CN103087995A - Method of preparing foot-and-mouth disease vaccine by using BHK-21 adherent cells - Google Patents
Method of preparing foot-and-mouth disease vaccine by using BHK-21 adherent cells Download PDFInfo
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Abstract
The invention discloses a method of producing a foot-and-mouth disease vaccine by using a large-scale high-density BHK-21 cell adherent culture technology. The method comprises the following steps of: 1) thawing and primarily amplifying cells; 2) conducting large-scale and high-density cell culture in a medium-volume bioreactor, and after the culture is completed, diluting the high-density cell sap in a large cell culture pot; and 3) inoculating foot-and-mouth disease seed virus, harvesting all virus suspension under proper conditions, and preparing the foot-and-mouth disease vaccine through the following steps. The process method is free from the process of acclimating the adherent cells into suspension cells; the necessarily gradual amplification process in the existing technology of preparing the foot-and-mouth disease vaccine by conducting large-scale suspension culture of the BHK-21 cell can be avoided, the technology flow can be obviously simplified, the production cycle can be shortened, the escape of the foot-and-mouth disease virus can be furthest reduced, and the pollution possibility can be decreased.
Description
Technical field
The invention belongs to field of biological pharmacy, specifically describe be a kind of method of aftosa vaccine for preparing through a step high-density large-scale adherent culture with the BHK-21 cell in bio-reactor.
Background technology
Foot and mouth disease (Foot and mouth disease, FMD) be by foot and mouth disease virus (Foot and mouth disease virus, FMDV) a kind of acute, hot, height contagious disease that the artiodactyl that causes is propagated, susceptible animal is kind more than 70 nearly.Foot and mouth disease epidemic disease infectivity is extremely strong, can cause that breeding performonce fo animals descends, in case the outburst meeting causes huge financial loss to the livestock industry in infected area, has a strong impact on international trade, classified as one of epidemic disease that must report an epidemic in the very first time by OIE.
FMDV belongs to Picornaviridae (Picornaviridae) Hostis (Aphthovirus), and serotype is numerous, comprises 0, A, C, Asia1, SAT1, SAT2 and SAT3 type totally 7 serotypes.Without the cross immunity protection, so for the vaccine of One serotype, other serotype viruses are difficult to the generation effect, cause epidemic disease to control difficulty larger between its serotype.
The FMD vaccine has experienced inactivated vaccine, living vaccine, inactivated vaccine, and this goes through development trend, also has at present part new generation vaccine (synthetic peptide vaccine) to appear on the market.Wherein, the vaccine that inactivated vaccine uses the tongue skin to organize the poisons formalin-inactivated to make at first through with Development In recent 20 years, is mainly made vaccine by cell culture and virus through the BEI deactivation now.The FMD virus antigen mode of production also mainly contains three kinds of modes: 1, and the cow tongue skin is organized the productive culture method; 2, rolling bottle BHK21 cell culture method; 3, BHK21 cell suspension culture method.Wherein, rolling bottle BHK21 cell culture processes needs more manual operation in process of production, and is easily loose malicious, the more difficult Biosafety of controlling fully; And Maitland culture has been used the macro-organism reactor in process of production, can well keep the production process Biosafety, is the main mode of production of present vaccine.
1962, Capstick etc. were domesticated for suspension cell with BHK21 monolayer adherence cell and are used for suspension culture, and nineteen sixty-five, Telling and Elsworth use large fermentation tank to be used for suspension culture BHK21 suspension cell.After this, this mode of production is used in inactivated foot-and-mouth disease vaccine production more and more widely.At present, the world main foot and mouth disease manufacturing enterprise, as Intervet, Merial, IndianImmunologicals Ltd., Bayer HealthCare, Wellcome Biotechnology Limited, and domestic main foot and mouth disease manufacturing enterprise protect spirit as golden space, the Inner Mongol is must prestige safe and sound etc., all adopts this large fermentation tank suspension culture BHK21 suspension cell to prepare the production method of inactivated foot-and-mouth disease vaccine.
The typical production technical process of aforementioned production method is: get the cell of preserving in the cell work storehouse, and through 0.2 liter, 2 liters, the elementary amplification of 5 liters of volume of culture and the domestication that suspends are seeded to the ascending some fermentor tanks of volume and amplify step by step, reach in large fermentation tank at last and cultivate, for example, through 30 liters, 100 liters, 300 liters, 1000 liters, to 3000 liters, perhaps through 10 liters, 120 liters, 650 liters, to 4000 liters.Then access the kind venom for preparing, cells infected, results virus liquid when condition is suitable, deactivation, then concentrated the and purifying through antigen carries out emulsification and packing and obtains the inactivated foot-and-mouth disease vaccine finished product.
Existing extensive suspension culture BHK-21 cell prepares the method for inactivated foot-and-mouth disease vaccine, after the elementary amplification of cell, comprises a necessary process of amplifying step by step.The amplification procedure step by step in these-5 steps of 4 step need to consume a large amount of substratum, increased the possibility of cultivating difficulty and being polluted, each step amplifies needs culturing cell 3-4 days, so the amplification process cycle is 15-20 days step by step, the whole production process cycle is 30-40 days.
