CN108865885A - One kind is for the efficient device for trapping of cell culture and its retention method - Google Patents
One kind is for the efficient device for trapping of cell culture and its retention method Download PDFInfo
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- CN108865885A CN108865885A CN201810733093.7A CN201810733093A CN108865885A CN 108865885 A CN108865885 A CN 108865885A CN 201810733093 A CN201810733093 A CN 201810733093A CN 108865885 A CN108865885 A CN 108865885A
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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
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
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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
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
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Abstract
One kind being used for the efficient device for trapping of cell culture, including reactor, acoustics trap, hollow fiber column and harvest tank, the reactor are successively connected with the inbound port of outer circulation pump, acoustics trap by pipeline；The sedimentation mouth of the acoustics trap is connected with reactor, and the exit port of acoustics trap is connected by pipeline with hollow fiber column, and is equipped with Pump for giving-out at the pipeline between exit port and hollow fiber column；The liquid outlet of the hollow fiber column is successively connected with harvest pump, harvest tank by pipeline, and the cell after retention is collected into the reactor being connected with hollow fiber column by hollow fiber column.Retention efficiency can be greatly improved in the present invention, can meet 90% or more retention efficiency.
Description
Technical field
The present invention relates to field of cell culture, and in particular to one kind is used for the efficient device for trapping of cell culture and its retention side
Method.
Background technique
Kind with zooblast producer gene engineering drug is more and more, and the especially appearance of engineered antibody, yield is
Bottleneck as production.Substituted with perfused culture system traditional batch production technology increasingly induced one pay close attention to.Using
Perfused culture system is greatly improved cell density and yield, greatly reduces the investment in equipment and delivery room.It is effective in order to carry out
Perfusion culture, core are that have a set of efficient, low cost and the perfusion by cell retention in reactor tank that can be used for a long time
System.So far device for trapping have sound wave retention, ATF retention etc., but have the shortcomings that between various methods it is respective, such as
Sound wave retention carries out convergence precipitating to cell using ultrasonic wave, but fails to complete entrapped cell, when perfusion is larger,
Still there is part loss cell and enters in supernatant, if cell content is larger in supernatant, downstream needs to increase filter progress,
More supernatant cell has been likely to increase host protein content simultaneously, increases the difficulty of downstream purification chromatography；And the retention side ATF
Although method carries out effectively catching to a large amount of cells, with using the time to extend, cell and product can attach filter membrane in culture solution, block up
Filter membrane is filled in, causes harvest liquid production concentration to decline, influences yield.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention be intended to provide it is a kind of both reduced single use acoustics and shut off be
System causes to harvest the increased downstream purification technology difficulty of residual cell in supernatant, and it is easy to solve single use ATF entrapped cell
The efficient device for trapping and its retention method of the problem of blocking.
To achieve the above object, the present invention adopts the following technical scheme that：
One kind being used for the efficient device for trapping of cell culture, including reactor, acoustics trap, hollow fiber column and harvest
Tank, the reactor are successively connected with the inbound port of outer circulation pump, acoustics trap by pipeline；The acoustics trap
Sedimentation mouth is connected with reactor, and the exit port of acoustics trap is connected by pipeline with hollow fiber column, and in exit port
Pump for giving-out is installed at pipeline between hollow fiber column；The liquid outlet of the hollow fiber column by pipeline successively with harvest
Pump, harvest tank are connected, and the cell after retention is collected into the reactor being connected with hollow fiber column by hollow fiber column.
Further, the harvest volume of the harvest pump is set as 0.1-3 times of volume of culture, according to density and nutrition need
It asks, target protein concentration adjustment harvest perfusion flow.Enough nutrition supplies should be kept, guarantee that suitable target protein is dense again
Degree.
Further, the outer circulation pump rate is set as 1-3 times of Pump for giving-out flow velocity；It is super to can guarantee that enough culture solutions enter
It is trapped in sound device for trapping, a part is trapped and inside refluxing reactor, another part culture solution and non-entrapped cell quilt
Pump for giving-out is pumped into hollow fiber column and is retained again.
Further, the irrigation rate of the Pump for giving-out is set as 1-3 times of harvest pump irrigation rate.
