CN100497583C - Safety high-efficient continuous enclosed type cell culture and virus production-inactivation system - Google Patents
Safety high-efficient continuous enclosed type cell culture and virus production-inactivation system Download PDFInfo
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- CN100497583C CN100497583C CNB2006100232324A CN200610023232A CN100497583C CN 100497583 C CN100497583 C CN 100497583C CN B2006100232324 A CNB2006100232324 A CN B2006100232324A CN 200610023232 A CN200610023232 A CN 200610023232A CN 100497583 C CN100497583 C CN 100497583C
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Abstract
The safe high-efficiency continual closed cell culture virus generation/deactivation system comprises: a cell amplification unit connected to a gas supplying unit, a steam generator, a supplying device for cell growth medium, a primary fill and collection device, a heat source supply device, and a acid/alkali liquid supply device; a virus amplification unit connected to a gas supplying unit, a basic medium supply device, a cell/carrier separator, a heat source supply device, and a acid/alkali liquid supply device; and a old carrier collector. This invention integrates cell and virus amplification processes for automation control and standard production with little pollution.
Description
Technical field
What the present invention relates to is a kind of system of technical field of bioengineering, specifically is a kind of safety high-efficient continuous enclosed type cell culture and virus production/deactivation system.
Technical background
The mode of tradition culturing cell production virus is to adopt the rolling bottle mode to cultivate.This mode yields poorly, and is difficult to realize automatization control, and repeatability is bad, and batch difference is big.Appearance along with bio-reactor, the problems referred to above have been solved greatly, general cell cultures is made up of sensing elements such as reactor tank body, the chuck that heat is provided, gas distributor, mechanical stirring device, the cover plate that is connected with numerous injection ports, thief hole, venting port, sensor interface and temperature sensor, pH electrode, dissolved oxygen electrodes with bio-reactor, mixing, mass transfer and oxygen supply problem have been solved well, for cell cultures and virus degree of increasing propagation provides adapt circumstance, general maximum cell density can reach 10
7More than the cell/ml, Bing Du output is also corresponding like this is improved.
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 the system that a kind of Fibra-Cel of use Disks produces encephalitis b virus as the fixed bed Cultivation of Vero of carrier, this system is made up of nutrient solution transport pipe, peristaltic pump, bio-reactor, air feeder etc.This system is made up of nutrient solution transport pipe, peristaltic pump, bio-reactor, air feeder etc.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, Y.Z.Ghendon etc. are at " Vaccine " 2005, " Development of cell culture (MDCK) live cold-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, proposed a kind of Cytodex-1 of use microcarrier suspension culture mdck cell and produced the system of influenza virus, this system is made up of nutrient solution transport pipe, peristaltic pump, bio-reactor, cell separator, air feeder etc.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.More than in two kinds of systems, 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
At " discontinuity and non-closure " this weak point in the present virus vaccines production, the invention provides a kind of safety high-efficient continuous enclosed type cell culture and virus production/deactivation system, make it be used for cell cultures, virus multiplication, the continuous sealing production of inactivation of virus, at first adopting the perfusion culture mode to carry out high-density cells cultivates, stream adds fresh culture cultured continuously cell then, the cell that flows out from reactor enters the virus multiplication unit and carries out virus multiplication, virus enters holding tank and collects, the sealing deactivation, can reduce like this and not disconnect, close microbiological contamination and the viral risk that leaks that reactor causes, simultaneously whole system is convenient to automatization control, can safety, efficiently, extensive virus of proliferation is used for production of vaccine.
The present invention is achieved by the following technical solutions, the present invention includes: gas supply unit, vapour generator, supplying device for cell growth medium, cell amplification unit, elementary perfusion collection device, basic medium feeding mechanism, virus multiplication unit, cell/carrier tripping device, collection virus device, heat source accommodation device, acid/alkali lye feeding mechanism and outmoded carrier collection device.Mode of connection is: gas supply unit divides two-way, and a road of exit end is connected with the unitary entrance end of cell amplification, satisfies the required gas requirement of cell amplification, and another road is connected with the unitary entrance end of virus multiplication, satisfies the required gas requirement of virus multiplication; The exit end of vapour generator is connected with the unitary entrance end of cell amplification, is used for the sterilization on the throne of total system; The exit end of supplying device for cell growth medium is connected with the unitary entrance end of cell amplification, and the inlet end of elementary perfusion collection device is connected with the unitary exit end of cell amplification, realizes the elementary perfusion culture and the cultured continuously of cell; Heat source accommodation device is connected with the virus multiplication unit with the cell amplification unit respectively, satisfies temperature required requirement; The exit end of acid/alkali lye feeding mechanism is connected with the unitary entrance end of cell amplification unit and virus multiplication respectively, satisfies the requirement of required pH value; The unitary exit end of cell amplification is connected with the unitary entrance end of virus multiplication; The exit end of basic medium feeding mechanism is connected with the unitary entrance end of virus multiplication; The entrance end of cell/carrier tripping device is connected with the unitary exit end of virus multiplication, and three exit end are connected with virus multiplication unit, collection virus device and the entrance end of outmoded carrier collection device respectively; The exit end of outmoded carrier collection device is connected with the entrance end of collection virus device.
Gas supply unit comprises: gas bomb, coarse filter, air compressor, threeway, first Self controlling valve, gas meter, gas mixer and gas humidifying device.Wherein, gas bomb has three, i.e. N
2Gas bomb, CO
2Gas bomb, O
2Gas bomb; Gas mixer is made by stainless material, is elliptical cylinder-shape, and its oval top one side is four gas inletes, and opposite side is a mixed gas outlet; The gas humidification apparatus main body is a cylinder shape hemisphere top Glass Containers, and its top is integrated with first inlet mouth and first air outlet, and first intake interface gos deep into place, nearly bottom in the container, and the gas that comes out from first air outlet is by the-0.2 μ m membrane filtration air feed.Its mode of connection is: coarse filter links to each other with the inlet of air compressor, three gas bombs, air compressor connect four gas inletes on first Self controlling valve, gas meter and the gas mixer in turn by four flat copper gas pipe lines, first inlet mouth of mixed gas outlet and gas humidifying device joins, and first air outlet and the one 0.2 μ m filter membrane join.