Summary of the invention
The invention provides that a kind of technical process is simple, the shorter method for preparing aftosa vaccine of production cycle.Processing method provided by the invention can use the BHK-21 attached cell for the preparation of aftosa vaccine, has removed the process that attached cell is domesticated for suspension cell from; Processing method provided by the invention has avoided existing BHK-21 cell large scale suspension culture to prepare amplification process step by step essential in the aftosa vaccine technology, and significantly simplification of flowsheet, shorten the production cycle; Processing method provided by the invention, large scale culturing obtains the adherent BHK-21 cell of very high-density at short notice, for the infection of foot and mouth disease virus with copy the environment that provides more excellent, thereby improves the antigen quality and quantity, creates remarkable benefit; Processing method provided by the invention connects malicious mode in conjunction with the maxicell tank of high efficiency bio-reactor perfusion training method and widespread use is disposable, has farthest reduced the escape of foot and mouth disease virus when enhancing productivity, and reduces opportunities for contamination.Higher and the steady quality of the aftosa vaccine output of the inventive method preparation.
The invention provides a kind of method that the BHK-21 of use attached cell prepares aftosa vaccine, comprise the following steps:
1) cell recovery and elementary amplification;
2) on a large scale cell is carried out amplification culture;
3) cell suspension that obtains after extensive amplification culture is transferred in the virus liquid preparation tank, during condition maturity, inoculation hoof-and-mouth disease seed culture of viruses venom and venom results.
The inventive method can also comprise the downstream procedures of preparation vaccine finished product.
Cell recovery adopts this area ordinary method to carry out, for example:
(1) take out frozen BHK-21 cell in liquid nitrogen, be placed in 37 ℃ of water-baths and recover, and inoculate to enter to contain in the nutrient solution 20ml of foetal calf serum or calf serum, at 37 ℃, logical 5%CO
2CO2gas incubator in cultivated 1-2 days.
(2) with cultivating the cell suspension that obtains in step (1), through trysinization, be divided into some bottles, every bottle of use contains the nutrient solution 40ml of foetal calf serum or calf serum, continues to cultivate 2-3 days by the condition in (1).
(3) step (2) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(4) carry out cell counting and vigor inspection.
Described nutrient solution can adopt the cell culture medium of this area routine, CMRL1415 substratum for example, F12 (HAM) substratum, the M199 substratum, MEM substratum, DMEM substratum etc., add foetal calf serum in the substratum of selecting, or calf serum makes its concentration reach 3%-15%.
Step (4) can adopt trypan blue staining to carry out.
Elementary amplification culture after the BHK-21 cell recovery can be used the circular culture dish of various volumes, T-shaped bottle, square vase, cylindrical roller bottle (or claim rolling bottle), cell factory (Cell Factory, NUC company) ex hoc genus anne product is (for example, Cell Stack, Corning company), small-sized biological reactor, and the disposable bioreactor device of various volumes.
In one embodiment, described elementary amplification culture adopts the cell factory mode, and step is as follows:
(1) cell suspension that cell recovery is obtained is seeded to cell factory incubator device after being diluted to suitable density with nutrient solution;
(2) resulting enchylema in step (1) being put into CO2gas incubator cultivates;
(3) step (2) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(4) carry out cell counting and vigor inspection.
Described nutrient solution can adopt the cell culture medium of this area routine, CMRL1415 substratum for example, F12 (HAM) substratum, the M199 substratum, the MEM substratum, DMEM substratum etc. add foetal calf serum or calf serum to make its concentration reach 3%-15% to the substratum of selecting.In step (1), cell factory incubator device used is the conventional device that uses in this area, the Cell Stack of for example Cell Factory of U.S. NUC company, or U.S. Corning (healthy and free from worry) company.The culture condition of step (2) is the cell culture condition of this area routine, for example 37 ℃, and logical 5%CO
2Carbonic acid gas cultivated 2-3 days.Step (4) can adopt trypan blue staining to carry out.
In another embodiment, the spinner culture mode is adopted in described elementary cultivation, and step is as follows:
(1) cell suspension that cell recovery is obtained is seeded in rolling bottle after being diluted to suitable density with nutrient solution
(2) the resulting rolling bottle that contains enchylema in step (1) is placed in Rotary Machine;
(3) step (2) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(4) carry out cell counting and vigor inspection.
Described nutrient solution can adopt the cell culture medium of this area routine, CMRL1415 substratum for example, F12 (HAM) substratum, the M199 substratum, the MEM substratum, DMEM substratum etc. add foetal calf serum or calf serum to make its concentration reach 3%-15% to the substratum of selecting.In step (1), rolling bottle used is the conventional equipment that uses in this area, and volume is for example 3 liters, 15 liters.The culture condition of step (2) is the cell culture condition of this area routine, and the rolling bottle that enchylema for example is housed is placed on Rotary Machine, and the setting rotating speed is 20-40rpm, 37 ℃, and logical 5%CO
2Carbonic acid gas, cultivated 2-3 days.Step (4) can adopt trypan blue staining to carry out.
Use bio-reactor and microcarrier on a large scale cell to be carried out amplification culture, comprise the steps:
I. debugging and preparation bio-reactor, make it be in good production status; To microcarrier, soak and the preparation of sterilizing;
Ii. nutrient solution is injected bio-reactor, and microcarrier is added, set suitable culture condition, rotating speed and temperature;
Iii. the cell suspension that described elementary amplification culture is obtained is seeded in bio-reactor with suitable inoculum density;
Iv. the mode culturing cell of cultivating with perfusion.
Carry out cell counting and vigor inspection with the reasonable time interval between incubation period.