Further, the sound wave trap is connected with ultrasonic controller, controls sound wave trap by ultrasonic controller
Ultrasonic power be 5~100w, the supersonic frequency of sound wave trap is 2.09-2.17MHz, the ultrasonic wave arteries and veins of sound wave trap
Punching interval is not limited to 0min, 3min, 5min, 10min, 15min, 999min, the recoil time of sound wave trap be not limited to 0min,
3sec、5sec、1min。
Further, the membrane aperture size of the hollow fiber column be 0.2um~1.0um, doughnut column length be 20,
41.5 or 65cm, the diaphragm area in hollow fiber column are 13cm2~11.3m2；The harvest volume of hollow fiber column harvest pump is set
It is set to 0.1-3 times of volume of culture, the flow velocity of hollow fiber column inner cell liquid is 1~2 times of harvest flow velocity.Harvest pump irrigation rate is logical
It is often dependant on target harvest value, can get preferable rejection effect within the scope of 0.1-3 times of volume of culture, in hollow fiber column
The flow velocity of cell liquid is 1~2 times of harvest flow velocity, it is ensured that is not evacuated, while the cell being adsorbed and target product being flowed back
In tank.
One kind efficiently retaining method, including step for cell culture：
1) it is pumped by outer circulation and enters culture solution containing cell from the inbound port of acoustics trap, it is a large amount of in culture solution
Cell aggregation is in the cavity of acoustics trap, and the cell of aggregation is returned in bioreactor by the sedimentation mouth of cavity, part
Cell with culture solution from the exit port of cavity flow out；
2) culture solution flowed out from acoustics trap exit port flows into hollow fiber column and does into one under the action of Pump for giving-out
The retention of step, part cell in culture solution after hollow fiber column retains in culture solution remaining cell aggregation to biological respinse
Device, and remaining culture solution is collected into harvest tank by harvest pump.
Further, the Pump for giving-out irrigation rate range of the acoustics trap is that 1~3 times of harvest pumps irrigation rate.
Further, the irrigation rate of the outer circulation pump is 1~3 times of Pump for giving-out irrigation rate, through ultrasonic retention part
Cell can be captured and in refluxing reactor, and ultrasonic rejection generally can be to 60% or more.Biosep Pump for giving-out irrigation rate
Target harvest value is generally depended on, the big then corresponding ultrasound retention of harvest opposite can be deteriorated, and outer circulation irrigation rate is harvested at 1-3 times
Irrigation rate range has ideal rejection effect.
The beneficial effects of the present invention are：
The efficient device for trapping of cell culture of the present invention passes through ultrasonic device for trapping biosep first and carries out first section
It stays, most cells can be retained, another part of loss is retained by hollow fiber column again, uses hollow fiber column merely relatively
Retention, due to having carried out first of retention, cell is relatively fewer, this trapped pressure is relatively low, even if ultrasonic device for trapping
Biosep is unstable or overload capacity, remains to preferably be retained, greatly improves retention efficiency, to guarantee high-density cells
Long time cultivation.Upper each peristaltic pump control has a significant impact to retention than regular meeting, can satisfy most in the range
Low requirement, on meeting each peristaltic pump control proportional basis described above, in conjunction with ultrasonic device for trapping biosep power control, root
Power of different sizes is selected according to different densities, retention efficiency can be greatly improved in exportable 5~100W etc., can meet 90%
Above retention efficiency.High density can be expanded within a short period of time for the cell for growing slow.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the efficient device for trapping of cell culture of the invention.
Appended drawing reference：1, reactor；2, acoustics trap；201, outer circulation pumps；202, Pump for giving-out；3, hollow fiber column；
301, harvest pump；4, tank is harvested.
Specific embodiment
In the following, being described further in conjunction with attached drawing and specific embodiment to the present invention：
As shown in Figure 1, a kind of be used for the efficient device for trapping of cell culture, including reactor, acoustics trap, doughnut
Column and harvest tank, the reactor are successively connected with the inbound port of outer circulation pump, acoustics trap by pipeline；The acoustics
The sedimentation mouth of trap is connected with reactor, and the exit port of acoustics trap is connected by pipeline with hollow fiber column, and
Pump for giving-out is installed at pipeline between exit port and hollow fiber column；The liquid outlet of the hollow fiber column by pipeline according to
It is secondary with harvest pump, harvest tank be connected, hollow fiber column the cell after retention is collected into be connected with hollow fiber column it is anti-
It answers in device.
