CN115074243B - Gao Mozhu flux cell perfusion culture membrane column system and method and application thereof - Google Patents
Gao Mozhu flux cell perfusion culture membrane column system and method and application thereof Download PDFInfo
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- CN115074243B CN115074243B CN202210554588.XA CN202210554588A CN115074243B CN 115074243 B CN115074243 B CN 115074243B CN 202210554588 A CN202210554588 A CN 202210554588A CN 115074243 B CN115074243 B CN 115074243B
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- 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
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Abstract
The invention discloses a high-flux membrane column system for cell perfusion culture, which comprises a buffer column and a main membrane column which are communicated, wherein a pulse cavity communicated with the main membrane column is arranged at the lower part of the main membrane column, a high-frequency pulser is arranged on the pulse cavity, and the high-frequency pulser is used for periodically compressing or expanding the pulse cavity by high-frequency pulses. When fluid in the system flows forward (from the lower end to the upper end of the membrane column), the filtered culture medium is mainly filtered out of the outer cavity of the lower main membrane column, and when fluid flows reversely (from the upper end to the lower end of the membrane column), the filtered culture medium is mainly filtered out of the outer cavity of the upper buffer column, part of filtrate enters the lower main membrane column through a three-way pipeline and reversely penetrates through the outer cavity of the main membrane column, so that the lower main membrane column is reversely flushed. The pulse unit can promote back flushing, cause membrane silk vibration, promote the membrane cleaning effect, improve the membrane column loading capacity. The invention can effectively clean the filter membrane in the use process of the membrane column, and improves the membrane column loading capacity and the membrane area utilization efficiency.
Description
Technical Field
The invention relates to the technical field of perfusion culture of animal cells, in particular to a technology for improving flux of a cell perfusion culture membrane column.
Background
When the hollow fiber membrane column is used as cell interception equipment for perfusion culture, the method has the advantages of large membrane area, easy process amplification, easy realization of perfusion flow and the like, and is widely applied to cell perfusion culture production of biological products such as vaccines, antibodies and the like. However, the hollow fiber membrane column is easy to block membrane holes in the using process, so that the effective membrane area is reduced, and the membrane flux is reduced. Mitigation is typically required by either replacing new membrane columns or employing larger membrane area membrane columns, but increases the risk of contamination and cost.
The filter cake on the surface of part of the membrane hole and the blocking object in the membrane hole can be reversely flushed away from the surface of the membrane by adopting reverse flushing, and the blocking membrane hole is re-exposed, so that the use efficiency and the flux of the membrane are improved. However, the existing back flushing solutions generally require complex auxiliary equipment or complex operations, and the effect is not obvious and are not widely adopted. The phenomena of low use efficiency of the membrane area of the hollow fiber membrane column and low flux of the membrane column become key bottlenecks for restricting the long-term stable operation of the cell perfusion culture.
Disclosure of Invention
The invention designs a high-membrane column flux cell perfusion culture membrane column aiming at the problems that a hollow fiber membrane column membrane hole is easy to block, the membrane column flux is low and the like in the cell perfusion culture process.
A high-flux membrane column system for cell perfusion culture mainly comprises a controller, a circulating system, a high-flux membrane column, a liquid inlet pump, a liquid outlet pump and a pulse unit.
Fresh culture medium is supplemented by the liquid inlet pump, and the waste culture medium is pumped out of the membrane column by the liquid outlet pump and discharged to the waste liquid bag.
The circulation system is used for driving the cell-containing culture medium to circulate between the reactor and the high-flux membrane column. Circulation systems include, but are not limited to, peristaltic pumps, syringe pumps, gear pumps, centrifugal pumps, diaphragm pumps, and the like. These pumps may be used alone or in combination to achieve both forward and reverse flow of fluid in the pipeline.
The high-flux membrane column comprises a buffer column at the upper end and a main membrane column at the lower end, wherein the buffer column and the main membrane column can be connected by using a chuck, and can be integrally formed into a complete column. The buffer column outer cavity interface is connected with the main membrane column outer cavity interface through a three-way pipeline and then is connected with a liquid outlet pipeline, and a liquid outlet pump is arranged on the liquid outlet pipeline.
The buffer column and the main membrane column each include at least one external cavity interface.
The lower end of the main membrane column is provided with a pulse unit, and the pulse unit consists of a high-frequency pulser and a pulse cavity. The high-frequency pulser is used for extruding the pulse cavity at high frequency and recovering the original position action, so that the high-frequency pulse of the liquid in the pulse cavity is realized. The pulse speed and the pulse frequency of the pulse unit are automatically controlled by the controller. Preferably, the pulse cavity is a cylindrical cavity, a spherical cavity and a special-shaped cavity which are made of elastic materials, and also comprises a blind end pipeline made of elastic materials. The elastic material is preferably food grade silica gel, rubber, etc.