Described bio-reactor can use this area internal structure support commonly used to use the different kind organism reactor of carrier, German Eppendorf (Ai Bende) the New Brunswick Scientific of company bio-reactor for example, Germany Satorious company bio-reactor, Holland Applicon company bio-reactor, Hangzhou An Pu company bio-reactor, Shanghai Ri Tai company bio-reactor, Guangzhou flag company bio-reactor.Preferred German Eppendorf (Ai Bende) the New Brunswick Scientific of the company bio-reactor that uses, Germany Satorious company bio-reactor, Holland Applicon company bio-reactor more preferably uses German Eppendorf (Ai Bende) the NewBrunswick Scientific of company bio-reactor.
Described carrier can be selected from the carrier of the conventional various material systems of using, and comprises the polypropylene of chemical materials, PLA, PGA, porous silicon, PLGA, PPF etc., and the dextran of natural materials, collagen protein, agar, com gluten protein etc.The preferred microcarrier that uses the dextran material more preferably uses the spherical microcarrier of the Cytodex1 of U.S. GE company.
The concentration of described microcarrier is for example the 5-50 grams per liter, preferred 10-40 grams per liter, more preferably 15-30 grams per liter, most preferably 20-25 grams per liter.
Cell culture condition is the cell culture condition of this area routine, for example.Rotating speed is 80-120rpm.Temperature is 37 degree.Described nutrient solution can adopt the cell culture medium of this area routine, CMRL1415 substratum for example, F12 (HAM) substratum, the M199 substratum, MEM substratum, DMEM substratum etc., the preferred M199 substratum that uses, the MEM substratum, the DMEM substratum more preferably uses MEM substratum or the DMEM substratum of GIBCO company.
Suitable inoculum density is for example 0.8x10
5Individual cell/ml to 9x10
5Individual cell/ml, preferred 1x10
5Individual cell/ml to 7x10
5Individual cell/ml, more preferably 3x10
5Individual cell/ml to 6x10
5Individual cell/ml most preferably is 5x10
5Individual cell/ml.The mode culturing cell of cultivating with perfusion 2-10 days, preferred 3-7 days, more preferably 3-4 days.Cell counting and vigor inspection can be adopted trypan blue staining.Cell counting and vigor inspection are carried out once every 4-24h, preferably carry out once every 6-18h, more preferably carry out once every 12 hours.
Foot and mouth disease virus inoculation and results are carried out as follows:
When A reached suitable high-density when cell density, the perfusion that stops on the protista reactor was cultivated.Complete soln in tank body is transferred under aseptic condition in the maxicell tank body, replenishing nutrient solution to cell density is 0.5x10
7Individual cell/ml.
It is 80-110rpm that B sets rotating speed, and more preferably 100rpm, keep being no more than 24 hours, makes cell solution be in steady state.
C connects poison by the virus kind venom concentration of setting, surpass 80% cell infected after, the results venom, through deactivation, dilution, emulsifying process prepares foot and mouth disease virus vaccine.
In steps A, suitable high-density is for example 1.0x10
7Individual cell/ml to 12x10
7Individual cell/ml is preferably 2x10
7Individual cell/ml to 10x10
7Individual cell/ml, more preferably 4x10
7Individual cell/ml to 10x10
7Individual cell/ml most preferably is 5x10
7Individual cell/ml to 8x10
7Individual cell/ml.
In step B, held time for example 6-24 hour, preferred 8-20 hour, more preferably 10-18 hour, most preferably 12 hours, make cell solution be in steady state.
The basic step of step C is used the ordinary method of this area:
Be 0.05 amount inoculation hoof-and-mouth disease seed culture of viruses venom according to virus infection plural number MOI, design temperature is for example 34 ℃-37 ℃, and PH7.4, dissolved oxygen are 30%-50%.Connect poison and rose in rear 6 hours, per hour carry out cell counting and vigor inspection with trypan blue staining, and examine under a microscope the Growth of Cells situation on microcarrier.When coming off from microcarrier greater than 80% cell, stop stirring.After standing 4 hours, gather in the crops whole viral supernatant liquors.
During the results virus liquid, virus liquid separates through the microcarrier device for trapping with microcarrier, and microcarrier is recovered, and destroys through Biosafety step process such as the poison that goes out.
Here " microcarrier device for trapping " can be to be built in the cell tank, can be also outer being connected on outside cell tank liquid outlet, can be made by the material through aseptically process of any permission, it can be column, netted, trilateral waits different shape, as long as the effect of this device is virus liquid is flow through smoothly and microcarrier is stopped and do not enter virus liquid results container.
Adopt the sucrose density gradient method to measure foot and mouth disease virus content, when greater than 1000 μ g/ml, carry out deactivation, dilution, emulsifying process prepares foot and mouth disease virus vaccine.
Present method can be carried out the production of multiple batches of product at short notice, foot and mouth disease epidemic situation with the reply burst, concrete steps are: after BHK-21 cell recovery and elementary amplification, cultivate through the bio-reactor microcarrier perfusions of 3-5 days, in enchylema, cell can reach high-density (1x10
7Individual cell/ml to 10x10
7Individual cell/ml), all enchylema is transferred to and connects the steps such as poison and results in the maxicell tank.At this moment, the protista reactor can through steps such as cleaning, sterilizations, restart new round production of vaccine.Present method is after BHK-21 cell recovery and elementary amplification, need not tame cell is pure suspension cell, do not need to contrast the cell cultivation process in traditional suspension culture technique through amplification process step by step yet, with the time shorten of mass cell culturing process nearly 70%.
