CN1807599A - Method for safe continuous enclosed cell culture, virus production/ inactivation - Google Patents
Method for safe continuous enclosed cell culture, virus production/ inactivation Download PDFInfo
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- CN1807599A CN1807599A CNA200610023231XA CN200610023231A CN1807599A CN 1807599 A CN1807599 A CN 1807599A CN A200610023231X A CNA200610023231X A CN A200610023231XA CN 200610023231 A CN200610023231 A CN 200610023231A CN 1807599 A CN1807599 A CN 1807599A
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Abstract
A method of cells culture, virus production/deactivation in safe, continuum and blocked style, belonging to bioengineering technical field, compring steps of: 1) Inoculate cells and suspension culture, therebycarry out the primary culture of cells; 2)Flowing add fresh medium, simultly pump out cells suspension to maintain cell culture biological reactor stable and guarantee cell amplificatation during residence time in reactor, accordingly carry out cell's continuous cultivation; 3) Pump out cells suspension into virus-containing virus breeding reactor to make virus infect and amplificate in cells preliminary; 4)As the inflow of cells suspension and outflow of virus-containing upper clean liquid, system dynamic balancing is maintained and virus continuous cultivation is achieved; 5) Collect the upper clean liquid pumped from virus breeding reactor and deactivate the virus, accordingly achieve continuously and obturately cells cultivation and virus deactivation. This method is convenient for automatic control and standardized production, also depresses the pollution chances and heightens production assurance coefficient.
Description
Technical field
The present invention relates to a kind of method of technical field of bioengineering, specifically, is the method for a kind of safe continuous enclosed cell cultures, virus production/deactivation.
Technical background
Utilize cell to produce virus, mainly contain dual mode: (1) cell and viral seed are together cultivated, and in the process of cell proliferation, replication-competent virus reaches the purpose of mass production virus, and this is a kind of method of natural survival condition in the simulation organism; (2) with the amplification procedure of cell and virus separately, promptly earlier a large amount of amplifying cells add viral seed again after acquiring a certain degree, keep cell activity, by the intercellular continuous infection of progeny virus with duplicate, reach the purpose of mass production virus.Along with deepening continuously to virus research, find that cell amplification process and virus optimization culture condition of breeding in cell have certain difference, therefore, main at present cell cultivation process and the virus method that breeding is separated in the cell preparation virus of adopting.When production virus, cultivation a large amount of, high-density cells is the prerequisite that produces infectious titer, and intermittently cultivation and feed supplement are cultivated the nutritive substance that can not fully provide enough and in time removed deleterious meta-bolites, are poured cultured method gradually and substitute.It is a kind of culturing process under continuous dynamic monitoring condition that perfusion is cultivated, can add fresh culture in time on demand and extract the expendable substratum that contains products of cellular metabolism simultaneously out, it is a kind of relatively stable that cell is remained at, have in the growing environment of sufficient nutrition and grow, good effect is all being arranged aspect the cell speed of growth, cell density and the cellular form, thereby be applicable to that high-density cells is cultivated and extensive virus production.
Find through literature search prior art, people such as Liu Jianqing are at " Shandong medicine " 2005,45 (23): in " fixed-bed bioreactor is produced high titre encephalitis b virus " literary composition of delivering on 7～8, proposed a kind of Fibra-Cel of use Disks produces encephalitis b virus as the fixed bed Cultivation of Vero of carrier method.At first, carry out the perfusion culture mode Vero cell that increases in a large number with 199 (Gibco) that are added with 10% new-born calf serum as cell amplification culture medium, high-density culture, after acquiring a certain degree, remove cell culture fluid, keep substratum with 199 (Gibco) that are added with 0.4% human serum albumin as cell, virus inoculation adopts a large amount of virus of proliferation of perfusion culture mode.Also find in the retrieval, people such as Y.Z.Ghendon are at " Vaccine " 2005, " Development of cell culture (MDCK) livecold-adapted (CA) the attenuated influenza vaccine " that delivers on the 23:4678～4684 (development of cell cultures cold-resistant Gripovax alive, " vaccine ") in the literary composition, the method that a kind of Cytodex-1 of use microcarrier suspension culture mdck cell is produced influenza virus has been proposed.At first, with the Axcevir-MDCK serum free medium by the perfusion culture mode mdck cell that increases in a large number, high-density culture, after acquiring a certain degree, remove cell culture fluid, use instead be added with 2 μ g/mL pancreatin the Axcevir-MDCK serum free medium as cell maintenance medium, virus inoculation adopts a large amount of virus of proliferation of batch culture mode.