The circulating system, the high flux membrane column, the liquid inlet pump, the liquid outlet pump and the pulse unit are automatically controlled and regulated by the controller. The controller automatically controls the liquid inlet pump and the liquid outlet pump to supplement fresh culture medium into the reactor, and the waste culture medium is filtered by the hollow fiber column and then discharged out of the reactor.
When the fluid flows forward (the lower end of the membrane column flows to the upper end), the filtering culture medium is filtered out from the outer cavity of the buffer column and the outer cavity of the main membrane column, is merged by the three-way pipeline and then enters the liquid outlet pipeline, and the membrane column is pumped out by the liquid outlet pump and is discharged to the waste liquid bag; when the fluid flows reversely (the upper end of the membrane column flows towards the lower end), the filtering culture medium is mainly filtered out from the outer cavity of the buffer column at the upper end, and part of filtrate enters the outer cavity of the main membrane column at the lower end through a three-way pipeline and reversely penetrates through the surface of the membrane to enter the inside of the membrane column, so that the main membrane column at the lower end is reversely flushed, and the flux of the membrane column is improved.
The pulse unit operates at high frequency to generate pulse flow, so that the back flushing of the main membrane column is promoted, the membrane wires vibrate, the membrane cleaning effect is promoted, and the flux of the membrane column is improved.
The technology for improving the flux of the cell perfusion culture membrane column is suitable for animal cell perfusion culture and is also suitable for other fields related to the use of the membrane column (hollow fiber membrane column) such as protein purification and the like.
The beneficial effects achieved by the invention are as follows:
according to the invention, the membrane column is subjected to online back flushing by adopting the high-flux membrane column and combining the pulse unit, and the membrane wire is subjected to high-frequency vibration by combining the pulse unit, so that the hollow fiber membrane column is subjected to efficient cleaning, the blocking of the membrane holes is slowed down, the flux of the membrane column is improved, and the use efficiency of the membrane column is further improved. The technology for improving the flux of the hollow fiber membrane column has the advantages of simple structure, easy realization and effective realization of continuous operation of cell perfusion culture.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a schematic diagram of the structure of the present invention;
FIG. 2 is a schematic illustration of a high flux membrane column of the present invention;
FIG. 3 is a schematic illustration of the flow direction of fluid in a pipeline when the circulation system of the present invention drives the fluid to flow in a forward direction;
FIG. 4 is a schematic illustration of the flow direction of fluid in a circuit when the circulation system of the present invention drives the fluid to flow in a reverse direction;
FIG. 5 is a schematic diagram of the backflushing principle and effect of the present discovery;
FIG. 6 is a schematic diagram of the fluid flow direction of the impulse chamber during operation of the impulse unit of the present invention;
FIG. 7 is a schematic diagram of membrane filament vibration during operation of the pulse unit of the present invention;
fig. 8 is a schematic diagram of the structure of the high frequency pulser.
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
Examples
As shown in fig. 1, a high-throughput column-load cell perfusion culture membrane column system comprises a controller 1, a circulation system 2, a high-throughput membrane column 3, a liquid inlet pump 4, a liquid outlet pump 5 and a pulse unit 6. As shown in fig. 2, the high flux membrane column 3 comprises an upper end interface 31, a buffer column 32, a main membrane column 33, a lower end interface 34, a buffer column outer cavity interface 35, a main membrane column outer cavity interface 36 and a pulse unit 6; the buffer column 32 and the main membrane column 33 are hollow fiber membrane columns. The buffer column 32 is provided with an upper port 31 and an outer chamber port 35. The buffer column 32 and the main membrane column 33 may be integrally formed; the buffer column outer cavity interface 35 is connected with the main membrane column outer cavity interface 36 through a three-way pipeline 37 and then is connected with a liquid outlet pipeline, and the liquid outlet pipeline is provided with a liquid outlet pump 5.
As shown in fig. 3, when the circulation system 2 drives the fluid to flow forward in the membrane column (the fluid flows from the lower end to the upper end of the membrane column), the circulation system 2 pushes the fluid upward, the cavity of the main membrane column 33 is positive pressure, the liquid in the buffer column 32 and the cavity of the main membrane column enters the respective outer cavities of the membrane column after being filtered by the membrane, and the filtered liquid in the outer cavities of the buffer column and the outer cavity of the main membrane column is discharged to the waste liquid bag by the liquid outlet pump 5 after being collected by the three-way pipeline.