Embodiment
Material and equipment
1, material
1) MEM substratum, DMEM substratum, GIBCO company
2) foetal calf serum, GIBCO company
2, major equipment
1) cell factory, U.S. NUC company
2) bio-reactor: German Eppendorf (Ai Bende) the New Brunswick 40L of company bio-reactor (working volume 30L), 75L bio-reactor (working volume 52L), 150L bio-reactor (working volume 105L); The fermentor tank that working volume is 300 liters, working volume are the fermentor tank of 550 liters, and working volume is the fermentor tank of 1100 liters
3) CO2gas incubator, SANYO GS company
4) supercentrifuge, Beckman company
5) ultracentrifuge, Beckman company
6) spectrophotometer, German Eppendorf company
Embodiment 1
One, cell recovery and elementary amplification
(1) take out frozen BHK-21 cell in liquid nitrogen, be placed in 37 ℃ of water-baths and recover, and inoculate in the square vase (T-shaped bottle) of the DMEM substratum 20ml that enters to contain 15% foetal calf serum, at 37 ℃, logical 5%CO
2CO2gas incubator in cultivated 2 days.
(2) cultivate the cell suspension that obtains in will step (1), through trysinization, evenly distribute in 5 T-shaped bottles, the DMEM substratum that every bottle of use contains 10% foetal calf serum mend to cumulative volume be 20ml, continue to cultivate 3 days by the condition in (1).
(3) step (2) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(4) adopt trypan blue staining to carry out cell counting and vigor inspection.
(5) cultivate the cell suspension that obtains in will step (3), through trysinization, evenly distribute in 25 T-shaped bottles, the DMEM substratum that every bottle of use contains 10% foetal calf serum mend to cumulative volume be 20ml, continue to cultivate 3 days by the condition in (1).
(6) step (5) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(7) adopt trypan blue staining to carry out cell counting and vigor inspection.
(8) will be seeded to the cell factory device of 20 10 layers as the cell suspension that obtains as described in step (6), each is 1x10 with 1500ml nutrient solution diluting cells suspension to inoculum density
5Individual cell/ml.Be placed in 37 ℃, logical 5%CO
2CO2gas incubator in cultivated 3 days.
(9) step (8) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(10) adopt trypan blue staining to carry out cell counting and vigor inspection.
Two, on a large scale cell is carried out amplification culture
I. according to the debugging of equipment operation instruction and preparation bio-reactor, carry out necessary cleaning, sterilization steps, and carry out sterility test, to confirm that bio-reactor is in good production status.The Cytodex1 microcarrier of selecting is soaked and the preparation of sterilizing.
Ii. take the DMEM substratum as basic medium, adding foetal calf serum is 8% to making its concentration, as the nutrient solution that uses in amplification culture.Nutrient solution is injected bio-reactor, and microcarrier is added.To produce in the bio-reactor of a 40L as example, inject 20 liters of substratum, add the 600 pretreated Cytodex1 microcarriers of gram by the concentration of 20 grams per liters.
Iii. will be seeded in bio-reactor as the cell suspension that obtains as described in step 1, to produce as example, press inoculum density 5x10 in the bio-reactor of a 40L
5Individual cell/ml inoculation BHK-21 cell replenishes the injection nutrient solution and makes volume reach 30 liters, sets oxygen, nitrogen, air, the ratio that passes into of carbonic acid gas is 20%, 5%, 60%, 15%, air flow is 1.0 liter/mins of clocks, and setting rotating speed is 110rpm/ minute, 37 ℃ of temperature.
Iv. according to the equipment operation instruction, with the mode that perfusion is cultivated, culturing cell 3 days.
V. adopted trypan blue staining to carry out cell counting and vigor inspection in every 12 hours.
Three, the cell suspension that obtains after extensive amplification culture is transferred in the virus liquid preparation tank, during condition maturity, inoculation foot and mouth disease virus seed culture of viruses liquid and venom results
A works as cell density and reaches 5x10
7During individual cell/ml, the perfusion that stops on the protista reactor is cultivated.Together with carrier, transferring to a working volume under aseptic condition is in the fermentor tank of 300L with the whole 30L enchylema in the 40L bio-reactor, injects nutrient solution and makes cumulative volume reach 300L.
It is 80rpm that B sets rotating speed, keeps 12 hours, makes cell solution be in steady state.
C connects poison and venom results
Be 0.05 amount inoculation hoof-and-mouth disease seed culture of viruses venom according to virus infection plural number MOI, design temperature is 34 ℃, and PH is 7.4, and dissolved oxygen is 50%.Connect poison and rose in rear 6 hours, per hour carry out cell counting and vigor inspection with trypan blue staining, and examine under a microscope the Growth of Cells situation on microcarrier.When coming off from microcarrier greater than 80% cell, stop stirring.After standing 4 hours, gather in the crops whole viral supernatant liquors.Adopt the sucrose density gradient method to measure foot and mouth disease virus content, when greater than 1000 μ g/ml, carry out deactivation, dilution, emulsifying process prepares foot and mouth disease virus vaccine.
Foot and mouth disease virus content (146S) acquired results that the detection results obtain in virus liquid is: the result (μ g/ml) of three inoculations is respectively: 1895,2001,2136.Test by the existing veterinary biologics quality standard of China and with the Ministry of Agriculture, the relevant bulletin of aftosa vaccine is tested, quality meets the requirements.