But in two kinds of above-mentioned methods, two processes of cell cultures and virus multiplication have quantity-produced separately, but whole process is intermittently and does not seal and carry out, after previous process unit finishes, just begin next process unit, be unfavorable for extensive, continuity, safety in production virus vaccines.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, at in the present virus vaccines production " discontinuity and non-closure ", the invention provides the method for a kind of safe continuous enclosed cell cultures, virus production/deactivation, be used for cell cultures, virus multiplication, inactivation of virus, make it can reduce microbiological contamination and the viral risk that leaks, simultaneously whole system is convenient to automatization control, can be safely, efficient, virus of proliferation is used for production of vaccine on a large scale.
The present invention is achieved by the following technical solutions, comprises following steps:
1) in biological reactor for cell culture, inoculates 1～2 * 10
5The cell of cells/mL, suspension culture is expanded to 0.2～1.0 * 10
7Cells/mL (during for microcarrier suspension culture, 80～90% converge full cell on the microcarrier) realizes the elementary cultivation of cell;
2) according to the growth characteristics of culturing cell and the size of bio-reactor volume, flow velocity stream with 0.3～1.0 times of reactor working volume/sky adds fresh culture, simultaneously pump cell suspending liquid with same flow velocity, keep biological reactor for cell culture stable the time, guarantee that cell is expanded to 0.2～1.0 * 10 in the residence time in reactor
7Cells/mL (during for microcarrier suspension culture, 80～90% converge full cell on the microcarrier) realizes the cultured continuously of cell;
3) pump cell suspending liquid to containing the virus multiplication reactor that infection multiplicity is 2～10 viruses, realize the elementary infection development of virus in cell;
4), in the process of keeping total system running balance, realize the continuous propagation of virus along with the continuous inflow of cell suspending liquid and continuously outflowing of viral supernatant liquor;
5) collect the viral supernatant liquor that pumps with the collection virus jar from the virus multiplication reactor, inactivation of viruses is realized sealing culturing cell and inactivation of viruses continuously.
In the described step 1), the elementary cultivation of cell, specifically be meant: adopt the suspension culture mode to carry out cell cultures, cultured cells can be the suspension known of technician, through suspension culture domestication or adherent cell, as chick embryo fibroblast, Chinese hamster ovary celI, mdck cell, BHK-21 cell, Vero cell, 293 cells etc.At different cell types, the dissimilar substratum that can adopt the technician to know, as the DMEM, Eagle MEM, RPMI1640,199 etc. that add 4% ~ 10% serum contain blood serum medium, or serum free medium, as NCTC135: F12 (1: 1), the combination of alpha MEM: F12 (1: 1) etc.At different cell types and suspension culture requirement, the dissimilar microcarrier that can adopt the technician to know, as microcarriers such as dextran, collagen, polystyrene, polyacrylamide, gelatin, glass, Mierocrystalline cellulose, polyethylene, consumption is between 2～10g/L.Temperature is controlled at 37 ± 0.2 ℃, and pH maintains between 7.0～7.2, dissolved oxygen (DO) 25%～60%.Inoculum density is 1～2 * 10
5Between the cells/mL, can by in batches, modes such as feed supplement or perfusion are cell amplification to 0.2～1.0 * 10
7Cells/mL (during for microcarrier suspension culture, 80～90% converge full cell on the microcarrier) finishes elementary cultivation stage.Preferred reperfusion mode can be realized quick cell amplification.According to the consumption of glucose and the generation situation of lactic acid, generally behind postvaccinal 48～72h, begin perfusion culture, according to the irrigation rate in 0.5～1.5 times of reactor working volume/sky of different choice of culturing cell kind.
Described step 2) in, the cultured continuously of cell specifically is meant: after elementary culturing cell density reaches requirement, enter high-density cultured continuously cell stage.At first finish elementary perfusion culture, then according to the kind of culturing cell, flow rate regulation stream with 0.3～1.0 reactor working volume/sky adds fresh culture (if microcarrier is cultivated, contain fresh microcarrier in the substratum), and pump cell suspending liquid with same speed, guarantee that again cell can be expanded to 0.2～1.0 * 10 in the residence time in reactor in stable keeping biological reactor for cell culture
7Cells/mL (during for microcarrier suspension culture, 80～90% converge full cell on the microcarrier) keeps the high-cell density state in the reactor, realizes the cultured continuously of cell.Carry out material with the mixing speed of 30～90rpm and mix, particularly during microcarrier suspension cultured continuously cell, for the benefit of by the transfer of " pearl ball " mode, rotating speed can not be too fast between microcarrier for cell.