Gas supply unit is divided into two paths with air feed, can make cell amplification unit and virus multiplication unit obtain different air feed demands, helps carrying out smoothly of cell amplification and virus multiplication process, and this process is by control N
2, CO
2, O
2Realize with the flow of air.
The cell amplification unit comprises: the first thermal source input aperture, the first thermal source delivery port, first chuck, the biological reactor for cell culture tank body, second inlet mouth, vapor inlet port, the cell inoculation mouth, the cell growth medium input aperture, first acid/alkali input aperture, second Self controlling valve, first electric motor and speed change gear, first sensor, elementary perfusion delivery port, first thief hole, the cell mixture delivery port, second air outlet, the 2 0.2 μ m filter membrane, first cover plate, the cell cut-off equipment, first whipping appts, first gas distributor and first controller and performer.Its mode of connection is: the unitary main body of cell amplification is made up of biological reactor for cell culture tank body and first cover plate that covers on it, the biological reactor for cell culture tank body is made by the erosion resistance high-boron-silicon glass, around it is first chuck, the first thermal source input aperture that is provided with above and the first thermal source delivery port; First cover plate is the circular steel prepared material, be connected with a lot of gangways on it, inlet comprises second inlet mouth, vapor inlet port, cell inoculation mouth, cell growth medium input aperture and first acid/alkali input aperture, and outlet comprises first sensor, elementary perfusion delivery port, first thief hole, cell mixture delivery port and second air outlet; Elementary perfusion delivery port is connected with cell cut-off equipment in the biological reactor for cell culture tank body by stainless steel tube; Second inlet mouth links to each other with first gas distributor by silicone rubber tube; Second air outlet links to each other with the 2 0.2 μ m filter membrane; First electric motor and speed change gear be positioned at first cover plate above, link to each other with first whipping appts; First controller and performer are connected with first sensor by data line one end, the other end and second Self controlling valve, first electric motor and speed change gear are connected first Self controlling valve in on-line automatic control cell amplification culture medium input aperture, first acid/alkali input aperture, first electric motor and speed change gear, cell mixture delivery port and the gas supply unit with first Self controlling valve in the gas supply unit.
In the cell amplification reaction member, vapor inlet port links to each other with vapour generator, can sterilize to total system with steam; First thermal source flows at the first chuck internal recycle, keeps the temperature of developing medium in the biological reactor for cell culture tank body; First thief hole can be used for the off-line sampling and measuring; Simultaneously, first controller and performer comprise temperature element by first sensor, pH electrode, dissolved oxygen electrode and cell density potential electrode, the input of on-line automatic control cell growth medium and first acid/alkali, the rotating speed of first whipping appts, the work output of cell mixture and air feed are formed and flow, for cell amplification provides suitable temperature, pH, environment and suitable cell growth medium flow rates such as dissolved oxygen, make from the cell amplification unit effusive cell mixture density moderate, be convenient to cell cultures and produce carrying out smoothly of virus multiplication step in the viral process.
At first in the cell amplification unit, inoculate certain cell, be connected with elementary perfusion collection device by cell growth medium input aperture, elementary perfusion delivery port, cell cut-off equipment, carry out elementary perfusion culture cell, when cell amplification stops elementary perfusion culture to suitable density, provide stream refinement intracellular growth substratum (if during microcarrier suspension culture by supplying device for cell growth medium, also in substratum, add microcarrier and mixed flow adds), cell mixture flows into the virus multiplication unit simultaneously, realizes the cultured continuously of cell.
The virus multiplication unit comprises: the second thermal source input aperture, the second thermal source delivery port, second chuck, virus multiplication reactor tank body, the 3rd inlet mouth, the cell mixture input aperture, cell/carrier backflow input aperture, the basic medium input aperture, second acid/alkali input aperture, the 3rd Self controlling valve, second electric motor and speed change gear, the virus inoculation mouth, two transmitters, second thief hole, virus mixed solution delivery port, the 3rd air outlet, the 3 0.2 μ m filter membrane, second cover plate, second whipping appts, second gas distributor and second controller and performer.Its mode of connection is: the unitary main body of virus multiplication is made up of virus multiplication reactor tank body and second cover plate that covers on it, virus multiplication reactor tank body is made by the erosion resistance high-boron-silicon glass, around it is second chuck, the second thermal source input aperture that is provided with above and the second thermal source delivery port; Second chuck is the circular steel prepared material, on be connected with a lot of gangways, inlet comprises the 3rd inlet mouth, cell mixture input aperture, cell/carrier backflow input aperture, basic medium input aperture, second acid/alkali input aperture and virus inoculation mouth, and outlet comprises second transmitter, second thief hole, viral mixed solution delivery port and the 3rd air outlet; The 3rd inlet mouth; By the silicone rubber tube and second gas distributor; Link to each other; The 3rd air outlet links to each other with the 3 0.2 μ m filter membrane; Second electric motor and speed change gear be positioned at second cover plate above, link to each other with second whipping appts; Second controller and performer are connected with second transmitter by data line one end, second Self controlling valve in the other end and the 3rd Self controlling valve, second electric motor and speed change gear, the cell amplification unit is connected first and second Self controlling valve of on-line automatic control, basic medium input aperture, second acid/alkali input aperture, second electric motor and speed change gear, cell/carrier backflow input aperture, viral mixed solution delivery port with first and second Self controlling valve in the gas supply unit.
