CN102660510B - A kind of method utilizing bio-reactor to produce transmissible gastro-enteritis virus - Google Patents
A kind of method utilizing bio-reactor to produce transmissible gastro-enteritis virus Download PDFInfo
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Abstract
The invention provides a kind of method utilizing bio-reactor to produce transmissible gastro-enteritis virus, comprise the steps: 1) prepare individual layer passage cell; 2) transmissible gastro-enteritis virus production kind of poison is prepared; 3) by step 1) the individual layer passage cell prepared is prepared into cell suspension, and be inoculated in bio-reactor, absorption on the microcarrier of passage cell in bio-reactor is cultivated; 4) when passage cell grows to the 80%-90% of microcarrier, empty ball rate is lower than 5%, and full ball rate is greater than 80%, and cell counting reaches 3-5 × 10
6individual/more than mL time, the inoculum size inoculation step 2 with 2-5%) prepare kind poison, carry out virus absorption cultivate; 4) when passage cell more than 80% pathology on microcarrier, results virus liquid.The inventive method can solve the problem that production efficiency is low, unstable product quality, virus titer are low, can improve viral units cultivation and tire 5-10 doubly, General Promotion vaccine quality and output, improve the security of vaccine.
Description
Technical field
The present invention relates to a kind of production method of virus, particularly, relate to the production method of a kind of transmissible gastro-enteritis virus, more specifically, relate to a kind of method utilizing bio-reactor to produce transmissible gastro-enteritis virus.
Background technology
Transmissible gastroenteritis of swine (PorcineTransmissibleGastroenteritis, TGE) be by the transmissible gastro-enteritis virus (PorcineTransmissibleGastroenteritisvirus of coronaviridae (Coronaviridae), coronavirus genus (Coronavirus), what TGEV) cause vomits with pig, suffers from diarrhoea, dewaters as the transmissible disease of feature, to newborn piglet, there is height lethality rate, be generally 100%.Although the pig of different ages is to the equal susceptible of this virus, after pig more than 5 week age infects, mortality ratio is very low.There is this after being ill in the report U.S. in 1945 in Doyle, many countries report transmissible gastroenteritis of swine all in succession in the world, and within 1956, China Guangdong this occurs first after being ill, and also there is generation in the whole nation in province mostly, has carried out serious harm to industrial belt of raising pigs first.
At present, domestic transmissible gastroenteritis of swine production of vaccine uniquely relies on cell spinner bottle culture method to realize.This traditional technology labour intensity is large, and length consuming time, efficiency are low, and production cost is high; Easily by environmental pollution; Difference between different production batch or between the different bottle of same production batch is large; Be difficult to expanding production; The vaccine quality hidden danger that easy appearance is caused by bacterium or other virus contamination, relates to Biosafety and public health problem.
Summary of the invention
In order to solve the problem, the invention provides a kind of method utilizing bio-reactor to produce transmissible gastro-enteritis virus.
Method provided by the invention, comprises the steps:
1) individual layer passage cell is prepared;
2) transmissible gastro-enteritis virus production kind of poison is prepared;
3) by step 1) after the digestion of the individual layer passage cell prepared, suspend with cell growth medium, being prepared into cell density is 1-3 × 10
5the cell suspension of individual/mL, is inoculated in bio-reactor, and absorption on the microcarrier of passage cell in bio-reactor is cultivated;
4) when passage cell grows to the 80%-90% of microcarrier, empty ball rate is lower than 5%, and full ball rate is greater than 80%, and cell counting reaches 3-5 × 10
6individual/more than mL time, the inoculum size inoculation step 2 with 2-5%) prepare kind poison, carry out virus absorption cultivate;
4) when passage cell more than 80% pathology on microcarrier, results virus liquid.
Wherein, described method is also included in step 3) in cell suspension inoculation to before bio-reactor, steril cell growth media is added by the 50-70% of bio-reactor cumulative volume, and add microcarrier by 4-10g/L concentration in often liter of steril cell growth media, start bio-reactor, 20-50 minute is stirred in the slow speed of revolution.