Embodiment 2
One, cell recovery and elementary amplification
Described identical with embodiment 1.
Two, on a large scale cell is carried out amplification culture
I. according to the debugging of equipment operation instruction and preparation bio-reactor, carry out necessary cleaning, sterilization steps, and carry out sterility test, to confirm that bio-reactor is in good production status.To selecting the Cytodex1 microcarrier to soak and the preparation of sterilizing.
Ii. take the DMEM substratum as basic medium, adding foetal calf serum is 8% to making its concentration, as the nutrient solution that uses in amplification culture.Nutrient solution is injected bio-reactor, and microcarrier is added.To produce in the bio-reactor of a 40L as example, inject 20 liters of substratum, add the 750 pretreated Cytodex1 microcarriers of gram by the concentration of 25 grams per liters.
Iii. will be seeded in bio-reactor as the cell suspension that obtains as described in step 1, to produce as example, press inoculum density 6x10 in the bio-reactor of a 40L
5Individual cell/ml inoculation BHK-21 cell replenishes the injection nutrient solution and makes volume reach 30 liters, sets oxygen, nitrogen, air, the ratio that passes into of carbonic acid gas is 25%, 5%, 55%, 15%, air flow is 1.0 liter/mins of clocks, and setting rotating speed is 110rpm/ minute, 37 ℃ of temperature.
Iv. according to the equipment operation instruction, with the mode that perfusion is cultivated, culturing cell 3 days.
V. adopted trypan blue staining to carry out cell counting and vigor inspection in every 12 hours.
Three, the cell suspension that obtains after extensive amplification culture is transferred in the virus liquid preparation tank, during condition maturity, inoculation foot and mouth disease virus seed culture of viruses liquid and venom results
A works as cell density and reaches 6x10
7During individual cell/ml, the perfusion that stops on the protista reactor is cultivated.Together with carrier, transferring to a working volume under aseptic condition is in the fermentor tank of 300L with the whole 30 liters of enchylema in the 40L bio-reactor, injects nutrient solution and makes cumulative volume reach 300L.
It is 80rpm that B sets rotating speed, keeps 12 hours, makes cell solution be in steady state.
C connects poison and venom results
Be 0.05 amount inoculation hoof-and-mouth disease seed culture of viruses venom according to virus infection plural number MOI, design temperature is 34 ℃, and PH is 7.4, and dissolved oxygen is 50%.Connect poison and rose in rear 6 hours, per hour carry out cell counting and vigor inspection with trypan blue staining, and examine under a microscope the Growth of Cells situation on microcarrier.When coming off from microcarrier greater than 80% cell, stop stirring.After standing 4 hours, gather in the crops whole viral supernatant liquors.Adopt the sucrose density gradient method to measure foot and mouth disease virus content, when greater than 1000 μ g/ml, carry out deactivation, dilution, emulsifying process prepares foot and mouth disease virus vaccine.
Foot and mouth disease virus content (146S) acquired results that the detection results obtain in virus liquid is: the result (μ g/ml) of three inoculations is respectively: 1745,1608,1889.Test by the existing veterinary biologics quality standard of China and with the Ministry of Agriculture, the relevant bulletin of aftosa vaccine is tested, quality meets the requirements.
Embodiment 3
One, cell recovery and elementary amplification
(1) take out frozen BHK-21 cell in liquid nitrogen, be placed in 37 ℃ of water-baths and recover, and inoculate in the square vase (T-shaped bottle) of the DMEM substratum 20ml that enters to contain 15% foetal calf serum, at 37 ℃, logical 5%CO
2CO2gas incubator in cultivated 2 days.
(2) cultivate the cell suspension that obtains in will step (1), through trysinization, evenly distribute in 5 T-shaped bottles, the DMEM substratum that every bottle of use contains 10% foetal calf serum mend to cumulative volume be 20ml, continue to cultivate 3 days by the condition in (1).
(3) step (2) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(4) adopt trypan blue staining to carry out cell counting and vigor inspection.
(5) cultivate the cell suspension that obtains in will step (3), through trysinization, evenly distribute in 25 T-shaped bottles, the DMEM substratum that every bottle of use contains 10% foetal calf serum mend to cumulative volume be 20ml, continue to cultivate 3 days by the condition in (1).
(6) step (5) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(7) adopt trypan blue staining to carry out cell counting and vigor inspection.
(8) will be seeded to the cell factory device of 20 10 layers as the cell suspension that obtains as described in step (6), each device is 1x10 with 1500ml nutrient solution diluting cells suspension to inoculum density
5Individual cell/ml.Be placed in 37 ℃, logical 5%CO
2CO2gas incubator in cultivated 3 days.
(9) step (8) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(10) adopt trypan blue staining to carry out cell counting and vigor inspection.
(11) will be seeded to the cell factory device of 70 10 layers as the cell suspension that obtains as described in step (9), each device is 1x10 with 1500ml nutrient solution diluting cells suspension to inoculum density
5Individual cell/ml.Be placed in 37 ℃, logical 5%CO
2CO2gas incubator in cultivated 3 days.
(12) step (11) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(13) adopt trypan blue staining to carry out cell counting and vigor inspection.