In the virus multiplication unit, second thermal source flows at the second chuck internal recycle, keeps the temperature of virus multiplication reactor tank culturing in vivo medium; Contain the required exogenous factor of virus multiplication in the basic medium, with cell mixture by a certain percentage mixed flow add, together form the virus multiplication substratum, play dilute serum and the effect of nutritive substance be provided; Second thief hole can be used for the off-line sampling and measuring; Simultaneously, second controller and performer comprise temperature element, pH electrode, dissolved oxygen electrode and nutritive substance potential electrode by second transmitter, the work output of on-line automatic control first Self controlling valve, basic medium and the input of second acid/alkali, the rotating speed of second whipping appts, viral mixed solution and air feed are formed and flow, for the cell amplification virus multiplication provides environment and suitable viral mixed solution output flow rates such as suitable temperature, pH, dissolved oxygen, keep carrying out smoothly of system stability and virus production.
In the virus multiplication unit, when cell mixture and basic medium by a certain percentage mixed flow add, when composition virus multiplication substratum flows into virus multiplication reactor tank body at first, carry out the elementary propagation of differential expression virus from virus inoculation mouth virus inoculation, when liquid volume reaches the effective volume of virus multiplication reactor tank body, extract viral mixed solution out, keep system balancing, carry out the continuous breeding of virus.
Cell/carrier tripping device comprises: cell/carrier backflow delivery port, viral mixed solution inlet opening, four selfs control valve, outer framework, viral supernatant liquor delivery port, the 3rd transmitter, deposition plate, the 3rd thief hole, outmoded carrier delivery port, carrier output valve and the 3rd controller and performer.
Cell/carrier tripping device is to utilize density variation, settlement separate cell/carrier, viral supernatant and outmoded carrier, cross section is " L " shape, is 15 °～45 ° with vertical direction, the top long-width ratio is used for the separation of viral supernatant liquor between 3～5; The lower section long-width ratio is used for the backflow of cell/carrier and the recovery of outmoded carrier between 5～10.Wherein, outer framework constitutes the shell of cell/carrier tripping device; Parallel cell/carrier tripping device the inside that is distributed in of deposition plate, spacing is between 0.5～1.5cm, and silicon oozes processing; The middle part is viral mixed solution inlet opening, and the upper end is viral supernatant liquor delivery port and the 3rd transmitter, and the lower end is cell/carrier backflow delivery port, the 3rd thief hole and outmoded carrier delivery port; Outmoded carrier delivery port links to each other with outmoded carrier collection device by the silicon rubber pipeline that the carrier output valve is housed; The 3rd controller and performer are connected with the 3rd transmitter by data line one end, second Self controlling valve in the other end and four selfs control valve, unitary the 3rd Self controlling valve of virus multiplication, the cell amplification unit is connected first, second and third Self controlling valve of on-line automatic control, viral mixed solution inlet opening, cell/carrier backflow delivery port and viral supernatant liquor delivery port with first Self controlling valve in the gas supply unit.
By cell/carrier tripping device, viral supernatant liquor is evacuated to the collection virus device to keep the balance of entire operation system, cell/the carrier that settles down is back to the virus multiplication unit by the backflow delivery port, reach the purpose that makes full use of the virus host cell, improves viral yield, simultaneously, can be according to the System Operation situation, outmoded carrier intermittently discharged that will the cell of overstand comes off in a large number in reactor is to outmoded carrier collection device, minimizing is to the influence of virus multiplication, and a small amount of viral supernatant liquor that wherein contains is incorporated the collection virus device into.
The collection virus device comprises: liquid dispenser inlet, liquid dispenser, liquid dispenser outlet, automatic control of liquid level opening-closing valve, viral liquid input aperture, liquid level sensor, viral liquid delivery port, inactivator input aperture, the 4 0.2 μ m filter membrane, the 4th air outlet, virus jar and the 4th controller and performer.Wherein, an end of liquid dispenser links to each other with viral supernatant liquor delivery port in cell/carrier tripping device by liquid dispenser inlet, and the outlet of the other end liquid dispenser links to each other with viral liquid input aperture on the virus jar by the automatic control of liquid level opening-closing valve; The virus jar links to each other with the 4th air outlet with viral liquid input aperture, liquid level sensor, viral liquid delivery port, inactivator input aperture; The 4th air outlet links to each other with the 4 0.2 μ m filter membrane; The 4th controller and performer are connected with liquid level sensor by data line one end, and the other end is connected with the automatic control of liquid level opening-closing valve, the collection of the viral liquid of on-line automatic control.
In the collection virus device, collection virus is to finish by a series of virus jars that have the control of automatic control of liquid level opening-closing valve, after the full virus of each virus jar collection, when closing this virus jar, open jar continuation of other virus by the automatic control of liquid level opening-closing valve and collect virus, then full virus jar is taken off, change empty virus jar, circulation so repeatedly realizes the continuous collection of virus.And, according to the virus production ability what, a papova jar and another papova jar can be separated, by the control of automatic control of liquid level opening-closing valve, collect the purpose of virus when realizing large scale continuous prod continuously.
After the virus jar of collecting full virus taken off, can be by adopting conventional virus inactivating agent such as inactivation of viruses 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.
When the present invention works, at first system is carried out integrated sterilization with vapour generator.Air feed comprises N
2, CO
2, O
2And air, carrying out pre-treatment by coarse filter, gas humidification device and 0.2 μ m filter membrane, gas supply unit is divided into two paths with air feed, regulates N
2, CO
2, O
2With the flow of four kinds of gases of air, can make cell amplification unit and virus multiplication unit obtain different air feed demands; Acid/alkali lye feeding mechanism is that cell amplification unit and virus multiplication unit add acid/alkali, regulates the pH value; Heat source accommodation device provides thermal source, attemperation for cell amplification unit and virus multiplication unit; Supplying device for cell growth medium and basic medium feeding mechanism are respectively the cell amplification unit and the virus multiplication unit provides cell cultures and required substratum of virus multiplication stage; The stream rate of acceleration of the online detection of transmitter, controller and performer and control cell amplification unit and the unitary temperature of virus multiplication, pH, dissolved oxygen, mixing speed and substratum makes that parameters reaches set(ting)value in the system, keeps stablizing of system.At first in the cell amplification unit, inoculate certain cell, elementary perfusion culture, cell amplification provides stream refinement intracellular growth substratum (during microcarrier suspension culture by supplying device for cell growth medium to suitable density, also to be mixed with microcarrier in the substratum), the cultured continuously cell, cell mixture flows into the virus multiplication unit, add basic medium by basic medium feeding mechanism stream in the virus multiplication unit simultaneously, the two mixes common composition virus multiplication substratum by a certain percentage, from virus inoculation mouth virus inoculation, carry out the elementary propagation of differential expression virus, when liquid volume reaches the effective volume of virus multiplication reactor tank body, effusive viral mixed solution enters cell/carrier tripping device from the virus multiplication unit, carry out the continuous breeding of virus, settlement separate by cell/carrier tripping device, cell/carrier refluxes and enters the virus multiplication unit, the virus supernatant liquor enters the collection virus device and carries out deactivation, realize continuous enclosed type cell culture and virus and deactivation, and can intermittently collect outmoded carrier to outmoded carrier collection device according to running situation, can reduce the influence of outmoded carrier to virus multiplication, a small amount of viral supernatant liquor that wherein contains is incorporated the together deactivation of collection virus device into.