Wherein, described method also comprise microcarrier joined cell growth medium before microcarrier carried out clean and the step of sterilizing, be followed successively by with PBS immersion microcarrier more than 3 hours; Microcarrier 3-5 time is cleaned with PBS; Microcarrier is soaked, 121 DEG C of steam sterilizing 20-50min with PBS.
Wherein, step 2) preparation of transmissible gastro-enteritis virus production kind poison specifically comprises the steps:
With virus-culturing fluid transmissible gastro-enteritis virus kind poison is inoculated in individual layer passage cell in 2-5% ratio and cultivates, gather in the crops virus liquid to during cell more than 80% pathology; Again using this virus liquid as kind of a poison, cell carries out cell adaptation continuous passage rejuvenation, and the product that at every turn goes down to posterity carries out viral TCID
50and Sterility testing, reach viral TCID
50stablize and reach 10
7tCID
50rejuvenation is stopped, as production kind of a poison during/more than mL.Wherein, described virus-culturing fluid can be the virus-culturing fluid of this area routine, and preferred formula is: massfraction is 98%-99%MEM liquid respectively, 1%-2% bovine serum, final concentration are the antibiotic of 100-200IU/mL, and pH value is 6.8-7.5.
Wherein, step 3) in individual layer passage cell EDTA-trysinization liquid digest, described EDTA-trysinization liquid is be Hank ' the S liquid of the pancreatin of 0.05%-0.25% and the EDTA of 0.02% containing quality volume fraction.
Wherein, step 3) described in cell suspension in the density of passage cell be 1-3 × 10
5individual/mL.
Wherein, step 3) described in the formula of cell growth medium be: massfraction is 95%-98%MEM liquid, 2%-5% bovine serum, final concentration are the antibiotic of 100-200IU/mL, and pH value is 6.8-7.5.
Wherein, described passage cell can be the conventional cell for virus culture in this area, is preferably ST cell or vero cell, most preferably is ST cell.
In the present invention, bio-reactor setting parameter be preferably: culture temperature 35-38 DEG C, pH value 6.8-7.5, dissolved oxygen 50%-70%, stirring velocity 50-80rpm.
Wherein, described transmissible gastro-enteritis virus can be the transmissible gastro-enteritis virus strain of any routine, is preferably floating plumage vaccine strain, h-5 vaccine strain or magnificent strain.
Wherein, described microcarrier can be any microcarrier of this area routine, common are Gelatin based Macroporous Microcarriers, polystyrene microcarrier, PHEMA microcarrier, chitin microcarrier, polyurethane foam microcarrier, alginate jelly microcarrier, magnetic microcarrier, Cytodex1, Cytodex2, Cytodex3, Cytopore and/or Cytoline, be preferably Cytodex1, Cytodex2, Cytodex3, Cytopore and/or Cytoline.
Compared with prior art, the present invention has following beneficial effect:
(1) rolling bottle cell culture technology is replaced to manufacture transmissible gastro-enteritis virus vaccine with bio-reactor Microcarrier Cell Culture Techniques, the problem that production efficiency is low, unstable product quality, virus titer are low can be solved, by the change of production technology and production technique, improve viral units cultivation and tire 5-10 doubly, General Promotion vaccine quality and output, improve the security of vaccine.For vero cell, the bio-reactor of advanced international level is produced and can be reached 10
7/ mL, and rolling bottle produces general 10
6/ mL has been the limit.
(2) the present invention can reduce production cost in a large number, and with short production cycle, and each production cycle only needs 5-6 days, and more than 6-7 days that compare existing rolling bottle cell culture method obviously shorten.
(3) low stain: 1 tank=100 ~ 2000 rolling bottle, more less contamination probability is lower for probability operation link.