Two, on a large scale cell is carried out amplification culture
I. according to the debugging of equipment operation instruction and preparation bio-reactor, carry out necessary cleaning, sterilization steps, and carry out sterility test, to confirm that bio-reactor is in good production status.To selecting the Cytodex1 microcarrier to soak and the preparation of sterilizing.
Ii. take the DMEM substratum as basic medium, adding foetal calf serum is 8% to making its concentration, as the nutrient solution that uses in amplification culture.Nutrient solution is injected bio-reactor, and microcarrier is added.To produce in the bio-reactor of a 150L as example, inject 80 liters of substratum, add the 2100 pretreated Cytodex1 microcarriers of gram by the concentration of 20 grams per liters.
Iii. will be seeded in bio-reactor as the cell suspension that obtains as described in step 1, to produce as example, press inoculum density 5x10 in the bio-reactor of a 150L
5Individual cell/ml inoculation BHK-21 cell replenishes the injection nutrient solution and makes volume reach 105 liters, sets oxygen, nitrogen, air, the ratio that passes into of carbonic acid gas is 25%, 5%, 55%, 15%, 1.0 liter/mins of clocks of air flow, setting rotating speed is 110rpm/ minute, 37 ℃ of temperature.
Iv. according to the equipment operation instruction, with the mode that perfusion is cultivated, culturing cell 4 days.
V. adopted trypan blue staining to carry out cell counting and vigor inspection in every 12 hours.
Three, the cell suspension that obtains after extensive amplification culture is transferred in the virus liquid preparation tank, during condition maturity, inoculation foot and mouth disease virus seed culture of viruses liquid and venom results
A works as cell density and reaches 5x10
7During individual cell/ml, the perfusion that stops on the protista reactor is cultivated.Together with carrier, transferring to a working volume under aseptic condition is in the fermentor tank of 1100L with the whole 105L enchylema in the 150L bio-reactor, injects nutrient solution and makes cumulative volume reach 1050L.
It is 80rpm that B sets rotating speed, keeps 12 hours, makes cell solution be in steady state.
C connects poison and venom results
Be 0.05 amount inoculation hoof-and-mouth disease seed culture of viruses venom according to virus infection plural number MOI, design temperature is 34 ℃, and PH is 7.4, and dissolved oxygen is 50%.Connect poison and rose in rear 6 hours, per hour carry out cell counting and vigor inspection with trypan blue staining, and examine under a microscope the Growth of Cells situation on microcarrier.When coming off from microcarrier greater than 80% cell, stop stirring.After standing 4 hours, gather in the crops whole viral supernatant liquors.Adopt the sucrose density gradient method to measure foot and mouth disease virus content, when greater than 1000 μ g/ml, carry out deactivation, dilution, emulsifying process prepares foot and mouth disease virus vaccine.
Foot and mouth disease virus content (146S) acquired results that the detection results obtain in virus liquid is: the result (μ g/ml) of three inoculations is respectively: 1535,1724,1666.Test by the existing veterinary biologics quality standard of China and with the Ministry of Agriculture, the relevant bulletin of aftosa vaccine is tested, quality meets the requirements.
Embodiment 4
One, cell recovery and elementary amplification
(1) take out frozen BHK-21 cell in liquid nitrogen, be placed in 37 ℃ of water-baths and recover, and inoculate in the square vase (T-shaped bottle) of the DMEM substratum 20ml that enters to contain 15% foetal calf serum, at 37 ℃, logical 5%CO
2CO2gas incubator in cultivated 2 days.
(2) cultivate the cell suspension that obtains in will step (1), through trysinization, evenly distribute in 4 T-shaped bottles, the DMEM substratum that every bottle of use contains 10% foetal calf serum mend to cumulative volume be 20ml, continue to cultivate 2 days by the condition in (1).
(3) step (2) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(4) adopt trypan blue staining to carry out cell counting and vigor inspection.
(5) cultivate the cell suspension that obtains in will step (3), through trysinization, evenly distribute in 16 T-shaped bottles, the DMEM substratum that every bottle of use contains 10% foetal calf serum mend to cumulative volume be 20ml, continue to cultivate 2 days by the condition in (1).
(6) step (5) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(7) adopt trypan blue staining to carry out cell counting and vigor inspection.
(8) will be seeded to the cell factory device of 10 10 layers as the cell suspension that obtains as described in step (6), each device is 1x10 with 1500ml nutrient solution diluting cells suspension to inoculum density
5Individual cell/ml.Be placed in 37 ℃, logical 5%CO
2CO2gas incubator in cultivated 3 days.
(9) step (8) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(10) adopt trypan blue staining to carry out cell counting and vigor inspection.
(11) will be seeded to the cell factory device of 32 10 layers as the cell suspension that obtains as described in step (9), each device is 1x10 with 1500ml nutrient solution diluting cells suspension to inoculum density
5Individual cell/ml.Be placed in 37 ℃, logical 5%CO
2CO2gas incubator in cultivated 3 days.
(12) step (11) is cultivated the cell that finishes rear gained and all use trysinization, collect.
(13) adopt trypan blue staining to carry out cell counting and vigor inspection.
Two, on a large scale cell is carried out amplification culture
I. according to the debugging of equipment operation instruction and preparation bio-reactor, carry out necessary cleaning, sterilization steps, and carry out sterility test, to confirm that bio-reactor is in good production status.To selecting the Cytodex3 microcarrier to soak and the preparation of sterilizing.