By above explanation, as seen, cell amplification and two procedure correlations of virus multiplication get up in system of the present invention, carry out cell cultures production/inactivation of viruses under the sealing continuously, 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 the new system that extensive high-efficiency and continuous is produced virus that very is suitable for.
Description of drawings
Fig. 1 is a structural representation of the present invention
Fig. 2 is a gas supply unit structural representation of the present invention
Fig. 3 is a cell amplification modular construction synoptic diagram of the present invention
Fig. 4 is a virus multiplication modular construction synoptic diagram of the present invention
Fig. 5 is cell of the present invention/carrier tripping device structural representation
Fig. 6 is a collection virus apparatus structure synoptic diagram of the present invention
,1. 2. 3. 4. 5. 6. 7.8./ 9. 10. 11./12. 13. 14. 15. 16. 17. 18. 19. 20. 21.22. 23. 24 25.0.2μm 26. 27. 28. 29.30. 31. 32. 33.34./ 35. 36. 37.38. 39. 40. 41.42.0.2μm 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54./ 55. 56./57. 58.59.60.61. 62. 63. 64.0.2μm65. 66. 67. 68. 69./ 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88.0.2μm 89. 90. 91.。
Embodiment
As shown in Figure 1, the present invention includes: gas supply unit 1, vapour generator 2, supplying device for cell growth medium 3, cell amplification unit 4, elementary perfusion collection device 5, basic medium feeding mechanism 6, virus multiplication unit 7, cell/carrier tripping device 8, collection virus device 9, heat source accommodation device 10, acid/alkali lye feeding mechanism 11, outmoded carrier collection device 12.Mode of connection is: 1 fen two-way of gas supply unit, a road of exit end is connected with the entrance end of cell amplification unit 4, satisfy the required gas requirement of cell amplification, another road is connected with the entrance end of virus multiplication unit 7, satisfies the required gas requirement of virus multiplication; The exit end of vapour generator 2 is connected with the entrance end of cell amplification unit 4, is used for the sterilization on the throne of total system; The exit end of supplying device for cell growth medium 3 is connected with the entrance end of cell amplification unit 4, the inlet end of elementary perfusion collection device 5 is connected with the exit end of cell amplification unit 4, realizes the elementary perfusion culture and the cultured continuously of cell; Heat source accommodation device 10 is connected with virus multiplication unit 7 with cell amplification unit 4 respectively, satisfies temperature required requirement; The exit end of acid/alkali lye feeding mechanism 11 is connected with the entrance end of cell amplification unit 4 with virus multiplication unit 7 respectively, satisfies the requirement of required pH value; The exit end of cell amplification unit 4 is connected with the entrance end of virus multiplication unit 7; The exit end of basic medium feeding mechanism 6 is connected with the entrance end of virus multiplication unit 7; The entrance end of cell/carrier tripping device 8 is connected with the exit end of virus multiplication unit 7, and three exit end are connected with virus multiplication unit 7, collection virus device 9 and the entrance end of outmoded carrier collection device 12 respectively; The exit end of outmoded carrier collection device 12 is connected with the entrance end of collection virus device 9.
As shown in Figure 2, gas supply unit 1 comprises: gas bomb 13, coarse filter 14, air compressor 15, threeway 16, first Self controlling valve 17, gas meter 18, gas mixer 19 and gas humidifying device 20.Wherein, gas bomb 13 has three, i.e. N
2Gas bomb, CO
2Gas bomb, O
2Gas bomb; Gas mixer 19 is made by stainless material, is elliptical cylinder-shape, and its oval top one side is four gas inletes 21, and opposite side is a mixed gas outlet 22; Gas humidification device 20 main bodys are cylinder shape hemisphere top Glass Containers, its top is integrated with first inlet mouth 23 and first air outlet 24, first intake interface 23 gos deep into place, nearly bottom in the container, and the gas that comes out from first air outlet 24 filters air feed by the one 0.2 μ m filter membrane 25.Mode of connection is: coarse filter 14 links to each other with the inlet of air compressor 15, three gas bombs 13, air compressor 15 connect four gas inletes 21 on first Self controlling valve 17, gas meter 18 and the gas mixer 19 in turn by four flat copper gas pipe lines, first inlet mouth 23 of mixed gas outlet 22 and gas humidifying device joins, and first air outlet 24 and the one 0.2 μ m filter membrane 25 join.Gas supply unit is divided into two paths with air feed, can make cell amplification unit and virus multiplication unit obtain different air feed demands, and this process is by control N
2, CO
2, O
2Realize with the flow of air.
