CN115449482A - Cell culture equipment and cell culture method - Google Patents
Cell culture equipment and cell culture method Download PDFInfo
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- CN115449482A CN115449482A CN202110641269.8A CN202110641269A CN115449482A CN 115449482 A CN115449482 A CN 115449482A CN 202110641269 A CN202110641269 A CN 202110641269A CN 115449482 A CN115449482 A CN 115449482A
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- 238000004113 cell culture Methods 0.000 title claims abstract description 49
- 101710106089 Protein cutoff Proteins 0.000 claims abstract description 45
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 39
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 39
- 239000006228 supernatant Substances 0.000 claims abstract description 29
- 239000000706 filtrate Substances 0.000 claims abstract description 12
- 238000007599 discharging Methods 0.000 claims abstract description 7
- 238000003306 harvesting Methods 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims abstract description 4
- 238000004891 communication Methods 0.000 claims description 18
- 239000012528 membrane Substances 0.000 claims description 15
- 239000012510 hollow fiber Substances 0.000 claims description 14
- 238000000108 ultra-filtration Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 8
- 238000000746 purification Methods 0.000 abstract description 7
- 239000007788 liquid Substances 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 51
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000011097 chromatography purification Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000010874 maintenance of protein location Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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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
- C12M47/10—Separation or concentration of fermentation products
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- Biomedical Technology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses cell culture equipment and a cell culture method, wherein the cell culture equipment comprises a first container, a bioreactor, a cell interception device, a second container and a protein interception device, wherein the first container is used for storing a culture medium; the bioreactor is communicated with the first container; the inlet of the cell cut-off device is communicated with the bioreactor; the second container is used for storing harvest liquid, and a first inlet of the second container is communicated with a first outlet of the cell stopping device; the inlet of the protein cut-off device is communicated with the outlet of the second container, the first outlet of the protein cut-off device is used for discharging the treated cell supernatant to the second container, and the second outlet of the protein cut-off device is used for discharging the filtrate. The rear end of the cell culture equipment is provided with a protein cut-off device, so that the cell supernatant is concentrated on line in real time, a part of impurity protein with small molecular weight is removed, and the workload of downstream impurity purification is reduced. The invention can be widely applied to the field of biotechnology.
Description
Technical Field
The invention relates to the technical field of biology, in particular to cell culture equipment and a cell culture method.
Background
Perfusion culture is one of the important processes of the prior mammalian cell culture, not only can provide a stable environment which is beneficial to cell culture by continuously discharging cell metabolic byproducts so as to solve the problems of unstable protein quality or low expression quantity and the like, but also can optimize the productivity utilization rate and improve the production efficiency by improving the yield of unit volume, but the impurity level in the culture supernatant obtained by the prior culture technology is higher, so that the difficulty of downstream chromatographic purification is increased, and the work load of chromatographic purification is increased.
Disclosure of Invention
In order to solve at least one of the above technical problems, the present invention provides a cell culture apparatus and a cell culture method, which adopts the following technical scheme:
the cell culture equipment provided by the invention comprises a first container, a bioreactor, a cell stopping device, a second container and a protein stopping device, wherein the first container is used for storing a culture medium; the bioreactor is in communication with the first container; the inlet of the cell shut-off device is communicated with the bioreactor; the second container is used for storing harvest liquid, and a first inlet of the second container is communicated with a first outlet of the cell stopping device; the inlet of the protein shut-off device is in communication with the outlet of the second vessel, the first outlet of the protein shut-off device is in communication with the second inlet of the second vessel, the first outlet of the protein shut-off device is for discharging the treated cell supernatant to the second vessel, and the second outlet of the protein shut-off device is for discharging a filtrate.
In certain embodiments of the invention, the second outlet of the protein shut-off device is provided with a delivery pump.
In certain embodiments of the invention, the cell culture apparatus comprises a third vessel for storing a filtrate, the third vessel being in communication with the second outlet of the protein shut-off device.
In certain embodiments of the invention, the first inlet of the second container is provided with a delivery pump and the inlet of the protein shut-off device is provided with a delivery pump.
In certain embodiments of the invention, the protein shut-off device comprises a protein shut-off hollow fiber column or an ultrafiltration membrane package.
In certain embodiments of the invention, the protein shut-off device comprises a plurality of protein shut-off hollow fiber columns arranged in parallel or a plurality of ultrafiltration membrane membranes arranged in parallel.
In some embodiments of the invention, a pressure gauge is disposed on a pipeline of the protein cut-off device, wherein the pipeline is communicated with the first outlet and the second container, and the pressure gauge monitors transmembrane pressure.
