CN111304067A - Intermittent cleaning and filtering system - Google Patents
Intermittent cleaning and filtering system Download PDFInfo
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- CN111304067A CN111304067A CN202010160562.8A CN202010160562A CN111304067A CN 111304067 A CN111304067 A CN 111304067A CN 202010160562 A CN202010160562 A CN 202010160562A CN 111304067 A CN111304067 A CN 111304067A
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- 238000001914 filtration Methods 0.000 title claims abstract description 47
- 238000004140 cleaning Methods 0.000 title claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 105
- 238000000926 separation method Methods 0.000 claims abstract description 59
- 238000004113 cell culture Methods 0.000 claims abstract description 15
- 238000007599 discharging Methods 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 16
- 238000011010 flushing procedure Methods 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 10
- 239000012510 hollow fiber Substances 0.000 claims description 9
- 239000012528 membrane Substances 0.000 claims description 7
- 239000002699 waste material Substances 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 230000010412 perfusion Effects 0.000 claims description 3
- 239000007789 gas Substances 0.000 claims 5
- 238000011143 downstream manufacturing Methods 0.000 claims 1
- 239000008188 pellet Substances 0.000 claims 1
- 239000002912 waste gas Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 241000700605 Viruses Species 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 3
- 239000007787 solid Substances 0.000 abstract description 3
- 230000002265 prevention Effects 0.000 abstract description 2
- 230000014616 translation Effects 0.000 abstract description 2
- 208000036828 Device occlusion Diseases 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 11
- 238000013461 design Methods 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000009295 crossflow filtration Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 239000011148 porous material Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000007792 addition Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000012444 downstream purification process Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- -1 recovered Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M39/00—Means for cleaning the apparatus or avoiding unwanted deposits of microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/12—Purification
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Abstract
The invention discloses an intermittent cleaning and filtering system which is used for discharging liquid, intercepting cells and separating solid from liquid and has a cleaning function to prevent a cell intercepting device from being blocked. The device can be used for various cell culture modes, and the trapped cells can be used for continuous culture, virus production, protein production, downstream separation and purification and the like, and can also be used for solid-liquid separation in the production process. The device has the advantages of continuous cell interception/solid-liquid separation, intermittent cell interception/solid-liquid separation, centralized high-speed cell interception/solid-liquid separation, prevention of device blockage, avoidance of pollution and guarantee of cell interception quality and required solid and liquid separation in the production process.
Description
Technical Field
The invention belongs to the field of cell culture, and particularly relates to a continuously-used device suitable for solid-liquid separation and cell interception.
Background
In cell culture in the field of biomedicine, particularly in continuous culture, cells are usually separated from a culture medium liquid, recovered, and a waste liquid is discharged, as required for liquid replacement. In the case of continuous culture, since fresh culture medium is continuously added during the cell culture process, part of the original culture medium needs to be discarded, but the cells in the original culture medium still need to be retained and returned to the bioreactor for continuous culture.
The process of separating solid from liquid is solid-liquid separation, and the methods are various, such as sedimentation, filtration, centrifuges and the like. In the cell retention process, the widely used technique is filtration. Filtration techniques for solid-liquid separation can generally be divided into dead-end filtration and tangential flow filtration.
Dead-end filtration devices used in cell culture processes are again generally classified into general filtration and rotary filtration. Dead-end filtration uses the pore size of the filter membrane and the pressure differential across the membrane to retain cells, while rotary filtration devices typically use the pressure differential created by centrifugation to retain cells and allow the culture fluid to permeate. The dead-end filtration principle and the operation are simple, and depending on the selection of the membrane pore size, the dead-end filtration equipment can ensure the interception of cells in the culture solution and can also protect the bioreactor from being polluted. However, the dead-end filtration device has a limited filtration capacity per unit area of the filter medium, and cell attachment growth on the surface of the filter medium is also likely to occur. Therefore, the dead-end filtration equipment often causes filter clogging in practical use and is difficult to continue production.