As shown in Figure 3, cell amplification unit 4 comprises: the first thermal source input aperture 26, the first thermal source delivery port 27, first chuck 28, biological reactor for cell culture tank body 29, second inlet mouth 30, vapor inlet port 31, cell inoculation mouth 32, cell growth medium input aperture 33, first acid/alkali input aperture 34, second Self controlling valve 35, first electric motor and speed change gear 36, first sensor 37, elementary perfusion delivery port 38, first thief hole 39, cell mixture delivery port 40, second air outlet 41, the 2 0.2 μ m filter membrane 42, first cover plate 43, cell cut-off equipment 44, first whipping appts 45, first gas distributor 46, first controller and performer 47.Mode of connection is: the main body of cell amplification unit 4 is made up of biological reactor for cell culture tank body 29 and first cover plate 43 that covers on it, biological reactor for cell culture tank body 29 is made by the erosion resistance high-boron-silicon glass, around it is first chuck 28, the first thermal source input aperture 26 that is provided with above and the first thermal source delivery port 27; First cover plate 43 is the circular steel prepared material, is connected with a lot of gangways on it, and suitable cell proliferation culture environment is provided.The inlet of first cover plate 43 comprises second inlet mouth 30, vapor inlet port 31, cell inoculation mouth 32, cell growth medium input aperture 33 and first acid/alkali input aperture 34, and outlet comprises first sensor 37, elementary perfusion delivery port 38, first thief hole 39, cell mixture delivery port 40 and second air outlet 41.Wherein, vapor inlet port 31 links to each other with vapour generator 2; Elementary perfusion delivery port 39 is connected with cell cut-off equipment 44 by stainless steel tube, is used for elementary perfusion culture; Second inlet mouth 30 links to each other with first gas distributor 46 by silicone rubber tube; Second air outlet 41 links to each other with the 2 0.2 μ m filter membrane 42.First electric motor and speed change gear 36 be positioned at first cover plate 43 above, link to each other with first whipping appts 45.First controller and performer 47 are connected with first sensor 37 by data line one end, the other end and second Self controlling valve 35, first electric motor and speed change gear 36 are connected on-line automatic control cell growth medium input aperture 33, first acid/alkali input aperture 34, first electric motor and speed change gear 36, cell mixture delivery port 40 and first Self controlling valve 17 with first Self controlling valve 17 in the gas supply unit 1.
As shown in Figure 4, virus multiplication unit 7 comprises: the second thermal source input aperture 48, the second thermal source delivery port 49, second chuck 50, virus multiplication reactor tank body 51, the 3rd inlet mouth 52, cell mixture input aperture 53, cell/carrier backflow input aperture 54, basic medium input aperture 55, second acid/alkali input aperture 56, the 3rd Self controlling valve 57, second electric motor and speed change gear 58, virus inoculation mouth 59, second transmitter 60, second thief hole 61, virus mixed solution delivery port 62, the 3rd air outlet 63, the 3 0.2 μ m filter membrane 64, second cover plate 65, second whipping appts 66, second gas distributor 67, second controller and performer 68.Mode of connection is: the main body of virus multiplication unit 7 is made up of virus multiplication reactor tank body 51 and second cover plate 65 that covers on it, virus multiplication reactor tank body 51 is made by the erosion resistance high-boron-silicon glass, around it is second chuck 50, offers virus multiplication unit 7 optimal temperatures by the second thermal source input aperture 48 and the second thermal source delivery port 49 that is provided with; Second cover plate 65 is the circular steel prepared material, is connected with a lot of gangways on it, and suitable virus multiplication culture environment is provided.The inlet of second cover plate 65 comprises the 3rd inlet mouth 52, cell mixture input aperture 53, cell/carrier backflow input aperture 54, basic medium input aperture 55, second acid/alkali input aperture 56 and virus inoculation mouth 59, and outlet comprises second transmitter 60, second thief hole 61, viral mixed solution delivery port 62 and the 3rd air outlet 63.Wherein, the 3rd inlet mouth 52 links to each other with second gas distributor 67 by silicone rubber tube; The 3rd air outlet 63 links to each other with the 3 0.2 μ m filter membrane 64.Second electric motor and speed change gear 58 be positioned at second cover plate 65 above, link to each other with second whipping appts 66.Second controller and performer 68 are connected with second transmitter 60 by data line one end, second Self controlling valve 35 in the other end and the 3rd Self controlling valve 57, second electric motor and speed change gear 58, the cell amplification unit 4 is connected on-line automatic control first Self controlling valve 17, second Self controlling valve 35, basic medium input aperture 55, second acid/alkali input aperture 56, second electric motor and speed change gear 58, cell/carrier backflow input aperture 54 and viral mixed solution delivery port 62 with first Self controlling valve 17 in the gas supply unit 1.
As shown in Figure 5, cell/carrier tripping device 8 comprises: cell/carrier backflow delivery port 69, viral mixed solution inlet opening 70, four selfs control valve 71, outer framework 72, viral supernatant liquor delivery port 73, the 3rd transmitter 74, deposition plate 75, the 3rd thief hole 76, outmoded carrier delivery port 77, carrier output valve 78, the 3rd controller and performer 79.The cross section of cell/carrier tripping device 8 is " L " shapes, is 15 °～45 ° with vertical direction, and the top long-width ratio is used for the separation of viral supernatant liquor between 3～5; The lower section long-width ratio is used for the backflow of cell/carrier and the recovery of outmoded carrier between 5～10.Mode of connection is: outer framework 72 constitutes the shell of cell/carrier tripping device 8, deposition plate 75 parallel cell/carrier tripping device 8 inside that are distributed in, spacing is between 0.5～1.5cm, the middle part of cell/carrier tripping device 8 is viral mixed solution inlet openings 70, the upper end is viral supernatant liquor delivery port 73 and the 3rd transmitter 74, and the lower end is cell/carrier backflow delivery port 69, the 3rd thief hole 76 and outmoded carrier delivery port 77; Outmoded carrier delivery port 77 links to each other with outmoded carrier collection device 12 by the silicon rubber pipeline of carrier output valve 78 controls; The 3rd controller and performer 79 are connected with the 3rd transmitter 74 by data line one end, the 3rd Self controlling valve 57 in the other end and four selfs control valve 71, the virus multiplication unit 7, second Self controlling valve 35 in the cell amplification unit 4 are connected on-line automatic control first Self controlling valve 17, second Self controlling valve 35, the 3rd Self controlling valve 57, viral mixed solution inlet opening 70, cell/carrier backflow delivery port 69 and viral supernatant liquor delivery port 73 with first Self controlling valve 17 in the gas supply unit 1.
