CN101851610A - Method for producing rabies virus antigens for animals at a large scale - Google Patents
Method for producing rabies virus antigens for animals at a large scale Download PDFInfo
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- CN101851610A CN101851610A CN201010135137A CN201010135137A CN101851610A CN 101851610 A CN101851610 A CN 101851610A CN 201010135137 A CN201010135137 A CN 201010135137A CN 201010135137 A CN201010135137 A CN 201010135137A CN 101851610 A CN101851610 A CN 101851610A
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Abstract
The invention discloses a method for producing rabies virus antigens for animals at a large scale, which produces the rabies virus antigens at a large scale by utilizing a bioreactor by a cell microcarrier suspension culture system. The method comprises the following steps: inoculating cells for preparing the antigens into a carrier tank containing a culture solution and microcarriers to enable the cells to be attached to the microcarriers; in a proper culture environment, enabling the cells to grow on the microcarriers until the quantity of the cells is 5-40 times more than inoculum density; making rabies viruses into a virus suspension, and enabling the virus suspension to be adsorbed on the cells; culturing the viruses in the proper culture environment by using a cell maintenance culture solution; continuously culturing for 3-5 days and then harvesting a virus solution for the first time, wherein a semicontinuous process is adopted and the ratio of a changed solution is 50 percent; continue culturing for 9-11 days, and harvesting the changed solution once every 24 hours; mixing the harvested virus solution with the virus solution of the bioreactor; and carrying out freeze thawing at the temperature of -20 DEG C and inactivation purification to obtain the rabies virus antigens. The method has large production scale, high single-scale yield and relatively low production cost.
Description
Technical field
The invention belongs to the veterinary biologics technical field, relate to animal virus, more specifically relate to the method for producing rabies virus antigens for animals.
Background technology
Rabies are that (Rabies virus RV) infects the natural epidemic disease source property people beast a kind of disease serious, that case fatality rate is high cause and suffers from transmissible disease altogether, and mortality ratio almost 100% does not still have therapy measure at present by rabies virus.Wildlife is the main reservoir host of RV, and in a single day the people is carried this type of animal bite of rabies virus, will infection morbidity, and its case fatality rate is 100%.According to the epidemic statistic data of the up-to-date announcement of the Ministry of Health, show that rabies continue to present dangerous sign in recent years.But rabies do not have special effective treatment means, and injecting pstudorabies vaccine is unique effective means.
Hydrophobia in the market substantially all is to adopt rolling bottle technology passage cell to cultivate, and scale operation needs a large amount of material resources manpower, and cost is higher, and differences between batches are big, the quality instability.
What production of vaccine need be a large amount of efficiently produces virus with high yield from host system.The culture condition of virus strain growth is significant for the acceptable high yield that obtains this strain system.Therefore, the output of the virus of wanting in order to maximize, system and culture condition all must specifically be suitable for providing advantageous environment for producing the virus of being wanted.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the weak point that existing technology exists, and a kind of method of tidal type cell microcarrier suspension culture system scale operation rabies virus antigens for animals is provided.This method production technique is simple and stable, easy to operate, virus antigen output height, and differences between batches are little, and system easy to control the quality can significantly improve vaccine output and quality, reduces cost.
For achieving the above object, the present invention utilizes bio-reactor, produces rabies virus antigens for animals with cell microcarrier suspension culture system, comprises following technological step:
(1) cultivate preparation antigen cell:
Preparation antigen is inoculated in the carrier tank that contains nutrient solution and microcarrier with host cell, and above-mentioned cell and microcarrier are mixed, start the attaching program, cell is attached on the microcarrier; Switch the cell cultures program, under suitable culture environment, provide enough nutrients of above-mentioned cell and suitable atmosphere surrounding, make cell on above-mentioned microcarrier, grow to 5～40 times of inoculum density.
(2) inoculation of venom and breeding:
Rabies virus is made viral suspension, it is adsorbed onto on the above-mentioned cell; Use cell maintenance culture solution instead, suitably cultivating virus under the culture environment; After the cultured continuously 3～5 days, gather in the crops viral liquid for the first time, adopt half-continuous process, changing liquid proportional is 50%; Continue to cultivate 9～11, every 24h results are changed liquid once, and changing liquid proportional is 50%.
(3) through after the assay was approved, the viral liquid that half-continuous process is gathered in the crops mixes with the viral liquid of bio-reactor, and in-20 ℃ of freeze thawing twice, the deactivation purifying obtains rabies virus antigen.
Virus liquid check: the relevant regulations by 15,19,20 pages of " People's Republic of China's veterinary drug allusion quotation " version appendix in 2005 is tested, and meets fully, and safety has no side effect, no bacterium, mould, mycoplasma and exogenous virus pollution.Measure the every 1.0ml viral level of viral liquid 〉=10 by inoculation method in the mouse brain
7.0LD
50
In the technique scheme, described host cell be for can breeding the clone of rabies virus, as hamster nephrocyte clone (BHK21) etc.