The harvest volume of the harvest pump is set as 3 times of volume of culture；The outer circulation pump rate is set as 3 times of Pump for giving-out
Flow velocity；The irrigation rate of the Pump for giving-out is set as 3 times of harvest pump irrigation rates.The sound wave trap is controlled by ultrasound
Device control, 5~100w of ultrasonic power, supersonic frequency are set as 2.09~2.17MHz, and ultrasonic pulse interval may be configured as
0min, 3min, 5min, 10min, 15min, 999min etc., recoil time settable 0min, 3sec, 5sec, 1min etc. should
Ultrasonic wave under parameter does not influence the growth of cell, and the suitable pulse spacing (3min~10min is advisable) can promote cell super
The aggregation of operatic tunes body is agglomerating, is conducive to sedimentation, advantageous ultrasound retention.
Above-mentioned each peristaltic pump control has a significant impact to retention than regular meeting, can satisfy minimum want in the range
It asks, on meeting each peristaltic pump control proportional basis described above, in conjunction with ultrasonic device for trapping biosep power control, according to not
Power of different sizes is selected with density, retention efficiency can be greatly improved in exportable 5~100W etc., can meet 90% or more
Retention efficiency.The cell for growing slow can be expanded within a short period of time.And the control of above-mentioned each peristaltic pump is in
Control equipment is uniformly controlled, and improves the degree of automation.
One kind efficiently retaining method, including step for cell culture：
1) it is pumped by outer circulation and enters culture solution containing cell from the inbound port of acoustics trap, it is a large amount of in culture solution
Cell aggregation is in the cavity of acoustics trap, and the cell of aggregation is returned in bioreactor by the sedimentation mouth of cavity, part
Cell with culture solution from the exit port of cavity flow out；
2) culture solution flowed out from acoustics trap exit port flows into hollow fiber column and does into one under the action of Pump for giving-out
The retention of step, part cell in culture solution after hollow fiber column retains in culture solution remaining cell aggregation to biological respinse
Device, and remaining culture solution is collected into harvest tank by harvest pump.
The Pump for giving-out irrigation rate range of the acoustics trap is that 1~3 times of harvest pumps irrigation rate.
The irrigation rate of the outer circulation pump is 1~3 times of Pump for giving-out irrigation rate.
The membrane aperture size of the hollow fiber column can select the specification of different manufacturers different pore size according to product demand, preferably
High-hydrophilic is selected, high-throughput, low protein binding rate, optional range 3kD (the kilodalton) -02um in aperture is differed, can be covered substantially
Different size of antibody and recombinant protein demand, optional fibre inner diameter 0.5mm~1.0mm are covered, doughnut column length is set as
20,41.5 or 65cm, the diaphragm area in hollow fiber column are set as 13cm2~11.3m2；The harvest of hollow fiber column harvest pump
Volume is set as 0.1-3 times of volume of culture, and the flow velocity of hollow fiber column inner cell liquid is 1~2 times of harvest flow velocity, it is ensured that not by
It evacuates, while will be in the cell that be adsorbed and target product return tank.
Carrying out practically example：Adopt in Chinese hamster ovary celI perfusion incubation in the reactor that capacity is 40L volume of culture
Cell is retained with device for trapping of the present invention.The membrane aperture size of the hollow fiber column of control fibre columns is in wherein
0.8mm, doughnut column length are 65cm, and the diaphragm area in hollow fiber column is set as 13cm2；Outer circulation pump rate is controlled
System is in 80L/d, and Pump for giving-out flow velocity is controlled in 60L/d, and the irrigation rate of the harvest pump of hollow fiber column is 80L/d, practical to receive
The amount of obtaining about 80L.Wherein the cell concentration in 40L culture solution is 10,000,000 cell/ml, and it is thin that tank is harvested after device for trapping retains
Born of the same parents' concentration retains efficiency up to 95% less than 500,000 cell/ml, compares and the side of ultrasonic wave retention and ATF retention is used alone
The retention efficiency of method, the present embodiment increases substantially, and guarantees concentration cultivation.
The efficient device for trapping of cell culture described in the present embodiment passes through ultrasonic device for trapping biosep first to carry out first
Retention can retain most cells, and another part of loss is retained by hollow fiber column again, use doughnut merely relatively
Column retention, due to having carried out first of retention, cell is relatively fewer, this trapped pressure is relatively low, even if ultrasonic device for trapping
Biosep is unstable or overload capacity, remains to preferably be retained, thus guarantee concentration cultivation, doughnut retention
Pressure is small, is not easy to plug, and use process reduces replacement operation risk without frequently replacement, while extending doughnut and using the longevity
Life reduces production cost.