Ii. take the DMEM substratum as basic medium, adding foetal calf serum is 8% to making its concentration, as the nutrient solution that uses in amplification culture.Nutrient solution is injected bio-reactor, and microcarrier is added.To produce in the bio-reactor of a 75L as example, inject 38 liters of substratum, add 1092 pretreated Cytodex 3 microcarriers of gram by the concentration of 21 grams per liters.
Iii. will be seeded in bio-reactor as the cell suspension that obtains as described in step 1, to produce as example, press inoculum density 3x10 in the bio-reactor of a 75L
5Individual cell/ml inoculation BHK-21 cell replenishes the injection substratum and makes volume reach 52 liters,, set oxygen, nitrogen, air, the ratio that passes into of carbonic acid gas is 25%, 5%, 55%, 15%, 1.0 liter/mins of clocks of air flow, setting rotating speed is 110rpm/ minute, 37 ℃ of temperature.
Iv. according to the equipment operation instruction, with the mode that perfusion is cultivated, culturing cell 4 days.
V. adopted trypan blue staining to carry out cell counting and vigor inspection in every 12 hours.
Three, the cell suspension that obtains after extensive amplification culture is transferred in the virus liquid preparation tank, during condition maturity, inoculation foot and mouth disease virus seed culture of viruses liquid and venom results
A works as cell density and reaches 5x10
7During individual cell/ml, the perfusion that stops on the protista reactor is cultivated.Together with carrier, transferring to a working volume under aseptic condition is in the fermentor tank of 550L with the whole 52L enchylema in the 75L bio-reactor, injects nutrient solution and makes cumulative volume reach 520L.
It is 80rpm that B sets rotating speed, keeps 12 hours, makes cell solution be in steady state.
C connects poison and venom results
Be 0.05 amount inoculation hoof-and-mouth disease seed culture of viruses venom according to virus infection plural number MOI, design temperature is 34 ℃, and PH is 7.4, and dissolved oxygen is 50%.Connect poison and rose in rear 6 hours, per hour carry out cell counting and vigor inspection with trypan blue staining, and examine under a microscope the Growth of Cells situation on microcarrier.When coming off from microcarrier greater than 80% cell, stop stirring.After standing 4 hours, gather in the crops whole viral supernatant liquors.Adopt the sucrose density gradient method to measure foot and mouth disease virus content, when greater than 1000 μ g/ml, carry out deactivation, dilution, emulsifying process prepares foot and mouth disease virus vaccine.
Foot and mouth disease virus content (146S) acquired results that the detection results obtain in virus liquid is: the result (μ g/ml) of three inoculations is respectively: 1438,1587,1802.Test by the existing veterinary biologics quality standard of China and with the Ministry of Agriculture, the relevant bulletin of aftosa vaccine is tested, quality meets the requirements.
Claims (10)
- One kind in bio-reactor large scale and high density cultivate the method that the BHK-21 attached cell prepares aftosa vaccine, the method comprises following key step: 1) BHK-21 cell recovery and elementary amplification culture; 2) BHK-21 cell large scale and high density in bio-reactor is cultivated; 3) enchylema after cultivation finishes is transferred in the maxicell tank and dilutes, inoculation hoof-and-mouth disease seed culture of viruses poison, results venom.
- 2. the method for claim 1, it is characterized in that: described BHK-21 cell is the attached cell strain, and without the suspension acclimation and screening, directly uses.
- 3. the method for claim 1 is characterized in that: the BHK-21 suspension cell strain of described BHK-21 cell strain for having suspended and tamed, these cell strains are adapted to the attached cell strain in the culturing process of described method.
- 4. the method for claim 1, be further characterized in that: described step 2), according to the bio-reactor of the various volumes of production-scale big or small choice for use (for example 1 liter, 5 liters, 7.5 liters, 14 liters, 40 liters, 75 liters, 150 liters, 300 liters, and more massive) and various known carrier; The carrier that these carriers are made including, but not limited to following various materials: polypropylene, PLA, PGA, porous silicon, PLGA, PPF etc., and the dextran of natural materials (Cytodex 1, Cytodex2, Cytodex3), collagen protein, agar, com gluten protein etc.; Cytodex1 and the spherical microcarrier of Cytodex3 that the preferred GE of use company produces.
- 5. application as claimed in claim 1 is further characterized in that: described step 2), use the concentration of microcarrier to be the 5-50 grams per liter, preferred 10-40 grams per liter, more preferably 15-30 grams per liter, most preferably 20-25 grams per liter; The inoculum density of cell is 0.8x10 5Individual cell/ml to 9x10 5Individual cell/ml, preferred 1x10 5Individual cell/ml to 7x10 5Individual cell/ml, more preferably 3x10 5Individual cell/ml to 6x10 5Individual cell/ml; Add 3%-15% foetal calf serum or calf serum in substratum; The culture condition of bio-reactor is set to: temperature 36.5-37.5 degree centigrade, and dissolved oxygen DO 30%-80%, pH value 7.0-7.2, rotating speed 80-120rpm.
- 6. the method for claim 1, be further characterized in that: described step 2), when cell reaches high-density (1x10 7Individual cell/ml to 10x10 7During individual cell/ml), finish the cell cultures in bio-reactor, whole enchylema be transferred to one under aseptic condition in the maxicell tank of sterilising treatment, with step 2) in 10 times of nutrient solution dilutions used, or more.