The present invention is owing to top condition that relates to cell cultures and virus multiplication top condition is related, for improving the turndown ratio of entire reaction system, the volume of virus multiplication reactor is 2～5 times of biological reactor for cell culture volume, simultaneously virus multiplication is divided into elementary infection development and continuous two stages of propagation, takes differential expression initial stage propagation mode to carry out respectively with the continuous propagation mode that has cell, carrier reflux.
In the described step 3), elementary infection development, specifically be meant: the virus of being bred comprises and can breed justacrine to extracellular virus in corresponding host cell, as rabies virus, poliovirus, influenza virus, hemorrhagic fever virus, Porcine epidemic diarrhea virus, encephalitis b virus, rotavirus etc.Culture condition such as the pH in the virus multiplication culturing process, temperature, dissolved oxygen can be selected suitable, the known culture condition of these those skilled in the art according to different virus, such as, in pH7.0～7.2,32 ℃～34 ℃ down with Vero cell cultures influenza viruses; In pH7.8,35 ℃ of following Vero cells produce rabies virus of using, dissolved oxygen control 25%～60%.If what the cell cultures stage selected for use is that the DMEM that contains 4%～10% serum, Eagle MEM, RPMI1640, Medium 199 etc. contain blood serum medium, in the virus multiplication stage, to serum content be diluted to 1%～2% with the corresponding base basal culture medium, as the virus multiplication substratum; If what the cell cultures stage selected for use is serum free medium, in the virus multiplication stage, can be according to the content of harmful meta-bolitess such as nutritive substances such as glucose in the virus multiplication reactor and lactic acid, proportional flow with 1～5 adds this substratum, as the virus multiplication substratum, keep good physiological status of cell and virus multiplication activity in order to the extra-nutrition material.Simultaneously can add known other material of these those skilled in the art,, generally will add the pancreatin of 1～3 μ g/mL as using Vero cell proliferation influenza virus according to different virus.
In the described step 3), elementary infection development adopts the elementary propagation mode of differential expression to realize, stream adds basic medium or serum free medium according to the above ratio when cell suspending liquid flows into the virus multiplication reactor, the two mixes the common virus multiplication substratum of forming, simultaneously, the inoculation infection multiplicity is 2～10 infectious titer in the virus multiplication reactor.Done two purposes like this, on the one hand, when cell suspending liquid flowed into the virus multiplication reactor at first, high virus concentration had increased the chance of viral contact infection cell, can play the effect of accelerating the virus multiplication process; On the other hand, continuous inflow along with cell suspending liquid, liquid volume in the virus multiplication reactor constantly increases, for keeping the stability of whole cell cultures and virus multiplication system, when liquid volume reaches the effective volume of virus multiplication reactor, must extract liquid out to keep system balancing, and in the ever-increasing process of liquid volume, given the time of virus replication propagation, like this, when initial extraction contains viral liquid, can not reduce the virus quantity in the virus multiplication reactor, make next viral continuous breeding smooth.
In the described step 4), the continuous propagation of virus, specifically be meant: contain cell when cultivating (microcarrier is the microcarrier that is attached with cell) and the viral suspension extracted out from the virus multiplication reactor enter a cell/carrier settlement separator that has the cell microcarrier of cell (or be attached with) reflux, the supernatant liquor that will contain virus is evacuated to the collection virus jar to keep the balance of entire operation system, the cell that the settles down microcarrier of cell (or be attached with) is back to the virus multiplication reactor by reflux, reach and make full use of the virus host cell, improve the purpose of viral yield, simultaneously, can be according to running situation, will be in reactor overstand, the outmoded microcarrier that cell comes off in a large number intermittently is collected into outmoded carrier holding tank, can reduce the influence of outmoded carrier to virus multiplication, a small amount of viral supernatant liquor that wherein contains is incorporated the collection virus jar into and is together collected, the sealing deactivation.General 30～the 90rpm that selects of mixing speed carries out the material mixing and realizes viral intercellular consecutive infection.