The tangential flow filtration can reduce the blockage of cells on the surface of the membrane, and is beneficial to increasing the loading capacity of the filtration equipment. However, since cell retention in cell culture is mostly intermittent operation, although the tangential flow filtration can reduce cell adhesion and blockage during operation, cells can still adhere to the surface of the filtration medium and grow during non-operation of the system, and the separation effect is influenced. An ATF (alternating tangential flow cell trapping system) system developed based on the trapping principle of a hollow fiber filter is a cell trapping and separating device capable of preventing blockage. The ATF system includes a forward and reverse diaphragm pump and a hollow fiber filter connected to the fermenter, the diaphragm pump alternately flowing the culture solution containing the cells through the hollow fiber filter in both forward and reverse directions. Because the system can make the culture solution flow uninterruptedly in the hollow fiber filter, the ATF not only can effectively retain the cells, but also can effectively prevent the cells from attaching and growing on the surface of the hollow fiber so as to keep the separation efficiency of the system. Since the ATF system should maintain the strength of the cell culture fluid repeatedly flowing in the hollow fiber, it should not damage the cells, and the setting of the process parameters requires time, experience, and money.
In order to simplify the design and operation of cell interception and solid-liquid separation equipment used in continuous production, the invention designs two different process links for cell interception and filter flushing; can effectively realize cell interception and solid-liquid separation, can fully clean the filter medium, avoid bacterial growth and reduce filter blockage.
Disclosure of Invention
In order to solve the existing problems of cell culture and solid-liquid separation at present, the invention provides an intermittent cleaning and filtering system which can be continuously used and can prevent blockage. The system is beneficial to cell interception, solid-liquid separation and waste liquid discharge, and has the functions of intermittent independent cleaning and blockage prevention. The invention has simple design principle, manufacture, assembly, operation and is easy to test and verify.
The invention provides an intermittent cleaning and filtering system which comprises a bioreactor 1, a first pump 2, a first two-way valve 3, a cell interception/solid-liquid separation component 4, a second two-way valve 5, a first valve 6, a second valve 7, a flushing liquid/gas tank 8 and a second pump 9.
Further, the batch cleaning filtration system of the present invention comprises: the liquid inlet end of the cell interception/solid-liquid separation component 4 is connected with the bioreactor 1 through a first pump 2 and a first two-way valve 3, and the liquid outlet end of the cell interception/solid-liquid separation component is connected with the bioreactor 1 through a second two-way valve 5; the flushing liquid/gas tank device 8 is connected with the liquid inlet end of the cell interception/solid-liquid separation component 4 through a second pump 9 and a first two-way valve 3; the filtering end of the cell interception/solid-liquid separation component 4 is connected with a first valve 6 and is used for discharging waste liquid; the second two-way valve 5 is connected to a second valve 7 for discharging the washing liquid/gas passing through the cell trapping/solid-liquid separating module 4.
The intermittent cleaning and filtering system can be used for separating, cleaning and filtering cell culture solution in various cell culture modes, including but not limited to batch culture, fed-batch culture, perfusion culture, semi-perfusion culture, continuous culture and semi-continuous culture.
Further, the cell trapping/solid-liquid separating member 4 of the batch cleaning filtration system of the present invention is generally a filter made of a membrane pack or hollow fibers.
One or more cell interception/solid-liquid separation components 4 can be connected to the bioreactor 1 in the intermittent cleaning and filtering system, and when a plurality of cell interception/solid-liquid separation components 4 are needed, the cell interception/solid-liquid separation components can be connected in series or in parallel or can be connected in series and parallel to coexist, so that the single work, the simultaneous work or the alternate work can be realized; further, switching or replacement of components during operation may be achieved. When a plurality of cell-trapping/solid-liquid separation modules 4 are used, the filter of each separation module 4 may be a filter of a different material, such as a membrane filter, or a hollow fiber filter.
The intermittent cleaning and filtering system is suitable for cell interception, solid-liquid separation and cleaning and filtering required in various cell culture processes, wherein the solid-liquid separation can be used for realizing solid-liquid separation in a biological process and is also suitable for solid-liquid separation in a downstream purification process. The cell culture includes, but is not limited to, the culture of single cells, lysed cells, fused cells, cell debris, cell aggregates, bacteria, lysed bacteria, homogenized bacteria, yeast, lysed yeast, homogenized yeast; the trapped cells can be cultured, used for virus production, protein production and the like, and can also be used for further separation and purification. The culture solution after separation can be discarded, or used for producing virus, producing protein, or used for downstream separation and purification, etc.
The intermittent cleaning and filtering system can be cleaned in the modes of liquid, gas, ultrasound, rays and the like, and all pipelines, valves, pumps, flushing liquid/gas tanks, interception devices, cleaning liquid, cleaning gas and the like must be subjected to aseptic treatment, so that exogenous pollution and cross pollution are prevented.
The cell culture mode in the bioreactor 1 can be batch culture, fed-batch culture, perfusion culture, semi-perfusion culture, continuous culture, semi-continuous culture and the like.