(4) production technique is controlled: high-accuracy cultivation, Real-Time Monitoring dissolved oxygen, aerogenesis, PH, cell state etc., and comprehensive parameters setting is cultivated curve, directly ensured the state of carrier cell; Batch is large, difference between batch is little; Save space, personnel's (reaching as high as 1: 75); Once moulding process will be followed the prescribed order very stable.
Accompanying drawing explanation
Fig. 1 is that the present invention utilizes bio-reactor to produce the method flow diagram of transmissible gastro-enteritis virus.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
(1) select bio-reactor as the means of cultivating: 7L Switzerland Lambda bio-reactor.
(2) carrier selecting microcarrier to grow as cell attachment: microcarrier Cytodex2.
(3) cleaning of microcarrier, sterilising method: 1) weigh Cytodex2 microcarrier 10g/L, use 2LPBS immersion to steep 3 hours.2) clean 3 times with 2LPBS liquid at every turn.3) 2LPBS immersion bubble microcarrier is added, 121 DEG C of steam sterilizing 30min.
(4) select ST cell as seedling cell.
(5) the going down to posterity and cultivation of seedling cell: above-mentioned cell is through EDTA-pancreatin (Hank ' the S liquid containing 0.25% pancreatin, 0.02%EDTA) had digestive transfer culture, with containing 95%MEM liquid, 5% calf serum, the benzylpenicillin sodium of 200IU/mL and Vetstrep, pH value is adjusted to the cell growth medium continuation cultivation of 7.2, and culture temperature is 37 DEG C.When forming good individual layer, carry out microcarrier suspension culture for being inoculated in bio-reactor.
(6) breeding of cell seed culture of viruses: (be benzylpenicillin sodium and the Vetstrep of 200IU/mL containing the MEM liquid of calf serum 2%, final concentration with virus-culturing fluid, by the kind poison of the transmissible gastro-enteritis virus China strain well-grown above-mentioned cell monolayer of 2% ratio inoculation by volume, continue to cultivate, culture temperature is 35 DEG C.Virus liquid is gathered in the crops to during cell more than 80% pathology; Measure the TCID of virus
50, treat that virus titer is stablized and reaches 10
7tCID
50rejuvenation is stopped, using this virus as production kind of a poison during/more than mL.
(7) microcarrier suspension culture of ST in bio-reactor: get well-grown ST cell on square vase, one time is cleaned with the PBS that pH value is 7.2, add the digestion of aforesaid EDTA-trysinization liquid, treat that cellular layer starts to occur that full wafer comes off, add described cell growth medium, blow to hang and make cell suspending liquid, cell counting density is by 2 × 10
5in/mL reaction of inoculation device.Bio-reactor is set as follows parameters of temperature 37 DEG C, pH value 7.2, stirring velocity 60rpm, dissolved oxygen content 50% carry out reactor and automatically control to cultivate.
(8) breeding of seedling venom: after cultivating, cells on microcarriers to be seen on the 3rd covers with (more than 95%) substantially, and cell counts reaches 4 × 10
6after/more than mL, be 2% virus inoculation by volume.The culture parameters such as design temperature 34 DEG C, pH value 6.8, stirring velocity 70rpm, dissolved oxygen content 30%, carry out reactor and automatically control to cultivate.Get microcarrier in reactor at regular intervals after connecing poison, by microscope observing cell pathology situation, and detect the TCID of sample
50.Cells on microcarriers to be seen substantially all comes off (about 36h), and the obvious ascendant trend of DO value, stopped reaction device stirs, and after microcarrier all sinks to reactor bottom, gathers in the crops supernatant liquor and microcarrier respectively.
The process of results virus liquid: results supernatant, after 3 multigelations, filters-20 DEG C of freezen protective after removing cell debris for subsequent use.
Embodiment 2
(1) select bio-reactor as the means of cultivating: 7L Switzerland Lambda bio-reactor.
(2) carrier selecting microcarrier to grow as cell attachment: microcarrier Cytodex3.