As shown in Figure 6, collection virus device 9 comprises: liquid dispenser inlet 80, liquid dispenser 81, liquid dispenser outlet 82, automatic control of liquid level opening-closing valve 83, viral liquid input aperture 84, liquid level sensor 85, viral liquid delivery port 86, inactivator input aperture the 87, the 4 0.2 μ m filter membrane 88, the 4th air outlet 89, virus jar the 90 and the 4th controller and performer 91.Mode of connection is: an end of liquid dispenser 81 links to each other with viral supernatant liquor delivery port 73 in cell/carrier tripping device 8 by liquid dispenser inlet 80, and the outlet 82 of the other end liquid dispenser links to each other with viral liquid input aperture 84 on the virus jar 90 by automatic control of liquid level opening-closing valve 83; Virus jars 90 links to each other with viral liquid input aperture 84, liquid level sensor 85, viral liquid delivery port 86, inactivator input aperture 87 and the 4th air outlet 89; The 4th air outlet 89 links to each other with the 4 0.2 μ m filter membrane 88; The 4th controller and performer 91 are connected with liquid level sensor 85 by data line one end, and the other end is connected with automatic control of liquid level opening-closing valve 83, and the collection of the viral liquid of on-line automatic control, each jar are collected full back, sealed inactivation of viruses with inactivator.
After native system fixedly mounts, open vapour generator 2, make steam fully enter cell amplification unit 4, supplying device for cell growth medium 3, elementary perfusion collection device 5, virus multiplication unit 7, basic medium feeding mechanism 6, cell/carrier tripping device 8, collection virus device 9, acid/alkali lye feeding mechanism 11 and outmoded carrier collection device 12, total system is sterilized.After the sterilization, steam off producer 2, the air compressor 15 of unlatching gas supply unit 1 dries up steam residual in the system and moisture.Then, open heat source accommodation device 10, thermal source is circulated at first chuck 28 and second chuck 50, guarantee the temperature of biological reactor for cell culture tank body 29 and virus multiplication reactor tank body 51.Cell amplification culture medium, basic medium, the acid/alkali of bacterium of will going out injects supplying device for cell growth medium 3, basic medium feeding mechanism 6 and acid/alkali lye feeding mechanism 11 respectively, then cell growth medium is pumped into biological reactor for cell culture tank body 29.The gas of gas supply unit 1 supply enters biological reactor for cell culture tank body 29 successively behind threeway 17, first Self controlling valve 18, gas meter 19, gas mixer 20, gas humidification device 21 and the one 0.2 μ m filter membrane 25 filtration sterilizations, form and flow for the cell amplification process provides suitable air feed.Regulate first whipping appts 45 and make gas and cell growth medium uniform mixing.Then, from cell inoculation mouth 32 inoculating cells, elementary perfusion culture, cell amplification stops elementary perfusion culture to suitable density, provide stream refinement intracellular growth substratum (if during microcarrier suspension culture by supplying device for cell growth medium 3, also in substratum, add microcarrier and mixed flow adds), the cultured continuously cell.Cell mixture flows into virus multiplication reactor tank body 51, add basic medium by basic medium feeding mechanism 6 streams simultaneously, the two mixes common composition virus multiplication substratum by a certain percentage, from virus inoculation mouth 59 virus inoculations, open the gas supply unit 1 and second whipping appts 66 according to the step identical again with the cell amplification process, carry out the elementary propagation of differential expression virus, when liquid volume reaches the effective volume of virus multiplication reactor tank body 51, effusive viral mixed solution enters cell/carrier tripping device 8 from virus multiplication unit 7, carries out the continuous breeding of virus.Settlement separate by cell/carrier tripping device 8, cell/carrier refluxes and enters virus multiplication reactor tank body 51, the virus supernatant liquor enters collection virus device 9 and collects, the sealing deactivation, realize continuous enclosed type cell culture and virus and deactivation, and can intermittently collect outmoded carrier to outmoded carrier collection device 12 according to running situation, can reduce the influence of outmoded carrier to virus multiplication, a small amount of viral supernatant liquor that wherein contains is incorporated collection virus device 9 into and is together collected, seals deactivation.
In system's operational process, inductor block comprises that temperature element, pH electrode, dissolved oxygen electrode and nutritive substance potential electrode, cell density potential electrode and liquid level sensor are connected with controller and performer, online auto monitoring control, regulate the flow and the air feed of thermal source, acid/alkali, substratum and form and flow, keep carrying out smoothly of system stability and cell culture and virus production/inactivation process.Simultaneously, also can carry out sampling analysis by thief hole.
Claims (10)
1, a kind of safety high-efficient continuous enclosed type cell culture and virus production/deactivation system, comprise: gas supply unit (1), vapour generator (2), supplying device for cell growth medium (3), cell amplification unit (4), elementary perfusion collection device (5), basic medium feeding mechanism (6), virus multiplication unit (7), cell/carrier tripping device (8), collection virus device (9), heat source accommodation device (10), acid/alkali lye feeding mechanism (11), outmoded carrier collection device (12), it is characterized in that, gas supply unit (1) divides two-way, a road of exit end is connected with the entrance end of cell amplification unit (4), another road is connected with the entrance end of virus multiplication unit (7), the exit end of vapour generator (2) is connected with the entrance end of cell amplification unit (4), the exit end of supplying device for cell growth medium (3) is connected with the entrance end of cell amplification unit (4), the inlet end of elementary perfusion collection device (5) is connected with the exit end of cell amplification unit (4), heat source accommodation device (10) is connected with virus multiplication unit (7) with cell amplification unit (4) respectively, the exit end of acid/alkali lye feeding mechanism (11) is connected with cell amplification unit (4) entrance end with virus multiplication unit (7) respectively, the exit end of cell amplification unit (4) is connected with the entrance end of virus multiplication unit (7), the exit end of basic medium feeding mechanism (6) is connected with the entrance end of virus multiplication unit (7), the entrance end of cell/carrier tripping device (8) is connected with the exit end of virus multiplication unit (7), three exit end respectively with virus multiplication unit (7), collection virus device (9) is connected with the entrance end of outmoded carrier collection device (12), and the exit end of outmoded carrier collection device (12) is connected with the entrance end of collection virus device (9).