As shown in fig. 4, when the circulation system 2 drives the fluid to flow in the reverse direction inside the membrane column (the fluid flows from the lower end to the upper end of the membrane column), the circulation system 2 draws the liquid from the inside of the main membrane column to cause the cavity of the main membrane column 33 to be negative pressure. The liquid in the buffer column inner cavity enters the outer cavity after membrane filtration, when the filtered liquid in the buffer column outer cavity flows to the three-way pipeline 37 under the driving of the main membrane column negative pressure and the liquid outlet pump, one part of the filtered liquid is discharged to the waste liquid bag by the liquid outlet pump 5, and the other part of the filtered liquid enters the main membrane column outer cavity and further reversely enters the main membrane column inner cavity to reversely wash the main membrane column.
As shown in fig. 5, in the use process of the high-flux membrane column 3, the particles gradually block the membrane holes of the membrane filaments in the membrane column, and when the membrane column is backwashed, liquid penetrates through the membrane holes from the outer cavity to enter the inner cavity, and the plugs in the membrane holes are flushed into the inner cavity and out of the membrane column by the liquid, so that the effect of backwashed cleaning is achieved.
As shown in fig. 6, the pulse unit 6 is composed of a high-frequency pulser and a pulse cavity 62. The high frequency pulser is used to periodically high frequency pulse compress the pulse cavity 62. In the initial state, the high-frequency pulser is in an open state, the pulse cavity is filled with culture medium, and when the pulse unit works, the high-frequency pulser is periodically opened and closed; when the high-frequency pulser is closed, the culture medium in the pulse cavity is extruded to the outer cavity of the main membrane column, part of the culture medium flows upwards along the membrane column, and part of the culture medium reversely enters the inner cavity of the main membrane column. When the pulse unit returns to its original state, the high frequency pulser returns to its open state, and the medium in the outer cavity of the main membrane column flows to and fills the pulse cavity 62.
As shown in FIG. 7, in the initial state, the high-frequency pulser is in an open state, the pulse cavity is filled with culture medium, and membrane wires inside the main membrane column are in a static state. When the pulse unit works, the high-frequency pulser is closed, the culture medium in the pulse cavity 62 is extruded to the outer cavity of the main membrane column, and impacts the membrane wires, so that the membrane wires vibrate, and the plugs in the membrane holes are reversely punched out of the membrane holes. When the pulse unit is restored to the initial state, the high-frequency pulser is restored to the open state, and the culture medium in the outer cavity of the main membrane column flows to the pulse cavity.
As shown in fig. 8, the high-frequency pulser is composed of an upper plate 61, a lower plate 65, a bracket 63, and a motor 64. The upper plate 61 is fixed on the bracket 63, the pulse cavity 62 is arranged between the upper plate 61 and the lower plate 65, the lower plate 65 is driven by the motor 64 to move upwards, and the pulse cavity 62 is periodically compressed/relaxed to realize high-frequency pulse. The speed and frequency of movement of the motor 64 are controlled by a controller.
CHO cell perfusion culture is carried out by utilizing the technology of the invention:
the high-flux membrane column flux cell perfusion culture membrane column system is adopted to carry out 10L-scale CHO cell perfusion culture, and the area of the high-flux membrane column is 0.2m 2 And (3) infiltrating the high-flux membrane column by using a proper amount of culture medium in advance, so that the inner cavity, the outer cavity and the pulse cavity of the membrane column are filled with the culture medium. To grow the cell density in the reactor to 3-5×10 6 When cells/mL, connecting a high-flux membrane column and a circulating system with a reactor through a pipeline, and starting perfusion culture after installing a pulse unit: setting culture system parameters at a controller interface as: the perfusion speed is 2VVD; the forward flowing time of the circulating system is 30min, the reverse flowing time of the circulating system is 30min, and the circulating system alternately operates; the flow in the hollow fiber column is 1L/min; the frequency of the pulse unit is 30cycles/min, and the amplitude is middle (the minimum gap is 1 cm); and starting automatic operation after the parameter setting is completed. After 7 days of perfusion culture, the cells were grown to a density of 1.8X10 6 The membrane flux of the membrane column is not reduced and reaches 250L/m when the cell/mL is higher than or equal to 2 The above. And adopts 0.2m 2 When the hollow fiber membrane column with the conventional structure is used for perfusion culture, the same circulation system is used, the perfusion culture is started at the same time, and the perfusion culture parameters are kept consistent. When the culture is carried out until the 6 th day, the hollow fiber membrane column is blocked, the experiment is stopped, and the membrane flux is about 200L/m 2 . When the membrane area is greater than 0.3m 2 The hollow fiber membrane column with the conventional structure can maintain cell perfusion culture for more than 7 days and support cell density growth to 1.8X10 6 cell/mL or more. The invention can effectively improve the flux of the fiber membrane column by 25 percent。
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. Gao Mozhu flux cell perfusion culture membrane column system, characterized by, including the high flux membrane column, the high flux membrane column includes buffer column and main membrane column that communicate, there is pulse cavity communicated with main membrane column in the inferior part of the main membrane column, there are high-frequency pulsers on the pulse cavity, the high-frequency pulser is used for compressing or opening the periodic high-frequency pulse of the pulse cavity;
the reactor, the circulating system and the liquid inlet pump are also included; the circulating system drives the cell-containing culture medium to circulate between the reactor and the high-flux membrane column, and the liquid inlet pump is used for supplementing fresh culture medium into the reactor;
the buffer column is arranged on the main membrane column, and the buffer column and the main membrane column are hollow fiber membrane columns; the outer cavity interface of the buffer column and the outer cavity interface of the main membrane column are connected with a liquid outlet pipeline through a three-way pipeline, and a liquid outlet pump is arranged on the liquid outlet pipeline;
the upper end interface of the buffer column and the lower end interface of the main membrane column are communicated with the reactor through pipelines, and the circulating system is arranged on the pipeline between the lower end interface of the main membrane column and the reactor.