(3) cleaning of microcarrier, sterilising method: 1) weigh Cytodex3 microcarrier 6g/L, use 2LPBS immersion to steep 3 hours.2) clean 3 times with 2LPBS liquid at every turn.3) 2LPBS immersion bubble microcarrier is added, 121 DEG C of steam sterilizing 30min.
(4) select vero cell as seedling cell.
(5) the going down to posterity and cultivation of seedling cell: above-mentioned cell is through EDTA-pancreatin (Hank ' the S liquid containing 0.25% pancreatin, 0.02%EDTA) had digestive transfer culture, with containing 96%MEM liquid, 4% calf serum, the benzylpenicillin sodium of 200IU/mL and Vetstrep, pH value is adjusted to the cell growth medium continuation cultivation of 7.2, and culture temperature is 37 DEG C.When forming good individual layer, carry out microcarrier suspension culture for being inoculated in bio-reactor.
(6) breeding of cell seed culture of viruses: (be benzylpenicillin sodium and the Vetstrep of 100IU containing the MEM liquid of calf serum 1%, final concentration with virus-culturing fluid, pH value is adjusted to 7.3), be the well-grown above-mentioned cell monolayer of 5% ratio inoculation by volume by the kind poison of transmissible gastro-enteritis virus China strain, continue to cultivate, culture temperature is 35 DEG C.Virus liquid is gathered in the crops to during cell more than 80% pathology; Measure the TCID of virus
50, treat that virus titer is stablized and reaches 10
7tCID
50rejuvenation is stopped, using this virus as production kind of a poison during/more than mL.
(7) microcarrier suspension culture of vero cell in bio-reactor: get well-grown vero cell on square vase, one time is cleaned with the PBS that pH value is 7.2, add the digestion of aforesaid EDTA-trysinization liquid, treat that cellular layer starts to occur that full wafer comes off, add described cell growth medium, blow to hang and make cell suspending liquid, cell counting density is by 1 × 10
5in/mL reaction of inoculation device.Bio-reactor is set as follows parameters of temperature 37 DEG C, pH value 7.2, stirring velocity 60rpm, dissolved oxygen content 50% carry out reactor and automatically control to cultivate.
(8) breeding of seedling venom: after cultivating, cells on microcarriers to be seen on the 3rd covers with (more than 95%) substantially, and cell counts reaches 5 × 10
6after/more than mL, be 2% carry out virus inoculation by volume.The culture parameters such as design temperature 38 DEG C, pH value 7.5, stirring velocity 50rpm, dissolved oxygen content 50%, carry out reactor and automatically control to cultivate.Gather in the crops virus liquid two days later.The process of results virus liquid: results supernatant, after 3 multigelations, filters-20 DEG C of freezen protective after removing cell debris for subsequent use.
Comparative example 1 spinner culture transmissible gastro-enteritis virus
(1) select rolling bottle as the means of cultivating: 3L cell-culturing rotating bottle, ZP-01-16 Rotary Machine.
(2) select vero cell as seedling cell.
(3) the going down to posterity and cultivation of seedling cell: above-mentioned cell is through EDTA-pancreatin (Hank ' the S liquid containing 0.25% pancreatin, 0.02%EDTA) had digestive transfer culture, with containing 95%MEM liquid, 5% calf serum, the benzylpenicillin sodium of 200IU/mL and Vetstrep, pH value is adjusted to the cell growth medium continuation cultivation of 7.2, and culture temperature is 37 DEG C.When forming good individual layer, for being inoculated in spinner culture.
(4) breeding of cell seed culture of viruses: (be benzylpenicillin sodium and the Vetstrep of 200IU containing the MEM liquid of serum 1%, final concentration with virus-culturing fluid, pH value is adjusted to 7.3), be the well-grown above-mentioned cell monolayer of 5% ratio inoculation by volume by the kind poison of transmissible gastro-enteritis virus China strain, continue to cultivate, culture temperature is 35 DEG C.Virus liquid is gathered in the crops to during cell more than 80% pathology; Measure the TCID of virus
50, treat that virus titer is stablized and reaches 10
7tCID
50rejuvenation is stopped, using this virus as production kind of a poison during/more than mL.