In the described step 5), the sealing deactivation of virus, specifically be meant: collection virus is to finish by a series of collection virus jars that have automatic liquid level control switch.After each holding tank is collected and is expired, opening other holding tank by the liquid automatic control switch when closing this holding tank collects, full holding tank is taken off, change empty holding tank, so repeatedly circulation, realize the continuous collection of virus, and, according to the virus production ability what, one group of holding tank and another group holding tank can be separated, open and close by the liquid automatic control on-off control, collect the purpose of virus when realizing large scale continuous prod continuously.After completely the holding tank of virus takes off with collection, adopt conventional as method deactivations such as β propiolactone, formalin, and then by purification process such as centrifugal, ultrafiltration, chromatography with add adjuvant and the stablizer supervisor prepares virus vaccines, this is that these those skilled in the art are known.
By above explanation, as seen, the present invention gets up cell amplification and two procedure correlations of virus multiplication, finishes cell cultures production/inactivation of viruses whole process under the sealing continuously, and adopts corresponding measure to improve turndown ratio, on the one hand, be convenient to automatization control, standardized production, on the other hand, having reduced opportunities for contamination and improved the production safety coefficient, is very to be suitable for the novel method that extensive high-efficiency and continuous is produced virus.
Description of drawings
Fig. 1 the inventive method schema
Embodiment
Embodiment:
Above description has illustrated content of the present invention haply, below several examples will more direct embodiment situation of the present invention, but should point out the purpose just at this example of enumerating, rather than limited in order to be illustrated.
Example 1 Vero cell cultures production/inactivated rabies virus
Substratum: Medium 199 (Gibco) adds 5% foetal calf serum, transfers to pH7.0～pH7.2 with sodium bicarbonate.
Microcarrier and processing thereof: adopt Cytodex-1 (Pharmacia) as microcarrier, matrix is sephadex, during use dried microcarrier is added in the container of dimethyldichlorosilane(DMCS) silication, at room temperature fully soaks with PBS solution, supernatant discarded, with PBS solution washing 2 times, the PBS that more renews is at 121 ℃ of sterilization 30min down, supernatant is removed in suction, with Medium 199 washings that contain 5% foetal calf serum 1 time, more renew substratum, put equilibrate overnight in the incubator.
1) the elementary cultivation of cell
Add treated microcarrier at the homemade 3g/L that presses in the 500mL of siliconizing stirring type bioreactor (Spinner Flasks), insert the Vero cell, inoculum density is 1 * 10
5Cells/mL supplies substratum to volume required, 37 ℃ of cultivations.In beginning 4h, adopt the alr mode at intermittence of 40rpm, stirring 5min, static 25min to cultivate, then stir speed (S.S.) is remained on 60rpm, cultivate, dissolved oxygen 50% air saturation, perfusion culture behind the 48h, irrigation rate is 400mL/day, timing sampling, counting, and continous pouring is after 4 days, the sampling counting, cell density reaches 4.35 * 10
6Cells/mL, 80%～90% compiles full cell on the microcarrier.
2) cultured continuously of cell
According to the amount of 3g/L treated microcarrier is joined in 2 liters of Medium 199 cell culture mediums that contain 5% foetal calf serum, mixing speed is 80rpm, the substratum that will contain microcarrier adds with the data rate stream of 250mL/day, with same speed collecting cell effluent liquid, timing sampling, counting in 3 days find that cell density can remain on 4.08 * 10
6About cells/mL, find that the cell culture of continuous cultivation is more steady.
3) Bing Du elementary propagation
With homemade through the 2000mL of siliconizing stirring type bioreactor (Spinner Flasks) as the virus multiplication reactor, data rate stream with 375mL/day when above-mentioned cell suspending liquid enters the virus multiplication reactor adds serum-free Medium 199 basic mediums, infection multiplicity inoculation aG rabies virus strain with 10, under 35 ℃, pH7.0, cultivate, dissolved oxygen 50%, stir speed (S.S.) remains on 60rpm.About 72 hours, liquid level begins to reach reactor and effectively demarcates liquid level, with the flow velocity of about 1000mL/day extract out contain microcarrier, cell, virus mixed solution to cell/carrier settlement separator, and extract the supernatant liquor that contains virus out with the speed of 625mL/day, beginning is propagation continuously.