Nutrient solution described in the inventive method is 95%～98%DMEM preferably, adds 2%～5% bovine serum, adds an amount of antibiotic, and the pH value is 7.0～7.4 better.
In the technique scheme, the rabies virus of described inoculation can be the LEP-Flury strain.
In the technique scheme, described suitable microcarrier of the present invention can be for having cancellated fiber, as trevira etc.
The mode of cell acquisition gaseous interchange can realize by the mode of nutrient solution liquid surface lifting in the inventive method.
In the technique scheme, in the step (1), generally start the attaching program after 4h change the cell cultures program into.Described cell attaches the program optimum parameter: up:2400mL/min hold 1min, Down:2400mL/min hold 1min, setting maximum are changed liquid measure 18500mL; Cell cultures program optimum parameter is: up:1800mL/min hold 1min, Down 1800mL/min hold 1min, setting maximum are changed liquid measure 18500mL.
In the technique scheme, microcarrier consumption described in the step (1) is that every 500ml nutrient solution interpolation 5.5g microcarrier is better, and the initial inoculation amount of cell is 1.8～3.6 * 10
7The cells/g microcarrier is better.
In the technique scheme, cell density is generally 1.1 * 10 during virus inoculation described in the step (2)
8～1.0 * 10
9The cells/g microcarrier.
In the technique scheme, during virus inoculation described in the step (2) be 0.01～1 ratio in virus infection plural number (M.O.I.).
In the technique scheme, in the step (2), 4h changes the virus culture program into after the general startup absorption program, and described viral absorption program optimum parameter is: up:1500mL/min hold 1min, Down:1500mL/min hold 1min, setting maximum are changed liquid measure 18500mL; Virus culture program optimum parameter is: up:1000mL/min hold 1min, Down 1000mL/min hold 1min, setting maximum are changed liquid measure 18500mL.
In the technique scheme, the envrionment temperature of cell and virus culture is generally 36 ℃～37 ℃, contains CO in the culture environment
2Concentration is 2.5%～5% better.
In the inventive method, the bio-reactor that tidal type microcarrier suspension culture technology adopts mainly is made up of carrier tank, container for storing liquid, pH controller, DO monitor, input and output system.Working process is as follows: cell is attached on the carrier in the carrier tank grows, and when the nutrient solution of container for storing liquid was pumped to carrier tank, the nutrient solution liquid level rose and supplies with nutrient and promote the removal of cell metabolism product to cell; When the nutrient solution of carrier tank pumps into container for storing liquid, cultivate liquid level and descend thereupon, cell is ventilated, promote to breathe, reduce the cell tangential pressure, no O
2The supply restriction, the non-foam worry.This multiple motion makes the cell on the carrier can access enough nutrition and O
2, produced simultaneously metabolic waste such as CO
2Can effectively be discharged from, thus amplifying cells that can be a large amount of and increment virus, and this kind technology is referred to as tidal type microcarrier suspension culture technology.
Method of producing rabies virus antigens for animals with the tidal type cell microcarrier suspension culture system provided by the present invention is compared with traditional rolling bottle passage cell culture technique, has more following advantage:
1. employing the inventive method, single batch of problem such as output is not high, differences between batches big, unstable product quality, labour intensity are big, production cost height when having solved traditional rolling bottle scale operation.
2. the carrier that adopts in the inventive method is netted trevira, possess hydrophilic property and biological innocuousness, and the 1g carrier accounts for the space of 15ml, and 2400cm can be provided
2Adherent area, in same space, increased the adherent area of cell greatly, increased the density of cell growth, cell count can reach 1.0 * 10
9More than, its usefulness is tens of times of traditional rolling bottle culture systems, can save many costs and manpower.
3. the cell inoculation amount of the inventive method preparation is low, and control is easy, and virus infection efficient height, and the antigen of the high titre that obtains can improve the immunological competence of vaccine greatly.
The invention provides a kind of new Technology, can greatly satisfy a large amount of breeding of virus conditions needed, and can prepare the antigen of high titre, it is required to satisfy vaccine production greatly, has significantly improved the output and the quality of vaccine, reduces production costs.
Embodiment
Employed bio-reactor is the TideCell-020 type that U.S. CE SCO company produces among the embodiment, and employed carrier is the BioNoc II trevira of the said firm, wide 5mm, and long 10mm, the 1g carrier accounts for the space of 15ml, and 2400cm is provided
2Adherent area, can provide 1.0 * 10
9The cell growth of above quantity.
Embodiment 1
1. virus and cell strain
The virus strain that is used to prepare rabies virus antigen is the LEP-Flury strain, and through pathogenic test, this strain virus does not have pathogenic.This virus strain is had the clone hamster nephrocyte (BHK21) of good sensitivity,, use and infect and a large amount of breeding rabies virus as seedling clone.