(5) cultivation of vero cell in rolling bottle: get well-grown vero cell pH value on square vase be 7.2 PBS clean one time, add described EDTA-trysinization liquid digestion, treat that cellular layer starts to occur that full wafer comes off, add described cell growth medium, blow to hang and make cell suspending liquid, cell counting density is by 3 × 10
5/ mL inoculates 3000mL rolling bottle, and spinner culture amount is 300mL, 37 DEG C, and 20rpm/h is cultured to cell and grows up to individual layer.
(6) breeding of seedling venom: cultivate after the 3rd day to be seen, on rolling bottle wall, cell covers with (more than 95%) substantially, and cell counts reaches 5 × 10
6virus inoculation is carried out after/more than mL.Be the inoculum size virus inoculation of 2% by volume, after 4 days, gather in the crops vial supernatant.The process of results virus liquid: results supernatant, after 3 multigelations, filters-20 DEG C of freezen protective after removing cell debris for subsequent use.
Test example 1 inspection of semifinished product
1. gather in the crops the mensuration of virus liquid poison valency: sample after the cytopathy venom mixing of gathering in the crops above, measure malicious valency.
Result shows, viral level>=10 of the virus that embodiment 1 and 2 is produced with bio-reactor
7tCID
50/ mL, and viral level≤10 that comparative example 1 spinner culture is produced
6tCID
50/ mL.
2. the inspection of semifinished product after deactivation
Deactivation: add formaldehyde solution, jolting 2 minutes by 0.2% of total amount in the virus liquid of embodiment 1 ~ 2 and comparative example 1, put 37 DEG C of condition deactivations 24 hours, period timing agitation mixing.
Steriling test: undertaken by " People's Republic of China's veterinary biologics quality standard " annex 301 pages, the equal asepsis growth of virus of the embodiment of the present invention and comparative example.
Deactivation is checked: after deactivation, sample with aseptic technique from inactivation of virus liquid, inoculate healthy ST cell monolayer in 1% ratio, cultivate 3 days for 37 DEG C, according to said method, continuous passage 3 generation, cell should not occur that pathology is qualified, and the embodiment of the present invention and comparative example are all without pathology, therefore product is qualified.
3. bioreactor culture virus and the comparing of spinner culture virus immunity effect
Respectively getting that 200 μ L tire is 1 × 10
6the virus immunity of/mL is without each 20 of the piglet of TGEV neutralizing antibody, and using 200 μ L virus-culturing fluids as blank, high 4 times than spinner culture of the NAT of result bioreactor culture virus immunity group, result is as following table.