On the other hand, the invention provides an intermittent cleaning and filtering method, which can conveniently intercept cells in the cell culture process, realize solid-liquid separation and waste liquid discharge, and simultaneously prevent the filter from being blocked by intermittent independent cleaning.
Further, the intermittent cleaning and filtering method is completed by the intermittent cleaning and filtering system.
Further, the intermittent cleaning and filtering method comprises the following steps:
(1) adding a corresponding culture medium into the bioreactor 1, inoculating cell seed liquid, and culturing cells to an ideal cell concentration according to the process requirement.
(2) When the liquid is changed, the first two-way valve 3 is opened, the bioreactor 1 is communicated with the cell interception/solid-liquid separation
Leaves the module 4 and opens the two-way valve 5, connecting to the bioreactor 1. The first pump 2 is started, and the culture medium in the bioreactor 1 returns to the bioreactor 1 through the first two-way valve 3, the cell interception/solid-liquid separation component 4 and the second two-way valve 5. The cells in the culture solution are intercepted by the cell interception medium, and part of the culture solution passes through the cell interception medium and is discharged out of the system through the first valve 6.
(3) When the removal volume meets the process requirement, the first pump 2 is stopped; the first two-way valve 3 is turned to the flushing liquid/gas tank 8, the second two-way valve 5 is turned to the second valve 7, the second valve 7 is opened, the second pump 9 is started. The liquid or gas in the flushing liquid/gas tank 8 flows through the cell trapping/solid-liquid separation component 4, the second two-way valve 5 and the second valve 7 through the first two-way valve 3 and is discharged out of the system, so that the purpose of cleaning residual cells on the surface of the trapping medium in the cell trapping device is achieved.
(4) The cell trap cleaning is completed and the second pump 9 is stopped.
(5) And when the liquid is changed again, repeating the steps to realize intermittent cleaning and filtering.
Compared with the prior art, the invention has the following advantages:
1. the cell interception and solid-liquid separation equipment has an independent cleaning function, can effectively prevent the system from polluting and blocking the filter, and improves the use efficiency.
2. The device has simple and clear flow path design, fully utilizes the prior filter medium to carry out cell interception and solid-liquid separation, has simple operation and is beneficial to test verification.
Drawings
FIG. 1: is a schematic structural flow diagram of the invention. Wherein: bioreactor 1, first pump 2, first two-way valve 3, cell interception/solid-liquid separation component 4, second two-way valve 5, first valve 6, second valve 7, flushing liquid/gas tank 8, second pump 9.
Detailed Description
The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described with reference to the drawings, but the present invention is not limited to these embodiments.
System overview: the invention relates to an intermittent cleaning and filtering system, which comprises a bioreactor 1 and a cell interception/solid-liquid separation component 4, wherein the liquid inlet end of the cell interception/solid-liquid separation component 4 is connected with the bioreactor 1 through a first pump 2 and a first two-way valve 3, and the liquid outlet end of the cell interception/solid-liquid separation component 4 is connected with the bioreactor 1 through a second two-way valve 5; the flushing liquid/gas tank 8 is connected with the liquid inlet end of the cell interception/solid-liquid separation component 4 through a second pump 9 and a first two-way valve 3; the second two-way valve 5 is connected with a second valve 7, and the second valve 7 can be used for discharging the washing buffer solution for washing the cell interception/solid-liquid separation component; the cell-trapping/solid-liquid-separating module 4 discharges the waste liquid/gas through the second valve 7.
The operation steps are as follows:
adding corresponding culture medium into the bioreactor 1, inoculating cell seed liquid, and culturing cells to ideal cell concentration according to process requirements. The cell culture process is carried out as follows:
1. when liquid needs to be changed, the first two-way valve 3 is opened to communicate the bioreactor 1 and the cell interception/solid-liquid separation component 4, and the two-way valve 5 is opened to be connected to the bioreactor 1. The first pump 2 is started, and the culture medium in the bioreactor 1 returns to the bioreactor 1 through the first two-way valve 3, the cell interception/solid-liquid separation component 4 and the second two-way valve 5. The cells in the culture solution are intercepted by the cell interception medium, and part of the culture solution passes through the cell interception medium and is discharged out of the system through the first valve 6.
2. When the removal volume meets the process requirement, the first pump 2 is stopped; the first two-way valve 3 is turned to the flushing liquid/gas tank 8, the second two-way valve 5 is turned to the second valve 7, the second valve 7 is opened, the second pump 9 is started. The liquid or gas in the flushing liquid/gas tank 8 flows through the cell trapping/solid-liquid separation component 4, the second two-way valve 5 and the second valve 7 through the first two-way valve 3 and is discharged out of the system, so that the purpose of cleaning residual cells on the surface of the trapping medium in the cell trapping device is achieved.