4) Bing Du the continuous multiplicative stage
Extract flow velocity out according to top cell sample introduction flow velocity, fresh serum-free Medium 199 basic medium sample introduction flow velocitys and the supernatant liquor that contains virus, keep the stable continuous virus multiplication, timing sampling in 10 days, cell detachment situation on check virus titer and the microcarrier finds that virus titer maintains 9lgLD
50About/mL, the virus multiplication process is more steady.Those microcarriers that cell of overstand comes off in a large number in reactor can drain into outmoded carrier holding tank by the discharge outlet below cell/carrier settlement separator.
5) the sealing deactivation of rabies virus
In step 2) in the viral supernatant of extracting out, collect with the receiving flask of 4 2000mL, add beta-propiolactone according to 1: 4000,4 ℃ are stirred placement 24 hours, 37 ℃ of water-bath hydrolysis 2 hours can the complete inactivation rabies virus.
Example 2 mdck cells are cultivated production/deactivation H3N2 influenza virus
With reference to carrying out present embodiment, adopt mdck cell to cultivate the H3N2 influenza virus with example 1 essentially identical step.
Substratum: (Excele Biotechnologies France) transfers to pH7.0～pH7.2 with sodium bicarbonate to the Axcevir-MDCK serum free medium
Microcarrier: Cytodex-3, consumption 5g/L.
Cell cultivation process: inoculum density 1.5 * 10
5Cells/mL, microcarrier Cytodex-3, consumption 5g/L, culture bioreactors is homemade through the 500mL of siliconizing stirring type bioreactor (SpinnerFlasks), 37 ℃ of cultivations, dissolved oxygen 40%, mixing speed 50rpm, just stage speed 600mL/day inoculated after 2 days, and cell density is 4.67 * 10
6About cells/mL, the beginning cultured continuously, sample rate 300mL/day, timing sampling, counting in 5 days, cell density maintains 4.21 * 10
6About cells/mL, find that the cell culture of continuous cultivation is more steady.
Virus breeding: with homemade through the 1500mL of siliconizing stirring type bioreactor (Spinner Flasks) as the virus multiplication reactor, data rate stream with 200mL/day when above-mentioned cell suspending liquid enters the virus multiplication reactor adds the Axcevir-MDCK serum free medium that contains 8 μ g/mL pancreatin, infection multiplicity inoculation H3N2 influenza virus with 5, under 33 ℃, pH7.1, cultivate, dissolved oxygen 50%, stir speed (S.S.) remains on 50rpm.Liquid level begins to reach reactor and effectively demarcates liquid level, extracts the supernatant liquor that contains virus out with the speed of 500mL/day from virus, cell settlement tripping device, the continuous propagation of beginning 9 days.
Collect the viral supernatant of above-mentioned extraction with the receiving flask of 3 2000mL, the Trisodium Citrate that adds 0.02% formalin and 0.05mol stirs at 4 ℃ to be placed 20 hours, can complete inactivation H3N2 influenza virus.
Example 3 Vero cell cultures production/deactivation method H3N2 influenza viruses
With reference to carrying out present embodiment, adopt Vero cell cultures H3N2 influenza virus with example 1 essentially identical step.
Substratum: Medium 199 (Gibco) adds 4% foetal calf serum, transfers to pH7.0～pH7.2 with sodium bicarbonate.
Microcarrier: Cytodex-3, consumption 10g/L.
Cell cultivation process: inoculum density 2.0 * 10
5Cells/mL, microcarrier Cytodex-3, consumption 10g/L, culture bioreactors is homemade through the 500mL of siliconizing stirring type bioreactor (SpinnerFlasks), 37 ℃ of cultivations, dissolved oxygen 60%, mixing speed 90rpm, just stage speed 700mL/day inoculated after 3 days, and cell density is 7.45 * 10
6About cells/mL, the beginning cultured continuously, sample rate 300mL/day, timing sampling, counting in 5 days, cell density maintains 7.13 * 10
6About cells/mL, find that the cell culture of continuous cultivation is more steady.
Virus breeding: with homemade through the 2500mL of siliconizing stirring type bioreactor (Spinner Flasks) as the virus multiplication reactor, data rate stream with 500mL/day when above-mentioned cell suspending liquid enters the virus multiplication reactor adds serum-free Medium 199 basic mediums, infection multiplicity inoculation hemorrhagic fever virus strain with 2, under 33 ℃, pH7.1, cultivate, dissolved oxygen 60%, stir speed (S.S.) remains on 90rpm.About 75 hours, liquid level begins to reach reactor and effectively demarcates liquid level, flow velocity extraction with about 1000mL/day contains the mixed solution of microcarrier, cell, virus to cell/carrier settlement separator, and extract the supernatant liquor that contains virus out with the speed of 800mL/day, beginning is propagation continuously, the continuous propagation of beginning 9 days.