2. The high membrane column flux cell perfusion culture membrane column system of claim 1, wherein the pulse cavity is an elastic cavity.
3. The high membrane column flux cell perfusion culture membrane column system of claim 1, wherein the high frequency pulser is composed of an upper plate, a lower plate, a bracket and a motor; the upper plate is fixed on the bracket, the pulse cavity is arranged between the upper plate and the lower plate, and the lower plate is driven by the motor to move upwards/downwards to periodically compress and reset the pulse cavity.
4. The use of the high membrane column flux cell perfusion culture membrane column system of claim 1 in animal cell perfusion culture and protein purification.
5. A method for cell culture by using the high-membrane column flux cell perfusion culture membrane column system as claimed in claim 1, characterized in that,
when the circulation system drives fluid to flow in the membrane column from bottom to top, liquid in the buffer column and the inner cavity of the main membrane column enters respective outer cavities after being filtered by the membrane, and filtered liquid in the outer cavities of the buffer column and the outer cavities of the main membrane column is collected by the three-way pipeline and then is discharged to the waste liquid bag by the liquid outlet pump;
when the circulation system drives fluid to flow from top to bottom in the membrane column, liquid in the inner cavity of the buffer column enters the outer cavity after being filtered by the membrane, and part of filtered liquid in the outer cavity of the buffer column is discharged to the waste liquid bag by the liquid outlet pump when the filtered liquid flows to the three-way pipeline under the driving of the negative pressure of the main membrane column and the liquid outlet pump, and the other part of the filtered liquid enters the outer cavity of the main membrane column and further reversely enters the inner cavity of the main membrane column to reversely wash the main membrane column, so that the flux of the membrane column is improved;
when the high-frequency pulser is closed, the culture medium in the pulse cavity is extruded to the outer cavity of the main membrane column, part of the culture medium flows upwards along the membrane column, and part of the culture medium reversely enters the inner cavity of the main membrane column; when the pulse unit is restored to the initial state, the high-frequency pulser is restored to the open state, and the culture medium in the outer cavity of the main membrane column flows to the pulse cavity and fills the pulse cavity.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN202390249U (en) * | 2011-12-22 | 2012-08-22 | 北京碧水源膜科技有限公司 | Membrane biological reaction tank for impacting and cleaning membrane modulus device |
| CN202415243U (en) * | 2011-12-22 | 2012-09-05 | 北京碧水源膜科技有限公司 | MBR (membrane bioreactor) membrane module instrument for descaling by adopting mechanical vibration |
| CN210394394U (en) * | 2018-12-19 | 2020-04-24 | 比欧联科供应链管理(北京)有限公司 | Ventilative bag is positive and negative perfusion cell culture device |
| CN113265330A (en) * | 2021-06-04 | 2021-08-17 | 河南大学 | Animal cell high-density culture system suitable for efficient production of vaccines and antibodies |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109415669B (en) * | 2016-07-19 | 2023-01-31 | 自动化合作关系（剑桥）有限公司 | Reversible liquid filtration system |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202390249U (en) * | 2011-12-22 | 2012-08-22 | 北京碧水源膜科技有限公司 | Membrane biological reaction tank for impacting and cleaning membrane modulus device |
| CN202415243U (en) * | 2011-12-22 | 2012-09-05 | 北京碧水源膜科技有限公司 | MBR (membrane bioreactor) membrane module instrument for descaling by adopting mechanical vibration |
| CN210394394U (en) * | 2018-12-19 | 2020-04-24 | 比欧联科供应链管理(北京)有限公司 | Ventilative bag is positive and negative perfusion cell culture device |
| CN113265330A (en) * | 2021-06-04 | 2021-08-17 | 河南大学 | Animal cell high-density culture system suitable for efficient production of vaccines and antibodies |
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