3. The cell-trap washing is finished and the second pump 9 is stopped.
4. And when the liquid is changed again, repeating the steps to realize intermittent cleaning and filtering.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (9)
1. An intermittent cleaning and filtering system comprises a bioreactor, a flushing liquid/gas tank and a cell interception/solid-liquid separation component; the liquid inlet end of the cell interception/solid-liquid separation component is connected with the bioreactor through a first pump and a first two-way valve; the liquid outlet end of the cell interception/solid-liquid separation component is connected with the bioreactor through a second two-way valve; the flushing liquid/gas tank is connected with the liquid inlet end of the cell interception/solid-liquid separation component through a second pump and a first two-way valve; the cell interception/solid-liquid separation component is also connected with a first valve, and the first valve is used for discharging waste liquid generated in the cell interception/solid-liquid separation component; the second two-way valve is also connected to a second valve for discharging the flushing waste/waste gas flowing through the cell trapping/solid-liquid separating assembly.
2. A batch cleaning filtration system according to claim 1, wherein the cell trapping/solid-liquid separating element is a filter, preferably a membrane, hollow fiber.
3. A batch cleaning filtration system according to any one of claims 1-2, wherein the cell culture in the bioreactor is batch culture, fed-batch culture, perfusion culture, semi-perfusion culture, continuous culture, semi-continuous culture.
4. A batch cleaning filtration system according to any one of claims 1 to 3, wherein one of the bioreactors is connected to one or more cell trapping/solid-liquid separation modules.
5. A batch cleaning filtration system according to claim 4, wherein the plurality of cell-trapping/solid-liquid-separating elements are of the same or different types.
6. A batch cleaning filtration system according to claim 4, wherein the plurality of cell trapping/solid-liquid separating modules are present in series, in parallel, or in series and in parallel.
7. A batch cleaning filtration system according to claim 5, wherein the plurality of cell trapping/solid-liquid separation modules are operated simultaneously or alternately.
8. The batch cleaning filtration system of one of claims 1-7, wherein the type of cells used for entrapment is single cell, lysed cell, fused cell, cell debris, cell pellet, bacteria, lysed bacteria, homogenized bacteria, yeast, lysed yeast, homogenized yeast; or for solid-liquid separation in downstream processes.
9. A batch cleaning and filtering method comprises the following steps:
(1) adding corresponding culture medium into the bioreactor 1, inoculating cell seed liquid, culturing cells to ideal cell concentration,
(2) and when the liquid is changed, opening the first two-way valve 3, communicating the bioreactor 1 with the cell interception/solid-liquid separation component 4, opening the two-way valve 5, connecting the two-way valve 5 to the bioreactor 1, starting the first pump 2, and returning the culture medium in the bioreactor 1 to the bioreactor 1 through the first two-way valve 3, the cell interception/solid-liquid separation component 4 and the second two-way valve 5. The cells in the culture solution are intercepted by the cell interception medium, part of the culture solution passes through the cell interception medium and is discharged out of the system through the first valve 6,
(3) when the removal volume meets the process requirement, the first pump 2 is stopped; the first two-way valve 3 is turned to the flushing liquid/gas tank 8, the second two-way valve 5 is turned to the second valve 7, the second valve 7 is opened, the second pump 9 is started. The liquid or gas in the flushing liquid/gas tank 8 flows through the cell interception/solid-liquid separation component 4, the second two-way valve 5 and the second valve 7 through the first two-way valve 3 and is discharged out of the system, so as to achieve the purpose of cleaning the residual cells on the surface of the interception medium in the cell interceptor,
(4) after the cell retention device is cleaned, the second pump 9 is stopped,
(5) and when the liquid is changed again, repeating the steps to realize intermittent cleaning and filtering.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010160562.8A CN111304067A (en) | 2020-03-10 | 2020-03-10 | Intermittent cleaning and filtering system |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010160562.8A CN111304067A (en) | 2020-03-10 | 2020-03-10 | Intermittent cleaning and filtering system |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113082822A (en) * | 2021-04-23 | 2021-07-09 | 承德石油高等专科学校 | Filtering liquid-changing device |
| WO2022129862A1 (en) * | 2020-12-15 | 2022-06-23 | Cellularevolution Ltd | A bioprocessing system and method |
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Application publication date: 20200619 |
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