Collect the viral supernatant of above-mentioned extraction with the receiving flask of 4 2000mL, the Trisodium Citrate that adds 0.02% formalin and 0.05mol stirs at 4 ℃ to be placed 20 hours, can complete inactivation H3N2 influenza virus.
Example 4 Vero cell cultures production/deactivation hemorrhagic fever viruss
With reference to carrying out present embodiment, adopt Vero cell cultures hemorrhagic fever virus with example 1 essentially identical step.
Substratum: Medium 199 (Gibco) adds 4% foetal calf serum, transfers to pH7.0～pH7.2 with sodium bicarbonate.
Microcarrier: Cytodex-1, consumption 2g/L.
Cell cultivation process: inoculum density 1.0 * 10
5Cells/mL, microcarrier Cytodex-1, consumption 2g/L, culture bioreactors is homemade through the 500mL of siliconizing stirring type bioreactor (SpinnerFlasks), 37 ℃ of cultivations, dissolved oxygen 30%, mixing speed 90rpm, just stage speed 250mL/day inoculated after 2.5 days, and cell density is 2.56 * 10
6About cells/mL, the beginning cultured continuously, sample rate 150mL/day, timing sampling, counting in 5 days, cell density maintains about 2.37 * 106cells/mL, finds that the cell culture of continuous cultivation is more steady.
Virus breeding: with homemade through the 2000mL of siliconizing stirring type bioreactor (Spinner Flasks) as the virus multiplication reactor, data rate stream with 450mL/day when above-mentioned cell suspending liquid enters the virus multiplication reactor adds serum-free Medium 199 basic mediums, infection multiplicity inoculation hemorrhagic fever virus strain with 2, under 33 ℃, pH7.0, cultivate, dissolved oxygen 30%, stir speed (S.S.) remains on 90rpm.About 80 hours, liquid level begins to reach reactor and effectively demarcates liquid level, flow velocity extraction with about 800mL/day contains the mixed solution of microcarrier, cell, virus to cell/carrier settlement separator, and extract the supernatant liquor that contains virus out with the speed of 600mL/day, beginning is propagation continuously, the continuous propagation of beginning 9 days.
Collect the viral supernatant liquor of stating extraction with the receiving flask of 3 2000mL, according to adding beta-propiolactone at 1: 4000,4 ℃ are stirred and placed 24 hours, and 37 ℃ of water-bath hydrolysis 2 hours can the complete inactivation hemorrhagic fever virus.
Claims (10)
1, the method for a kind of safe continuous enclosed cell cultures, virus production/deactivation is characterized in that, comprises following steps:
1) in biological reactor for cell culture, inoculates 1～2 * 10
5The cell of cells/mL, suspension culture is expanded to 0.2～1.0 * 10
7Cells/mL, during for microcarrier suspension culture, the full cell of 80～90% remittances is realized the elementary cultivation of cell on the microcarrier;
2) according to the growth characteristics of culturing cell and the size of bio-reactor volume, flow velocity stream with 0.3～1.0 times of reactor working volume/sky adds fresh culture, simultaneously pump cell suspending liquid with same flow velocity, keep biological reactor for cell culture stable the time, guarantee that cell is expanded to 0.2～1.0 * 10 in the residence time in reactor
7Cells/mL, during for microcarrier suspension culture, the full cell of 80～90% remittances on the microcarrier, the cultured continuously of realization cell;
3) pump cell suspending liquid to containing the virus multiplication reactor that infection multiplicity is 2～10 viruses, realize the elementary infection development of virus in cell;
4), in the process of keeping total system running balance, realize the continuous propagation of virus along with the continuous inflow of cell suspending liquid and continuously outflowing of viral supernatant liquor;
5) collect the viral supernatant liquor that pumps with the collection virus jar from the virus multiplication reactor, inactivation of viruses is realized sealing culturing cell and inactivation of viruses continuously.
