with the working method of HAV: said continuous cell line is the Vero cell.
5. a kind of production of vaccine according to claim 1 is characterized in that with the working method of HAV: the microcarrier of said step 1 is that particulate state solid microsphere carrier, porousness are microsphere supported, in reticular fiber structure carrier and the sheet-like fiber structure carrier any.
6. a kind of production of vaccine according to claim 1 is characterized in that with the working method of HAV: the hepatitis A vaccine production of said step 2 uses viral seed to be hepatitis A virus HM175 strain.
7. a kind of production of vaccine according to claim 1 is characterized in that with the working method of HAV: it is that MEM keeps liquid that said step 3 is kept liquid.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103590914A CN102492659A (en) | 2011-11-14 | 2011-11-14 | Production method for hepatitis A virus for vaccine production |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011103590914A CN102492659A (en) | 2011-11-14 | 2011-11-14 | Production method for hepatitis A virus for vaccine production |
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| CN102492659A true CN102492659A (en) | 2012-06-13 |
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| CN2011103590914A Pending CN102492659A (en) | 2011-11-14 | 2011-11-14 | Production method for hepatitis A virus for vaccine production |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602639A (en) * | 2013-08-16 | 2014-02-26 | 科兴（大连）疫苗技术有限公司 | Method of harvesting viruses by low-osmotic-pressure harvest liquid |
| CN114350624A (en) * | 2021-12-28 | 2022-04-15 | 艾美康淮生物制药（江苏）有限公司 | Method for preparing hepatitis A inactivated vaccine |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1147833A (en) * | 1994-03-08 | 1997-04-16 | 麦克公司 | Hepatitis A virus culture process |
| US20030108860A1 (en) * | 2001-12-10 | 2003-06-12 | Manfred Reiter | Method for large scale production of virus antigen |
| CN100374552C (en) * | 2001-12-10 | 2008-03-12 | 巴克斯特健康护理股份有限公司 | Method of large scale production of hepatitis a virus |
-
2011
- 2011-11-14 CN CN2011103590914A patent/CN102492659A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1147833A (en) * | 1994-03-08 | 1997-04-16 | 麦克公司 | Hepatitis A virus culture process |
| US20030108860A1 (en) * | 2001-12-10 | 2003-06-12 | Manfred Reiter | Method for large scale production of virus antigen |
| CN100374552C (en) * | 2001-12-10 | 2008-03-12 | 巴克斯特健康护理股份有限公司 | Method of large scale production of hepatitis a virus |
Non-Patent Citations (1)
| Title |
|---|
| 孙明波等: "应用生物反应器培养Vero细胞制备甲肝病毒", 《第三军医大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602639A (en) * | 2013-08-16 | 2014-02-26 | 科兴（大连）疫苗技术有限公司 | Method of harvesting viruses by low-osmotic-pressure harvest liquid |
| CN103602639B (en) * | 2013-08-16 | 2016-04-27 | 科兴（大连）疫苗技术有限公司 | A kind of method adopting Hyposmolality harvest liquid to gather in the crops virus |
| CN114350624A (en) * | 2021-12-28 | 2022-04-15 | 艾美康淮生物制药（江苏）有限公司 | Method for preparing hepatitis A inactivated vaccine |
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Application publication date: 20120613 |