2, safety high-efficient continuous enclosed type cell culture and virus production according to claim 1/deactivation system, it is characterized in that, gas supply unit (1) comprising: gas bomb (13), coarse filter (14), air compressor (15), threeway (16), first Self controlling valve (17), gas meter (18), gas mixer (19) and gas humidifying device (20), wherein, gas mixer (19) top one side is four gas inletes (21), and opposite side is a mixed gas outlet (22); Gas humidification device (20) top is integrated with first inlet mouth (23) and first air outlet (24), and first intake interface (23) gos deep into place, nearly bottom in the container; Coarse filter (14) links to each other with the inlet of air compressor (15), and gas bomb (13), air compressor (15) join with the one 0.2 μ m filter membrane (25) by first inlet mouth (23), first air outlet (24) that the copper gas pipe line connects first Self controlling valve (17), gas meter (18), gas mixer (19) and gas humidifying device (20) in turn.
3, safety high-efficient continuous enclosed type cell culture and virus production according to claim 2/deactivation system is characterized in that, gas bomb (13) has three, for: N
2Gas bomb, CO
2Gas bomb, O
2Gas bomb.
4, safety high-efficient continuous enclosed type cell culture and virus production according to claim 2/deactivation system is characterized in that, gas mixer (19) is elliptical cylinder-shape, and its oval top one side is four gas inletes (21); Gas humidification device (20) main body is a cylinder shape hemisphere top Glass Containers.
5, safety high-efficient continuous enclosed type cell culture and virus production according to claim 1/deactivation system, it is characterized in that, cell amplification unit (4) comprising: the first thermal source input aperture (26), the first thermal source delivery port (27), first chuck (28), biological reactor for cell culture tank body (29), second inlet mouth (30), vapor inlet port (31), cell inoculation mouth (32), cell growth medium input aperture (33), first acid/alkali input aperture (34), second Self controlling valve (35), first electric motor and speed change gear (36), first sensor (37), elementary perfusion delivery port (38), first thief hole (39), cell mixture delivery port (40), second air outlet (41), the 2 0.2 μ m filter membrane (42), first cover plate (43), cell cut-off equipment (44), first whipping appts (45), first gas distributor (46) and first controller and performer (47), wherein, the main body of cell amplification unit (4) is made up of biological reactor for cell culture tank body (29) and first cover plate (43) that covers on it, around the biological reactor for cell culture tank body (29) is first chuck (28), the first thermal source input aperture (26) that is provided with above and the first thermal source delivery port (27); First cover plate connects the gangway on (43), inlet comprises second inlet mouth (30), vapor inlet port (31), cell inoculation mouth (32), cell growth medium input aperture (33) and first acid/alkali input aperture (34), exports to comprise first sensor (37), elementary perfusion delivery port (38), first thief hole (39), cell mixture delivery port (40) and second air outlet (41); Vapor inlet port (31) links to each other with vapour generator (2); Elementary perfusion delivery port (38) is connected with cell cut-off equipment (44) by stainless steel tube; Second inlet mouth (30) links to each other with first gas distributor (46) by silicone rubber tube; Second air outlet (41) links to each other with the 2 0.2 μ m filter membrane (42); First electric motor and speed change gear (36) be positioned at first cover plate (43) above, link to each other with first whipping appts (45); First controller and performer (47) are connected with first sensor (37) by data line one end, the other end and second Self controlling valve (35), first electric motor and speed change gear (36) are connected first Self controlling valve (17) in on-line automatic control cell growth medium input aperture (33), first acid/alkali input aperture (34), first electric motor and speed change gear (36), cell mixture delivery port (40) and the gas supply unit (1) with first Self controlling valve (17) in the gas supply unit (1).
6, safety high-efficient continuous enclosed type cell culture and virus production according to claim 1/deactivation system, it is characterized in that, virus multiplication unit (7) comprising: the second thermal source input aperture (48), the second thermal source delivery port (49), second chuck (50), virus multiplication reactor tank body (51), the 3rd inlet mouth (52), cell mixture input aperture (53), cell/carrier backflow input aperture (54), basic medium input aperture (55), second acid/alkali input aperture (56), the 3rd Self controlling valve (57), second electric motor and speed change gear (58), virus inoculation mouth (59), second transmitter (60), second thief hole (61), virus mixed solution delivery port (62), the 3rd air outlet (63), the 3 0.2 μ m filter membrane (64), second cover plate (65), second whipping appts (66), second gas distributor (67), second controller and performer (68), wherein, the main body of virus multiplication unit (7) is made up of virus multiplication reactor tank body (51) and second cover plate (65) that covers on it, around the virus multiplication reactor tank body (51) is second chuck (50), the second thermal source input aperture (48) that is provided with above and the second thermal source delivery port (49); Second cover plate is connected with the gangway on (65), inlet comprises the 3rd inlet mouth (52), cell mixture input aperture (53), cell/carrier backflow input aperture (54), basic medium input aperture (55), second acid/alkali input aperture (56) and virus inoculation mouth (59), exports to comprise second transmitter (60), second thief hole (61), viral mixed solution delivery port (62) and the 3rd air outlet (63); The 3rd inlet mouth (52) links to each other with second gas distributor (67) by silicone rubber tube; The 3rd air outlet (63) links to each other with the 3 0.2 μ m filter membrane (64); Second electric motor and speed change gear (58) be positioned at second cover plate (65) above, link to each other with second whipping appts (66); Second controller and performer (68) one ends are connected with second transmitter (60), the other end and the 3rd Self controlling valve (57), second electric motor and speed change gear (58), second Self controlling valve (35) in the cell amplification unit (4) is connected on-line automatic control first Self controlling valve (17) with first Self controlling valve (17) in the gas supply unit (1), second Self controlling valve (35), basic medium input aperture (55), second acid/alkali input aperture (56), second electric motor and speed change gear (58), cell/carrier backflow input aperture (54) and viral mixed solution delivery port (62).