In certain embodiments of the invention, the second outlet of the cell shut-off device is provided with a diaphragm pump.
In certain embodiments of the invention, the second outlet of the cell shut-off device is in communication with the bioreactor; and a delivery pump is arranged at the inlet of the cell interception device.
In certain embodiments of the invention, the cell culture apparatus comprises an ultrasonic shut-off device, an inlet of the ultrasonic shut-off device is in communication with the bioreactor, a first outlet of the ultrasonic shut-off device is in communication with an inlet of the cell shut-off device, and a second outlet of the ultrasonic shut-off device is in communication with the bioreactor.
In the cell culture method provided by the invention, the cell culture equipment is adopted, the protein cut-off device processes the cell supernatant in the second container and then returns the processed cell supernatant to the second container, and the cell supernatants from the protein cut-off device and the cell cut-off device are mixed in the second container.
In certain embodiments of the invention, the flow rate at the first inlet of the second vessel is controlled to coincide with the flow rate at the second outlet of the protein stop.
The embodiment of the invention has at least the following beneficial effects: the protein cut-off device is arranged at the rear end of the cell culture equipment, the concentration of cell supernatant is carried out on line in real time, the downstream harvesting and culturing period can be prolonged, the pressure of downstream purification is reduced, the downstream purification production preparation is more flexible and sufficient, a part of impurity protein with small molecular weight is removed, and the workload of downstream purification impurities is reduced. The invention can be widely applied to the field of biotechnology.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a schematic structural view of a first embodiment of a cell culture apparatus;
FIG. 2 is a schematic structural view of a second embodiment of the cell culture apparatus;
FIG. 3 is a schematic structural view of a third embodiment of the cell culture apparatus.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention and are not to be construed as limiting the present invention.
In the description of the present invention, it is to be understood that if the terms "center", "middle", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "axial", "radial", "circumferential", etc., are used in an orientation or positional relationship indicated based on the drawings, it is merely for convenience of description and simplicity of description, and it is not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and therefore, is not to be considered as limiting the present invention. The features defined as "first" and "second" are used to distinguish feature names rather than having a special meaning, and further, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "a plurality" means two or more unless otherwise specified.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in a specific case to those of ordinary skill in the art.
Example one
The invention relates to a cell culture device, which comprises a first container 101 and a bioreactor 102, wherein an agitator is arranged in the bioreactor 102, the bioreactor 102 is communicated with the first container 101, the first container 101 is used for storing culture medium, and a delivery pump is arranged on a pipeline between the first container 101 and the bioreactor 102 so as to replenish liquid from the first container 101 to the bioreactor 102.
The cell culture equipment comprises a cell interception device 103 and a second container 104, wherein the second container 104 is used for storing harvest liquid, the inlet of the cell interception device 103 is communicated with the bioreactor 102, the first inlet of the second container 104 is communicated with the first outlet of the cell interception device 103, the first inlet of the second container 104 is provided with a delivery pump, and the delivery pump delivers cell supernatant obtained by processing the cell interception device 103 to the second container 104. Specifically, the cell shut-off device 103 comprises a cell shut-off hollow fiber column.
Referring to the figures, the second outlet of the cell stopping device 103 is provided with a diaphragm pump 108, and it will be understood that the diaphragm pump 108 provides power when the cell fluid enters the cell stopping device 103 from the bioreactor 102.
The cell culture equipment comprises a protein cut-off device 105, the protein cut-off device 105 is used for separating impurity proteins with small molecular weights in cell supernatant, an inlet of the protein cut-off device 105 is communicated with an outlet of the second container 104, a delivery pump is arranged at an inlet of the protein cut-off device 105, and the delivery pump provides power for the cell supernatant in the second container 104 to enter the protein cut-off device 105.
Referring to the figures, the first outlet of the protein shut-off device 105 is in communication with the second inlet of the second vessel 104, the first outlet of the protein shut-off device 105 is used to discharge the treated cell supernatant to the second vessel 104, and the second outlet of the protein shut-off device 105 is used to discharge the filtrate. It will be appreciated that the filtrate contains the contaminating proteins of lower molecular weight, and the portion of the cell supernatant discharged from the first outlet of the protein cut-off 105 is removed of the contaminating proteins of lower molecular weight, and is returned to the second vessel 104 as purified cell supernatant.
Further, the cell culture apparatus comprises a third vessel 106, the third vessel 106 being adapted to store a filtrate, the third vessel 106 being in communication with the second outlet of the protein shut-off device 105. Referring to the drawing, a second outlet of the protein cut-off device 105 is provided with a transfer pump to transfer the filtrate separated by the protein cut-off device 105 to the third vessel 106. Of course, it will be appreciated that the cell culture apparatus may also be designed without the third vessel 106, but with an external vessel to receive the filtrate.