2. preparation method
(1) will prepare antigen is inoculated into clone hamster nephrocyte (BHK21) and is added with the DMEM liquid nutrient medium and (comprises 5% bovine serum, 0.01mol/LNaHCO
3, 0.1mg/ml sulphuric acid kanamycin (kanamycinsulfate), 100,000IU penicillin G sodium salt (Penicillin G Sodium), pH value are 7.2) with the carrier tank of BioNoc II trevira in; The microcarrier consumption is that every 500ml nutrient solution adds the 5.5g microcarrier, and cell initial inoculation amount is 2.7 * 10
7The cells/g microcarrier.
(2) in 37 ℃, 5%CO
2Under the culture environment, cell attaches 4h, above-mentioned preparation antigen is mixed with cell and microcarrier, and cell is attached on the microcarrier.The attaching program parameter is: up:2400mL/minhold 1min, Down:2400mL/min hold 30s, setting maximum are changed liquid measure 18500mL; Switch to the cell cultures program behind the 4h, the cell cultures program parameter is: up:1800mL/min hold 1min, Down:1800mL/min hold 1min, setting maximum are changed liquid measure 18500mL.
(3) in 37 ℃, 5%CO
2Under the culture environment, cell is grown on above-mentioned microcarrier, treat that cell reaches 4.0 * 10
10During cell, use the DMEM cell maintenance culture solution of adding 2% bovine serum instead, in virus infection plural number (M.O.I.) is 0.01 ratio inoculation rabies virus LEP-Flury strain, and make it cells infected, make the virus increment, viral absorption program is: up:1500mL/min hold 1min, Down:1500mL/min hold 1min, setting maximum are changed liquid measure 18500mL; The virus culture program: up:1000mL/min hold 1min, Down 1000mL/min hold 1min, setting maximum are changed liquid measure 18500mL;
(4) in 37 ℃, 5%CO
2Continue under the culture environment to cultivate; Cultivate after 5 days, results adopt half-continuous process for the first time, and changing liquid proportional is 50%; Cultured continuously 11 days, every 24h gathers in the crops once, and change liquid proportional is 50% at every turn;
(5) the viral liquid of the viral liquid of half-continuous process results and bio-reactor is mixed after twice of-20 ℃ of freeze thawing, and carries out following check:
(a) pure property check: test by 15,19,20 pages of relevant regulations of " People's Republic of China's veterinary drug allusion quotation " 2005 editions appendix, the result does not have bacterium, mould, mycoplasma and exogenous virus and pollutes.
(b) viral level is measured: carry out according to conventional mouse intracranial inoculation method.The rabies venom that is about to results carries out 10 times of serial dilutions with sterile saline, and each viral liquid gets 10
-3～10
-6Each 4 extent of dilution, each extent of dilution is by the kunming mouse of 4 11～13g of intracranial inoculation injection, and 0.03ml/ only observed 14 days continuously, write down dead mouse situation after 14 days, and calculated viral level according to the Reed-Muench method, and every 1.0ml contains virus 〉=10
7.0LD
50
(c) specificity: with sterile saline dilution the becoming viral suspension that every 1ml contains the minimal infecting dose of 100 mouse,, put in 10～15 ℃ and 1h, jolting therebetween 2～3 times seed culture of viruses (vaccine) with the rabies poison specific serum thorough mixing of equivalent.Set up positive controls (virus control) and negative control group (physiological saline) simultaneously.Neutralization is inoculated 2 of mouse respectively, every intracerebral injection 0.03ml after finishing.Except that the whole death of positive controls, all the other two groups after inoculation 2 the week in the mental status good, have no adverse reaction.
(6) viral level of every milliliter through being up to the standards 〉=10
7.0LD
50Rabies virus stoste obtain rabies virus antigen through the deactivation centrifugal purification.
Comparative Examples suspension culture technology and traditional rolling bottle culture technique are relatively
By extensive tidal type cell microcarrier suspension culture system and rolling bottle culture systems related production performance index commonly used are compared, the result is as shown in table 1.
The relevance ratio of the different culture systems propagation of table 1 rabies virus
Remarks: the adherent area 1g=2400cm of BioNOC II carrier
2, 1 TideCell-020 microcarrier suspension culture system adds BioNOC II carrier 220g.
Above-listed detailed description system specifies at one of the present invention possible embodiments, and only this embodiment is not the claim in order to restriction the present invention, all do not break away from skill spirit of the present invention institute for it equivalence implement or change, all should be contained in the claim of this case.