7, safety high-efficient continuous enclosed type cell culture and virus production according to claim 1/deactivation system, it is characterized in that, cell/carrier tripping device (8) comprising: cell/carrier backflow delivery port (69), virus mixed solution inlet opening (70), four selfs control valve (71), outer framework (72), virus supernatant liquor delivery port (73), the 3rd transmitter (74), deposition plate (75), the 3rd thief hole (76), outmoded carrier delivery port (77), carrier output valve (78), the 3rd controller and performer (79), wherein, outer framework (72) constitutes the shell of cell/carrier tripping device (8), deposition plate (75) is located at cell/carrier tripping device (8) inside, the middle part of cell/carrier tripping device (8) is viral mixed solution inlet opening (70), the upper end is viral supernatant liquor delivery port (73) and the 3rd transmitter (74), and the lower end is cell/carrier backflow delivery port (69), the 3rd thief hole (76) and outmoded carrier delivery port (77); Outmoded carrier delivery port (77) links to each other with outmoded carrier collection device (12) by the silicon rubber pipeline of carrier output valve (78) control; The 3rd controller and performer (79) one ends are connected with the 3rd transmitter (74), the 3rd Self controlling valve (57) in the other end and four selfs control valve (71), virus multiplication unit (7), second Self controlling valve (35) in cell amplification unit (4) are connected on-line automatic control first Self controlling valve (17), second Self controlling valve (35), the 3rd Self controlling valve (57), viral mixed solution inlet opening (70), cell/carrier backflow delivery port (69) and viral supernatant liquor delivery port (73) with first Self controlling valve (17) in the gas supply unit (1).
8, according to claim 1 or 7 described safety high-efficient continuous enclosed type cell culture and virus production/deactivation systems, it is characterized in that, cell/carrier tripping device (8) cross section is down L shaped shape, be 15 °～45 ° with vertical direction, the top long-width ratio is between 3～5, and the lower section long-width ratio is between 5～10.
9, safety high-efficient continuous enclosed type cell culture and virus production according to claim 7/deactivation system is characterized in that, parallel cell/carrier tripping device (8) inside that is distributed in of deposition plate (75), and spacing is between 0.5～1.5cm, and silicon oozes processing.
10, safety high-efficient continuous enclosed type cell culture and virus production according to claim 1/deactivation system, it is characterized in that, collection virus device (9) comprising: liquid dispenser inlet (80), liquid dispenser (81), liquid dispenser outlet (82), automatic control of liquid level opening-closing valve (83), virus liquid input aperture (84), liquid level sensor (85), virus liquid delivery port (86), inactivator input aperture (87), the 4 0.2 μ m filter membrane (88), the 4th air outlet (89), virus jar (90) and the 4th controller and performer (91), wherein, one end of liquid dispenser (81) links to each other with viral supernatant liquor delivery port (73) in cell/carrier tripping device (8) by liquid dispenser inlet (80), the other end liquid dispenser outlet (82) links to each other with viral liquid input aperture (84) on the virus jar (90) by automatic control of liquid level opening-closing valve (83), virus jar (90) and viral liquid input aperture (84), liquid level sensor (85), virus liquid delivery port (86), inactivator input aperture (87) links to each other with the 4th air outlet (89), the 4th air outlet (89) links to each other with the 4 0.2 μ m filter membrane (88), the 4th controller and performer (91) one ends are connected with liquid level sensor (85), and the other end is connected with automatic control of liquid level opening-closing valve (83).
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| GB2495934A (en) * | 2011-10-25 | 2013-05-01 | Tap Biosystems Phc Ltd | Bioreactor outlet air conditioning systems and associated methods |
| CN103992936B (en) * | 2014-04-22 | 2015-10-07 | 北京科兴生物制品有限公司 | A kind of closed formalin-inactivated virus tubing system and application thereof |
| BE1024230B1 (en) * | 2016-11-08 | 2017-12-20 | Univercells Sa | Contained production of cells and / or cellular products |
| CN106399083B (en) * | 2016-11-22 | 2019-01-08 | 金宇保灵生物药品有限公司 | A kind of suspension cell lesion terminal automatic identification and control system and method |
| CN107541464B (en) * | 2017-10-31 | 2021-04-30 | 山东亦度生物技术有限公司 | Enhanced cell microcarrier perfusion culture interception method |
| CN108034583B (en) * | 2018-01-25 | 2024-04-02 | 吉林冠界生物技术有限公司 | Cell process vaccine manufacturing system |
| CN111235030A (en) * | 2020-03-10 | 2020-06-05 | 北京好思康科技有限公司 | System for producing viruses by two-stage mixed culture |
| CN114561270B (en) * | 2022-03-21 | 2023-04-28 | 广州齐志生物工程设备有限公司 | High pathogenic virus culture container system |
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Assignee: Yangzhou VAC BIO Engineering Co., Ltd. Assignor: Shanghai Jiao Tong University Contract record no.: 2010320000607 Denomination of invention: Safety high-efficient continuous enclosed type cell culture and virus production-inactivation system Granted publication date: 20090610 License type: Exclusive License Open date: 20061004 Record date: 20100514 |
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Effective date of registration: 20200120 Address after: 201499 room 17, No. 2165, Lane 258, hope road, Shanghai, Fengxian District Patentee after: Shanghai Li Kun biological Polytron Technologies Inc Address before: 200240 Dongchuan Road, Shanghai, No. 800, No. Patentee before: Shanghai Jiaotong University |