It will be appreciated that the flow rate a of the cell supernatant delivered by the cell shut-off device 103 to the second vessel 104 and the flow rate B of the filtrate discharged from the second outlet of the protein shut-off device 105 are controlled to be the same, so that the volume of the cell supernatant in the second vessel 104 is maintained constant, and the desired protein concentration of the cell supernatant in the second vessel 104 is gradually increased as the protein shut-off device 105 is purified in successive cycles.
Specifically, flow rate a is controlled by controlling the power of the delivery pump at the first inlet of the second vessel 104, and flow rate B is controlled by controlling the power of the delivery pump at the second outlet of the protein cut-off device 105. In some examples, flow rate A and flow rate B are controlled to be equal by two delivery pumps.
It will be appreciated that the addition of a protein shut-off device 105 to effect shut-off at the back end of the cell culture apparatus allows for the concentration of the harvested cell supernatant to be performed on-line in real time and the filtrate to be removed. The protein cut-off device 105 is added to continuously concentrate cell supernatant continuously harvested in the perfusion culture process, so that the downstream sample collection period can be prolonged, downstream purification production preparation can be more flexible and sufficient, the pressure of downstream purification is relieved, a part of impurity proteins with small molecular weights can be removed, and the workload of downstream purification impurities is reduced.
The protein cutoff device 105 includes a protein cutoff hollow fiber column or an ultrafiltration membrane module for purifying cell supernatant. In some examples, a protein-sequestering hollow fiber column or ultrafiltration membrane module is provided as one. In some examples, the protein cutoff device 105 includes a plurality of protein cutoff hollow fiber columns arranged in parallel or a plurality of ultrafiltration membranes arranged in parallel, each of which is used in sequence, in order not to affect the operation of the protein cutoff device 105 in consideration of clogging or damage of the protein cutoff hollow fiber columns or the ultrafiltration membranes. With reference to the drawings, a pressure gauge 107 is arranged on a pipeline of the protein cut-off device 105, which is communicated with the second container 104, the pressure gauge 107 is in communication connection with a control module of the cell culture equipment, the pressure gauge 107 is used for monitoring transmembrane pressure, and the pressure gauge 107 is arranged on a pipeline of a first outlet of the protein cut-off device 105.
In some examples, the protein cut-off device 105 includes a plurality of protein-cut-off hollow fiber columns, each of which is used in sequence, and when the pressure gauge 107 tests that the transmembrane pressure is higher than the process set value, the next protein-cut-off hollow fiber column is automatically switched in the protein cut-off device 105, and the used protein-cut-off hollow fiber column is put into a cleaning procedure for recycling and reuse. Specifically, an electric valve is arranged at an inlet of each protein interception hollow fiber column, and a control module of the cell culture equipment controls the opening and closing of the electric valve according to a signal of a pressure gauge 107, so that each protein interception hollow fiber column is sequentially switched to be opened and closed.
In some examples, the protein shut-off device 105 includes a plurality of ultrafiltration membrane modules, each of which is used in sequence, and when the pressure gauge 107 measures a transmembrane pressure above a process set point, the next ultrafiltration membrane module is automatically switched in the protein shut-off device 105, and the used ultrafiltration membrane module is subjected to a cleaning procedure for recycling. Specifically, an electric valve is arranged at the inlet of each ultrafiltration membrane package, and the control module of the cell culture equipment controls the opening and closing of the electric valve according to the signal of the pressure gauge 107, so that each ultrafiltration membrane package is sequentially switched to be opened and closed.
Example two
The present embodiment is different from the first embodiment in that the diaphragm pump 108 is not provided at the second outlet of the cell shutoff device 103, instead, the second outlet of the cell shutoff device 103 is designed to communicate with the bioreactor 102, and further, a delivery pump is provided at the inlet of the cell shutoff device 103.
EXAMPLE III
The present embodiment is different from the first embodiment in that the diaphragm pump 108 is not provided at the second outlet of the cell shutoff device 103, instead, the second outlet of the cell shutoff device 103 is designed to communicate with the bioreactor 102, and further, a delivery pump is provided at the inlet of the cell shutoff device 103.