- 7. the method for claim 1, be further characterized in that: described step 3), " maxicell tank " is selected from various macro-organism reactors with common Stirring oar, can be also disposable bioreactor; The tank body volume of described reactor is selected from 10 liters, and is 50 liters, 75 liters, 150 liters, 400 liters, 800 liters, 1500 liters, 3000 liters, even more massive.
- 8. the method for claim 1, be further characterized in that: described step 3), set the slow speed of revolution (not higher than 150rpm) and make the cell carrier suspension reach metastable state (being no more than 24 hours); Then check cell density and cell viability, when cell density more than or equal to target cell density (0.5x10 for example 7Individual cells/ml) time, inoculation hoof-and-mouth disease seed culture of viruses poison.
- 9. the method for claim 1 is further characterized in that: described step 3) after inoculation hoof-and-mouth disease seed culture of viruses poison, when coming off from carrier greater than 80% cell, begin to gather in the crops virus liquid; Carrier separates with virus liquid suppressed by vector device for trapping; Described " carrier device for trapping " is selected from and is built in the cell tank, or be connected on the outer various device for trapping of cell tank liquid outlet outward, the material through aseptically process by any permission is made, be selected from different shape, condition is that this device for trapping is virus liquid is flow through smoothly and microcarrier is stopped and do not enter virus liquid results container.
- 10. the method for claim 1, be further characterized in that: can carry out at short notice the production of multiple batches of product, foot and mouth disease epidemic situation with the reply burst, concrete steps are: after BHK-21 cell recovery and elementary amplification, cultivate through the bio-reactor microcarrier perfusions of 7-10 days, in enchylema, cell reaches high-density (1x10 7Individual cell/ml to 10x10 7Individual cell/ml), all enchylema is transferred to and connects the steps such as poison and results in the maxicell tank; At this moment, the protista reactor restarts new round production of vaccine through steps such as cleaning, sterilizations, and batch production cycle that makes a set of equipment is 8-14 days.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103289949A (en) * | 2013-06-28 | 2013-09-11 | 四川省华派生物制药有限公司 | Cell culture method direct from Kolle flask to bioreactor |
| CN104491855A (en) * | 2014-11-07 | 2015-04-08 | 吕宏亮 | Large-scale preparation method for foot-and-mouth disease totivirus marked vaccine with high yield, high purity and high safety and product thereof |
| CN106399261A (en) * | 2016-09-29 | 2017-02-15 | 天信和(苏州)生物科技有限公司 | Method for producing foot-and-mouth disease seed virus by suspension cultivation on BHK21 cells through microcarriers |
| CN107988143A (en) * | 2017-11-22 | 2018-05-04 | 中牧实业股份有限公司 | One plant of BHK-21 cells Gs cell line |
| CN108396006A (en) * | 2018-01-25 | 2018-08-14 | 武汉珈创生物技术股份有限公司 | A kind of BHK-21 cell strains and its cultural method and purposes |
| CN109529031A (en) * | 2019-01-25 | 2019-03-29 | 付显东 | A kind of preparation method and its preparation facilities of aftosa vaccine |
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| CN103289949A (en) * | 2013-06-28 | 2013-09-11 | 四川省华派生物制药有限公司 | Cell culture method direct from Kolle flask to bioreactor |
| CN104491855A (en) * | 2014-11-07 | 2015-04-08 | 吕宏亮 | Large-scale preparation method for foot-and-mouth disease totivirus marked vaccine with high yield, high purity and high safety and product thereof |
| CN104491855B (en) * | 2014-11-07 | 2016-05-25 | 吕宏亮 | Method of the aftosa whole virus particles marker vaccine of a kind of extensive preparation high yield, high-purity, high safety and products thereof |
| CN106399261A (en) * | 2016-09-29 | 2017-02-15 | 天信和(苏州)生物科技有限公司 | Method for producing foot-and-mouth disease seed virus by suspension cultivation on BHK21 cells through microcarriers |
| CN107988143A (en) * | 2017-11-22 | 2018-05-04 | 中牧实业股份有限公司 | One plant of BHK-21 cells Gs cell line |
| CN108396006A (en) * | 2018-01-25 | 2018-08-14 | 武汉珈创生物技术股份有限公司 | A kind of BHK-21 cell strains and its cultural method and purposes |
| CN109529031A (en) * | 2019-01-25 | 2019-03-29 | 付显东 | A kind of preparation method and its preparation facilities of aftosa vaccine |
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Effective date of registration: 20191106 Address after: 214000 no.8410, zone B, science and technology entrepreneurship Park, No.16, Changjiang Road, Xinwu District, Wuxi City, Jiangsu Province Patentee after: Jiangsu Taiyi Biotechnology Co., Ltd. Address before: 230601 Hefei Chuang Hua Industrial Park, Anhui, China, 2F218 351 Patentee before: Hefei Level Biotechnology Co., Ltd. |
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Effective date of registration: 20200812 Address after: Room 501, building 1, No. 619, BINKANG Road, Binjiang District, Hangzhou City, Zhejiang Province Patentee after: HANGZHOU GUOMU BIOLOGICAL SCIENCE & TECHNOLOGY Co.,Ltd. Address before: 214000 no.8410, zone B, science and technology entrepreneurship Park, No.16, Changjiang Road, Xinwu District, Wuxi City, Jiangsu Province Patentee before: JIANGSU TAIYI BIOTECHNOLOGY Co.,Ltd. |