2, the method for safe continuous enclosed cell cultures according to claim 1, virus production/deactivation, it is characterized in that, in the described step 1), the elementary cultivation of cell specifically is meant: adopt the suspension culture mode to carry out cell cultures, cultured cells be suspend, through suspension culture domestication or adherent cell, the microcarrier consumption is between 2～10g/L, and temperature is controlled at 37 ± 0.2 ℃, and pH maintains between 7.0～7.2, dissolved oxygen 25%～60%, inoculum density is 1～2 * 10
5Between the cells/mL, by in batches, feed supplement or reperfusion mode be cell amplification to 0.2～1.0 * 10
7Cells/mL, during for microcarrier suspension culture, the full cell of 80～90% remittances is finished elementary cultivation stage on the microcarrier.
3, according to the method for claim 1 or 2 described safe continuous enclosed cell cultures, virus production/deactivation, it is characterized in that cultured cells is a kind of in chick embryo fibroblast, Chinese hamster ovary celI, mdck cell, BHK-21 cell, Vero cell, 293 cells.
4, according to the method for claim 1 or 2 described safe continuous enclosed cell cultures, virus production/deactivation, it is characterized in that, in the described step 1), the elementary cultivation of cell, by reperfusion mode with cell amplification to 0.2～1.0 * 10
7Cells/mL begins perfusion culture behind postvaccinal 48～72h, select the irrigation rate in 0.5～1.5 times of reactor working volume/sky according to the culturing cell kind.
5, safe continuous enclosed cell cultures according to claim 1, the method of virus production/deactivation, it is characterized in that, described step 2) in, the cultured continuously of cell, specifically be meant: at first finish elementary perfusion culture, then according to the kind of culturing cell, flow rate regulation stream with 0.3～1.0 reactor working volume/sky adds fresh culture, if microcarrier is cultivated, contain fresh microcarrier in the substratum, and pump cell suspending liquid with same speed, guarantee that cell can be expanded to 0.2～1.0 * 10 in the residence time in reactor in stable keeping biological reactor for cell culture
7Cells/mL, during for microcarrier suspension culture, the full cell of 80～90% remittances is kept the high-cell density state in the reactor on the microcarrier, realizes the cultured continuously of cell.
6, according to the method for claim 1 or 5 described safe continuous enclosed cell cultures, virus production/deactivation, it is characterized in that described step 2) in, the cultured continuously of cell is carried out material with the mixing speed of 30～90rpm and is mixed.
7, the method for safe continuous enclosed cell cultures according to claim 1, virus production/deactivation, it is characterized in that, in described step 3) and the step 4), the volume of virus multiplication reactor is 2～5 times of biological reactor for cell culture volume, elementary infection development of while and continuous two stages of propagation, take differential expression initial stage propagation mode to carry out respectively with the continuous propagation mode that has cell, carrier reflux.
8, the method for safe continuous enclosed cell cultures according to claim 7, virus production/deactivation, it is characterized in that, in the described step 3), if cell cultivation process uses the substratum that contains serum, when virus multiplication, add the serum-free basic medium, realize that virus multiplication substratum serum content is 1%～2%; If cell cultivation process uses serum free medium, when virus multiplication, the proportional flow by 1～5 adds fresh serum free medium.
9, safe continuous enclosed cell cultures according to claim 7, the method of virus production/deactivation, it is characterized in that, in the described step 4), the continuous propagation of virus, specifically be meant: the suspension of extracting out from the virus multiplication reactor that contains cell and virus enters a cell/carrier settlement separator that has the cell reflux, the supernatant liquor that will contain virus is evacuated to the collection virus jar to keep the balance of entire operation system, the cell that settles down is back to the virus multiplication reactor by reflux, reach and make full use of the virus host cell, simultaneously, will be in reactor overstand, the outmoded microcarrier that cell comes off in a large number intermittently is collected into outmoded carrier holding tank, the a small amount of viral supernatant liquor that wherein contains is incorporated the collection virus jar into and is together collected, the sealing deactivation is carried out the material mixing and is realized viral intercellular consecutive infection with 30～90rpm mixing speed.
10, safety high-efficient continuous enclosed type cell cultures according to claim 7, the method of virus production/deactivation, it is characterized in that, in the described step 5), the sealing deactivation of virus, specifically be meant: collection virus is to finish by a series of collection virus jars that have automatic liquid level control switch, after each holding tank is collected and is expired, opening other holding tank by the liquid automatic control switch when closing this holding tank collects, full holding tank is taken off, change empty holding tank, so repeatedly circulation, realize the continuous collection of virus, after completely the holding tank of virus takes off with collection, carry out deactivation, and then the preparation virus vaccines.
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