CN115074243A - High-membrane column flux cell perfusion culture membrane column system and method and application thereof - Google Patents

High-membrane column flux cell perfusion culture membrane column system and method and application thereof Download PDF

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CN115074243A
CN115074243A CN202210554588.XA CN202210554588A CN115074243A CN 115074243 A CN115074243 A CN 115074243A CN 202210554588 A CN202210554588 A CN 202210554588A CN 115074243 A CN115074243 A CN 115074243A
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membrane column
column
membrane
cavity
pulse
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CN115074243B (en
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聂简琪
孙杨
白仲虎
盛磊
刘俊凯
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Taige Saier Life Technology Wuxi Co ltd
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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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/10—Hollow fibers or tubes
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/12—Pulsatile flow
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation

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Abstract

The invention discloses a high-flux membrane column cell perfusion culture membrane column system, wherein a high-flux membrane column comprises a buffer column and a main membrane column which are communicated, a pulse cavity communicated with the main membrane column is arranged at the lower part of the main membrane column, and a high-frequency pulser is arranged on the pulse cavity and used for carrying out periodic high-frequency pulse compression or expansion on the pulse cavity. When the fluid in the system flows in the forward direction (from the lower end to the upper end of the membrane column), the filtered culture medium is mainly filtered out from the outer cavity of the lower main membrane column, when the fluid flows in the reverse direction (from the upper end to the lower end of the membrane column), the filtered culture medium is mainly filtered out from the outer cavity of the upper buffer column, and part of the filtrate enters the lower main membrane column through the three-way pipeline and reversely penetrates through the outer cavity of the main membrane column to reversely flush the lower main membrane column. The pulse unit can promote the back flush, causes the membrane silk vibration, promotes membrane cleaning effect, improves membrane column load capacity. The invention can effectively clean the filter membrane in the use process of the membrane column, and improve the carrying capacity of the membrane column and the utilization efficiency of the membrane area.