Claims (9)
1. the preparation method of a rabies virus antigen utilizes bio-reactor, with cell microcarrier suspension culture system scale operation rabies virus antigen, comprises following technological step:
(1) cultivates preparation antigen cell
The clone that can breed rabies virus is inoculated in the carrier tank that contains nutrient solution and microcarrier, and above-mentioned cell and microcarrier are mixed, and starts the attaching program, and cell is attached on the microcarrier; Switch the cell cultures program, under suitable culture environment, provide enough nutrients of above-mentioned cell and suitable atmosphere surrounding, make cell on above-mentioned microcarrier, grow to 5～40 times of inoculum density;
(2) inoculation of venom and breeding
Rabies virus is made viral suspension, it is adsorbed onto on the above-mentioned cell; Use cell maintenance culture solution instead, suitably cultivating virus under the culture environment; After the cultured continuously 3～5 days, gather in the crops viral liquid for the first time, adopt half-continuous process, changing liquid proportional is 50%; Continue to cultivate 9～11, every 24h results are changed liquid once, and changing liquid proportional is 50%;
(3) through after the assay was approved, the viral liquid that half-continuous process is gathered in the crops mixes with the viral liquid of bio-reactor, and in-20 ℃ of freeze thawing twice, the deactivation purifying obtains rabies virus antigen.
2. the preparation method of rabies virus antigen according to claim 1 is characterized in that, described cell microcarrier suspension culture system is a tidal type.
3. the preparation method of rabies virus antigen according to claim 2 is switched the cultivation program after it is characterized in that starting attaching program 4h in the step (1); Described cell attaches program parameter: up:2200～2600mL/min hold 40～100s, Down:2200～2600mL/min hold 40～75s, setting maximum are changed liquid measure 18500mL; The cell cultures program parameter is: up:1600～2000mL/min hold40～100s, Down 1600～2000mL/min hold 40～100s, setting maximum are changed liquid measure 18500mL.
4. method according to claim 2,4h changes the virus culture program into after it is characterized in that starting the absorption program in the step (2), and described virus absorption program parameter is: up:1300～1700mL/min hold40～100s, Down:1300～1700mL/min hold 40～75s, setting maximum are changed liquid measure 18500mL; The virus culture program parameter is: up:1000～1400mL/min hold 40～100s, Down1000～1400mL/min hold 40～100s, setting maximum are changed liquid measure 18500mL.
5. the preparation method of rabies virus antigen according to claim 2 is characterized in that described cell of breeding rabies virus is the hamster kidney cell line; Nutrient solution is 95%～98%DMEM, adds 2%～5% bovine serum, adds an amount of antibiotic, and the pH value is 7.0～7.4; Described microcarrier is a trevira.
6. the preparation method of rabies virus antigen according to claim 2 is characterized in that 36～37 ℃ of cell cultures temperature, contains 2.5%～5% carbonic acid gas in the culture environment.
7. the preparation method of rabies virus antigen according to claim 2 is characterized in that the microcarrier consumption is that every 500ml nutrient solution adds the 5.5g microcarrier, and cell initial inoculation amount is 1.8～3.6 * 10
7The cells/g microcarrier.
8. the preparation method of rabies virus antigen according to claim 2, the cell density when it is characterized in that the described inoculation rabies virus of step (2) is 1.1 * 10
8～1.0 * 10
9The cells/g microcarrier.
9. the preparation method of rabies virus antigen according to claim 2 is characterized in that, step (2) is 0.01～1 ratio virus inoculation in virus infection plural number (M.O.I.).
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| CN201010135137A CN101851610A (en) | 2010-03-30 | 2010-03-30 | Method for producing rabies virus antigens for animals at a large scale |
| CN 201010294954 CN101979515B (en) | 2010-03-30 | 2010-09-26 | Animal rabies virus and vaccine and production method thereof |
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| CN102205119A (en) * | 2011-04-12 | 2011-10-05 | 广州市华南农大生物药品有限公司 | Inactivated rabies antigen and preparation method thereof |
| CN102240400A (en) * | 2011-05-25 | 2011-11-16 | 长春生物制品研究所 | Method for producing rabies vaccine by applying bioreactor and sheet carrier |
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- 2010-03-30 CN CN201010135137A patent/CN101851610A/en active Pending
- 2010-09-26 CN CN 201010294954 patent/CN101979515B/en active Active
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| CN102205119B (en) * | 2011-04-12 | 2012-05-30 | 广州市华南农大生物药品有限公司 | Inactivated rabies antigen and preparation method thereof |
| CN102240400A (en) * | 2011-05-25 | 2011-11-16 | 长春生物制品研究所 | Method for producing rabies vaccine by applying bioreactor and sheet carrier |
| CN106834237A (en) * | 2016-12-26 | 2017-06-13 | 成都康华生物制品有限公司 | A kind of technique of human diploid cell culture rabies viruses |
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| CN101979515B (en) | 2013-01-30 |
| CN101979515A (en) | 2011-02-23 |
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Open date: 20101006 |