Table 1 bioreactor culture virus compares with spinner culture virus immunity effect
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (5)
1. utilize bio-reactor to produce a method for transmissible gastro-enteritis virus, it is characterized in that, comprise the steps:
1) individual layer passage cell is prepared;
2) transmissible gastro-enteritis virus production kind of poison is prepared, specifically comprise the steps: with virus-culturing fluid transmissible gastro-enteritis virus kind poison to be inoculated in individual layer passage cell in 2-5% ratio to cultivate, gather in the crops virus liquid to during cell more than 80% pathology; Again using this virus liquid as kind of a poison, cell carries out cell adaptation continuous passage rejuvenation, and the product that at every turn goes down to posterity carries out viral TCID
50and Sterility testing, reach viral TCID
50stablize and reach 10
7tCID
50rejuvenation is stopped, as production kind of a poison during/more than mL;
3) before cell suspension inoculation to bio-reactor, steril cell growth media is added by the 50-70% of bio-reactor cumulative volume, and add microcarrier by 4-10g/L concentration in often liter of steril cell growth media, start bio-reactor, 20-50 minute is stirred in the slow speed of revolution, by step 1) after the individual layer passage cell EDTA-trysinization liquid prepared digests, suspend with cell growth medium, being prepared into cell density is 1-3 × 10
5the cell suspension of individual/mL, be inoculated in bio-reactor, absorption on the microcarrier of passage cell in bio-reactor is cultivated, described EDTA-trysinization liquid is be Hank ' the S liquid of the pancreatin of 0.05%-0.25% and the EDTA of 0.02% containing quality volume fraction, the formula of described cell growth medium is: massfraction is 95%-98%MEM liquid, 2%-5% bovine serum, final concentration is the antibiotic of 100-200IU/mL, pH value is 6.8-7.5, the parameter of bio-reactor setting is: culture temperature 35-38 DEG C, pH value 6.8-7.5, dissolved oxygen 50%-70%, stirring velocity 50-80rpm,
4) when passage cell grows to the 80%-90% of microcarrier, empty ball rate is lower than 5%, and full ball rate is greater than 80%, and cell counting reaches 3-5 × 10
6individual/more than mL time, the inoculum size inoculation step 2 with 2-5%) prepare kind poison, carry out virus absorption cultivate;
5) when passage cell more than 80% pathology on microcarrier, results virus liquid.
2. method according to claim 1, is characterized in that, also comprises and to be carried out by microcarrier before microcarrier joined cell growth medium cleaning and the step of sterilizing, be followed successively by with PBS immersion microcarrier more than 3 hours; Microcarrier 3-5 time is cleaned with PBS; Microcarrier is soaked, 121 DEG C of steam sterilizing 20-50min with PBS.
3. method according to claim 1 and 2, is characterized in that, described passage cell is ST cell or vero cell.
4. method according to claim 1 and 2, is characterized in that, described transmissible gastro-enteritis virus is floating plumage vaccine strain, h-5 vaccine strain or magnificent strain.
5. method according to claim 1 and 2, it is characterized in that, described microcarrier is Gelatin based Macroporous Microcarriers, polystyrene microcarrier, PHEMA microcarrier, chitin microcarrier, polyurethane foam microcarrier, alginate jelly microcarrier, magnetic microcarrier, Cytodex1, Cytodex2, Cytodex3, Cytopore and/or Cytoline.
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| CN103550771B (en) * | 2013-10-31 | 2016-05-25 | 成都天邦生物制品有限公司 | The production method of transmissible gastro-enteritis virus vaccine |
| CN103877571A (en) * | 2014-03-21 | 2014-06-25 | 吉林正业生物制品股份有限公司 | Preparation method and product of swine transmissible gastroenteritis inactivated vaccine |
| US11667906B2 (en) | 2014-11-24 | 2023-06-06 | Corning Incorporated | Magnetic microcarriers |
| CN104651320A (en) * | 2015-03-05 | 2015-05-27 | 成都天邦生物制品有限公司 | Method for producing porcine transmissible gastroenteritis Hua strain virus by low-serum culture of ST cells |
| CN105400743A (en) * | 2015-12-08 | 2016-03-16 | 天津瑞普生物技术股份有限公司 | Preparation method of TGEV |
| CN106754749B (en) * | 2016-12-14 | 2019-12-10 | 四川省华派生物制药有限公司 | Method for improving virus content of virus liquid by adopting cell culture process twice |
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Effective date of registration: 20200629 Address after: 211102, 999 Ting Dong Road, Jiangning economic and Technological Development Zone, Nanjing, Jiangsu Co-patentee after: Zhaofenghua Biotechnology (Fuzhou) Co.,Ltd. Patentee after: Zhaofenghua Biotechnology (Nanjing) Co.,Ltd. Address before: 100080 Beijing City, Haidian District Zhongguancun street, No. 27 Zhongguancun building 14 DaBeiNong group Co-patentee before: Zhaofenghua Biotechnology (Fuzhou) Co.,Ltd. Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. |
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