Description

High-membrane column flux cell perfusion culture membrane column system and method and application thereof
Technical Field
The invention relates to the technical field of animal cell perfusion culture, in particular to a technology for improving the flux of a cell perfusion culture membrane column.
Background
When a hollow fiber membrane column is used as cell interception equipment for perfusion culture, the hollow fiber membrane column is widely applied to cell perfusion culture for producing biological products such as vaccines, antibodies and the like due to the advantages of large membrane area, easy process amplification, easy realization of perfusion process and the like. However, the membrane pores of the hollow fiber membrane column are easy to block in the using process, so that the effective membrane area is reduced, and the membrane flux is reduced. Mitigation is often required by replacing the membrane column with a new one or using a larger membrane area, but the risk of contamination and the cost are increased.
By adopting the back flushing, part of filter cakes on the surface of the membrane pores and the blockage in the membrane pores can be flushed away from the surface of the membrane in a reverse direction, and the blocked membrane pores are exposed again, so that the use efficiency of the membrane and the membrane flux are improved. However, the existing back flushing implementation scheme usually requires complex auxiliary equipment or complex operation, has no obvious effect and is not widely adopted. The phenomena of low utilization efficiency of hollow fiber membrane column membrane area and low membrane column flux become key bottlenecks which restrict long-term stable operation of cell perfusion culture.
Disclosure of Invention
The invention designs a high-membrane column flux cell perfusion culture membrane column aiming at the problems that the membrane pores of a hollow fiber membrane column are easy to block, the flux of the membrane column is low and the like in the cell perfusion culture process.
A high-membrane column flux cell perfusion culture membrane column system mainly comprises a controller, a circulating system, a high-flux membrane column, a liquid inlet pump, a liquid outlet pump and a pulse unit.
And supplementing a fresh culture medium through a liquid inlet pump, and pumping the waste culture medium out of the membrane column through a liquid outlet pump and discharging the waste culture medium to a waste liquid bag.
The circulating system is used for driving the cell-containing culture medium to circulate between the reactor and the high-flux membrane column. The circulation system includes, but is not limited to, forms of peristaltic pumps, syringe pumps, gear pumps, centrifugal pumps, diaphragm pumps, and the like. These pumps may be used alone or in combination as a unit to achieve both forward and reverse fluid flow 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, the buffer column and the main membrane column can be connected by using a chuck, and the buffer column and the main membrane column can be integrally formed into a complete column. The buffer column outer cavity interface and the main membrane column outer cavity interface are connected through a three-way pipeline and then connected with a liquid outlet pipeline, and a liquid outlet pump is installed on the liquid outlet pipeline.
Both the buffer column and the main membrane column comprise at least one outer chamber 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 high-frequency extrusion pulse cavity and normal position action, thereby realizing the high-frequency pulse of liquid in the pulse cavity. 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 the pulse cavity also comprises a blind end pipeline made of elastic materials. The elastic material is preferably food-grade silica gel, rubber and the like.
The circulating system, the high-flux membrane column, the liquid inlet pump, the liquid outlet pump and the pulse unit are automatically controlled and adjusted by the controller. Fresh culture medium is supplemented into the reactor by automatically controlling the liquid inlet pump and the liquid outlet pump through the controller, and the waste culture medium is discharged out of the reactor after being filtered by the hollow fiber column.
When the fluid flows in the forward direction (the lower end of the membrane column flows towards the upper end), the filtered culture medium is filtered from the buffer column outer cavity and the main membrane column outer cavity, is merged by the three-way pipeline and then enters the liquid outlet pipeline, and is pumped out of the membrane column by the liquid outlet pump and is discharged to a waste liquid bag; when the fluid flows reversely (the upper end of the membrane column flows to the lower end), the filtered culture medium is mainly filtered out from the outer cavity of the upper buffer column, and part of the filtrate enters the outer cavity of the lower main membrane column through the three-way pipeline and reversely penetrates through the surface of the membrane to enter the interior of the membrane column, so that the lower main membrane column 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 main membrane column is promoted to be reversely washed, the membrane filaments are enabled to 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 perfusion culture of animal cells and other fields related to the use of the membrane column (hollow fiber membrane column) such as protein purification and the like.
The invention has the following beneficial effects:
according to the invention, the high-flux membrane column is combined with the pulse unit to realize on-line reverse washing of the membrane column, and the pulse unit is combined to enable the membrane filaments to generate high-frequency vibration to efficiently clean the hollow fiber membrane column, so that the membrane hole blockage is slowed down, the membrane column flux 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 is simple in structure, easy to realize and capable of effectively realizing continuous operation of cell perfusion culture.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic structural view of the present invention;
FIG. 2 is a schematic view of a high throughput membrane column of the present invention;
FIG. 3 is a schematic view of the flow of fluid in the circuit with the forward flow of fluid driven by the circulation system of the present invention;
FIG. 4 is a schematic view of the flow of fluid in the circuit with the reverse flow of fluid driven by the circulation system of the present invention;
FIG. 5 is a schematic diagram of the back flushing principle and effect of the present discovery;
FIG. 6 is a schematic diagram of the pulse chamber fluid flow direction during operation of the pulse 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 in conjunction with the accompanying drawings, and it should be understood that they are presented herein only to illustrate and explain the present invention and not to limit the present invention.
Examples
As shown in fig. 1, a high-flux cell perfusion culture membrane column system comprises a controller 1, a circulation system 2, a high-flux 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 both hollow fiber membrane columns. The buffer column 32 is provided with an upper port 31 and an outer port 35. The buffer column 32 and the main membrane column 33 may be integrated; the buffer column outer cavity interface 35 and the main membrane column outer cavity interface 36 are connected through a three-way pipeline 37 and then connected with a liquid outlet pipeline, and a liquid outlet pump 5 is installed on the liquid outlet pipeline.
As shown in fig. 3, when the circulation system 2 drives the fluid to flow in the membrane column in the forward direction (the fluid flows from the lower end to the upper end of the membrane column), the circulation system 2 pushes the fluid upward, the pressure in the cavity of the main membrane column 33 is positive, the liquids in the buffer column 32 and the inner cavity of the main membrane column enter the outer cavity of the respective membrane column after being filtered by the membrane, and the filtered liquids in the outer cavity of the buffer column and the outer cavity of the main membrane column are collected by the three-way pipe and then discharged to the waste liquid bag by the liquid outlet pump 5.