Referring to the drawings, the cell culture apparatus includes an ultrasonic cut-off device 110, and it can be understood that the ultrasonic cut-off device 110 aggregates cells by means of ultrasonic cut-off, and separates the cells from the supernatant to some extent. Specifically, the ultrasonic cut-off device 110 is provided with an ultrasonic cut-off controller 112, an inlet of the ultrasonic cut-off device 110 is communicated with the bioreactor 102, a first outlet of the ultrasonic cut-off device 110 is communicated with an inlet of the cell cut-off device 103, a second outlet of the ultrasonic cut-off device 110 is communicated with the bioreactor 102, and a second outlet of the ultrasonic cut-off device 110 is provided with a delivery pump.
The present invention relates to a cell culture method using a cell culture apparatus as described in the previous embodiments. In the cell culture method, the protein cut-off device 105 processes the cell supernatant in the second vessel 104 and then returns the processed cell supernatant to the second vessel 104, the cell supernatants from the protein cut-off device 105 and the cell cut-off device 103 are mixed in the second vessel 104, and the concentration of the cell supernatant in the second vessel 104 is gradually increased as the protein cut-off device 105 is recycled and purified.
Further, the flow rate at the first inlet of the second vessel 104 and the flow rate at the second outlet of the protein shut-off device 105 are controlled to be the same to maintain the volume of the cell supernatant in the second vessel 104 constant.
In the description herein, references to the terms "one embodiment," "some examples," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" or the like, if any, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While the embodiments of the present invention have been described in detail with reference to the drawings, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Claims (10)
1. A cell culture apparatus, characterized by: comprises that
A first container (101), said first container (101) being for storing a culture medium;
a bioreactor (102), the bioreactor (102) being in communication with the first container (101);
a cell shut-off device (103), an inlet of the cell shut-off device (103) being in communication with the bioreactor (102);
a second container (104), the second container (104) for storing harvest fluid, a first inlet of the second container (104) being in communication with a first outlet of the cell shut-off device (103);
a protein cut-off device (105), an inlet of the protein cut-off device (105) being in communication with an outlet of the second vessel (104), a first outlet of the protein cut-off device (105) being in communication with a second inlet of the second vessel (104), the first outlet of the protein cut-off device (105) being for discharging the treated cell supernatant to the second vessel (104), the second outlet of the protein cut-off device (105) being for discharging a filtrate.
2. The cell culture apparatus of claim 1, wherein: a second outlet of the protein cut-off device (105) is provided with a delivery pump.
3. The cell culture apparatus of claim 2, wherein: the cell culture apparatus comprises a third vessel (106), the third vessel (106) being for storing a filtrate, the third vessel (106) being in communication with the second outlet of the protein shut-off device (105).
4. The cell culture apparatus of claim 1, wherein: the first inlet of the second container (104) is provided with a delivery pump, and the inlet of the protein cut-off device (105) is provided with a delivery pump.
5. The cell culture apparatus of claim 1, wherein: the protein interception device (105) comprises a protein interception hollow fiber column or an ultrafiltration membrane package;
preferably, the protein cut-off device (105) comprises a plurality of protein cut-off hollow fiber columns arranged in parallel or a plurality of ultrafiltration membrane membranes arranged in parallel;
preferably, a pressure gauge (107) is arranged on a pipeline of the first outlet of the protein cut-off device (105) communicated with the second container (104), and the pressure gauge (107) monitors transmembrane pressure.
6. The cell culture apparatus of any one of claims 1 to 5, wherein: and a diaphragm pump (108) is arranged at a second outlet of the cell interception device (103).
7. The cell culture apparatus according to any one of claims 1 to 5, wherein: a second outlet of the cell shut-off device (103) is in communication with the bioreactor (102); and a delivery pump is arranged at the inlet of the cell interception device (103).
8. The cell culture apparatus of claim 7, wherein: the cell culture equipment comprises an ultrasonic cut-off device (110), wherein the inlet of the ultrasonic cut-off device (110) is communicated with the bioreactor (102), the first outlet of the ultrasonic cut-off device (110) is communicated with the inlet of the cell cut-off device (103), and the second outlet of the ultrasonic cut-off device (110) is communicated with the bioreactor (102).
9. A cell culture method using a cell culture apparatus according to any one of claims 1 to 8, wherein: the protein shut-off device (105) processes the cell supernatant in the second container (104) and returns the processed cell supernatant to the second container (104), and the cell supernatants from the protein shut-off device (105) and the cell shut-off device (103) are mixed in the second container (104).
10. The cell culture method according to claim 9, wherein: controlling the flow rate of the first inlet of the second container (104) to be consistent with the flow rate of the second outlet of the protein cut-off device (105).
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| CN202110641269.8A CN115449482A (en) | 2021-06-09 | 2021-06-09 | Cell culture equipment and cell culture method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110641269.8A CN115449482A (en) | 2021-06-09 | 2021-06-09 | Cell culture equipment and cell culture method |
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