As shown in fig. 4, when the circulation system 2 drives the fluid to flow in the reverse direction in 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 main membrane column to make the cavity of the main membrane column 33 be at negative pressure. Liquid in the inner cavity of the buffer column enters the outer cavity after being filtered by the membrane, when the filtered liquid in the outer cavity of the buffer column flows to the three-way pipeline 37 under the negative pressure of the main membrane column and the driving of the liquid outlet pump, one part of the filtered liquid is discharged to a waste liquid bag by the liquid outlet pump 5, 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 flush the main membrane column.
As shown in figure 5, in the use process of the high-flux membrane column 3, the particles can gradually block the membrane holes of the membrane filaments in the membrane column, when the membrane column is reversely flushed, liquid penetrates through the membrane holes from the outer cavity to enter the inner cavity, and a blockage in the membrane holes is flushed into the inner cavity and is flushed out of the membrane column by the liquid, so that the effect of reversely flushing and cleaning is achieved.
As shown in fig. 6, the pulse unit 6 is composed of a high frequency pulser and a pulse chamber 62. The high frequency pulser is used to periodically high frequency pulse compress the pulse chamber 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 opened and closed periodically; 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 the initial state, the high frequency pulser returns to the 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 the open state, the pulse cavity is filled with the culture medium, and the membrane filaments inside the main membrane column are in the 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, the membrane wire is impacted, and the membrane wire vibrates, so that the blockage in the membrane hole is favorably and reversely punched out of the membrane hole. When the pulse unit returns to the initial state, the high-frequency pulser returns 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 support 63, and a motor 64. The upper plate 61 is fixed on the support 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, the pulse cavity 62 is compressed/relaxed periodically, and high-frequency pulse is realized. The speed and frequency of the motor 64 are controlled by the controller.
CHO cell perfusion culture is carried out by utilizing the technology of the invention:
the high-flux membrane column 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 infiltrating the high-flux membrane column with a proper amount of culture medium in advance to fill the inner cavity, the outer cavity and the pulse cavity of the membrane column with the culture medium. When the cell density in the reactor is 3-5X 10 6 When cells/mL, connecting the high-flux membrane column and the circulating system with the reactor through a pipeline, and starting perfusion culture after installing a pulse unit: setting culture system parameters at the controller interface as: the perfusion rate is 2 VVD; the forward flow time of the circulation system is 30min, and the reverse flow time of the circulation system isPerforming alternate operation for 30 min; the flow rate in the hollow fiber column is 1L/min; the pulse unit frequency is 30cycles/min, the amplitude is midle (the minimum clearance is 1 cm); and starting automatic operation after parameter setting is finished. After 7 days perfusion culture, the cell density is 1.8 multiplied by 10 6 Above cells/mL, the membrane flux of the membrane column is not reduced, and the membrane flux reaches 250L/m 2 The above. And 0.2m 2 When the hollow fiber membrane column with the conventional structure is used for perfusion culture, the same circulation system is used, and the perfusion culture is started at the same time, so that the perfusion culture parameters are kept consistent. When the culture is carried out till 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 using a membrane area of more 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 1.8 multiplied by 10 6 cells/mL or more. The invention can effectively improve the flux of the fiber membrane column and improve the flux of the membrane column by 25 percent.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. The high-membrane column flux cell perfusion culture membrane column system is characterized by comprising a high-membrane column, wherein the high-flux membrane column comprises a buffer column and a main membrane column which are communicated, a pulse cavity communicated with the main membrane column is arranged at the lower part of the main membrane column, and a high-frequency pulser is arranged on the pulse cavity and is used for compressing or expanding the pulse cavity by periodic high-frequency pulses.
2. The high membrane column flux cell perfusion culture membrane column system of claim 1, wherein the buffer column is disposed on the main membrane column, an outer cavity interface of the buffer column and an outer cavity interface of the main membrane column are connected with the liquid outlet pipe through a three-way pipe, and the liquid outlet pipe is provided with a liquid outlet pump.
3. The high membrane column flux cell perfusion culture membrane column system of claim 1, wherein the pulse chamber is an elastic chamber.
4. The high membrane column flux cell perfusion culture membrane column system of claim 2, further comprising a reactor, a circulation system and a liquid inlet pump; 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.
5. The high membrane column flux cell perfusion culture membrane column system according to claim 4, wherein the upper end interface of the buffer column and the lower end interface of the main membrane column are both communicated with the reactor through a pipeline, and the circulating system is arranged on the pipeline between the lower end interface and the reactor.
6. The high membrane column flux cell perfusion culture membrane column system of claim 1, wherein the high frequency pulser is comprised of an upper plate, a lower plate, a support, and a motor; the upper plate is fixed on the support, 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.
7. The high membrane column flux cell perfusion culture membrane column system of claim 1 is applied to animal cell perfusion culture and protein purification.
8. The method for cell culture by using the high membrane column flux cell perfusion culture membrane column system of claim 1,
when the circulating system drives fluid to flow in the membrane column from bottom to top, the liquid in the inner cavities of the buffer column and the main membrane column enters the outer cavities of the buffer column and the main membrane column after being filtered by the membranes, and the filtered liquid in the outer cavities of the buffer column and the main membrane column is collected by a three-way pipeline and then discharged to a waste liquid bag by a liquid outlet pump;
when the circulating system drives fluid to reversely 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, one part of the liquid is discharged to a waste liquid bag by the liquid outlet pump while the other part of the liquid enters the outer cavity of the main membrane column and further reversely enters the inner cavity of the main membrane column under the drive of the negative pressure of the main membrane column and the liquid outlet pump, and the main membrane column is reversely washed, 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 returns to the initial state, the high-frequency pulser returns 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.
CN202210554588.XA 2022-05-19 2022-05-19 Gao Mozhu flux cell perfusion culture membrane column system and method and application thereof Active CN115074243B (en)

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Citations (5)

* Cited by examiner, † Cited by third party
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
US20190241856A1 (en) * 2016-07-19 2019-08-08 The Automation Partnership (Cambridge) Limited Reversible liquid filtration system
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

Patent Citations (5)

* Cited by examiner, † Cited by third party
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
US20190241856A1 (en) * 2016-07-19 2019-08-08 The Automation Partnership (Cambridge) Limited Reversible liquid filtration system
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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