It will be apparent to those skilled in the art that can make various other according to the above description of the technical scheme and ideas
Corresponding change and deformation, and all these changes and deformation all should belong to the protection scope of the claims in the present invention
Within.
Claims (9)
1. one kind is used for the efficient device for trapping of cell culture, which is characterized in that including reactor, acoustics trap, doughnut
Column and harvest tank, the reactor are successively connected with the inbound port of outer circulation pump, acoustics trap by pipeline；The acoustics
The sedimentation mouth of trap is connected with reactor, and the exit port of acoustics trap is connected by pipeline with hollow fiber column, and
Pump for giving-out is installed at pipeline between exit port and hollow fiber column；The liquid outlet of the hollow fiber column by pipeline according to
It is secondary with harvest pump, harvest tank be connected, hollow fiber column the cell after retention is collected into be connected with hollow fiber column it is anti-
It answers in device.
2. being used for the efficient device for trapping of cell culture shown according to claim 1, which is characterized in that the harvest of the harvest pump
Volume is set as 0.1-3 times of volume of culture.
3. being used for the efficient device for trapping of cell culture shown according to claim 1, which is characterized in that the outer circulation pump rate
It is set as 1-3 times of Pump for giving-out flow velocity.
4. being used for the efficient device for trapping of cell culture shown according to claim 1, which is characterized in that the perfusion of the Pump for giving-out
Rate is set as 1-3 times of harvest pump irrigation rate.
5. according to claim 1 be used for the efficient device for trapping of cell culture, which is characterized in that the sound wave trap connects
It is connected to ultrasonic controller, is 5~100w by the ultrasonic power that ultrasonic controller controls sound wave trap, sound wave trap
Supersonic frequency is 2.09-2.17MHz, the ultrasonic pulse interval of sound wave trap be not limited to 0min, 3min, 5min, 10min,
The recoil time of 15min, 999min, sound wave trap are not limited to 0min, 3sec, 5sec, 1min.
6. according to claim 1 be used for the efficient device for trapping of cell culture, which is characterized in that the hollow fiber column
Membrane aperture size is 0.2um~1.0um, and doughnut column length is set as 20,41.5 or 65cm, the diaphragm in hollow fiber column
Area is 13cm2~11.3m2；The harvest volume of hollow fiber column harvest pump is set as 0.1-3 times of volume of culture, hollow fiber column
The flow velocity of inner cell liquid is 1~2 times of harvest flow velocity.
7. one kind efficiently retains method for cell culture, which is characterized in that including step：
1) it is pumped by outer circulation and enters culture solution containing cell from the inbound port of acoustics trap, a large amount of cells in culture solution
It is gathered in the cavity of acoustics trap, the cell of aggregation is returned in bioreactor by the sedimentation mouth of cavity, part cell
As culture solution is flowed out from the exit port of cavity；
2) culture solution flowed out from acoustics trap exit port flows into hollow fiber column and does further under the action of Pump for giving-out
Retention, part cell in culture solution after hollow fiber column retains in culture solution remaining cell aggregation to bioreactor,
And remaining culture solution is collected into harvest tank by harvest pump.
8. according to claim 7 efficiently retain method for cell culture, which is characterized in that the acoustics trap
Pump for giving-out irrigation rate range is that 1~3 times of harvest pumps irrigation rate.
9. according to claim 7 efficiently retain method for cell culture, which is characterized in that the filling of the outer circulation pump
Note rate is 1~3 times of Pump for giving-out irrigation rate.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810733093.7A CN108865885B (en) | 2018-07-05 | 2018-07-05 | Cell culture interception device and cell culture interception method |
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|---|---|---|---|
| CN201810733093.7A CN108865885B (en) | 2018-07-05 | 2018-07-05 | Cell culture interception device and cell culture interception method |
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| CN108865885A true CN108865885A (en) | 2018-11-23 |
| CN108865885B CN108865885B (en) | 2021-03-16 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113846017A (en) * | 2021-10-13 | 2021-12-28 | 无锡药明生物技术股份有限公司 | Selective entrapment device and method of reducing entrapment efficiency |
| CN115449482A (en) * | 2021-06-09 | 2022-12-09 | 佛山汉腾生物科技有限公司 | Cell culture equipment and cell culture method |
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Cited By (3)
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| CN115449482A (en) * | 2021-06-09 | 2022-12-09 | 佛山汉腾生物科技有限公司 | Cell culture equipment and cell culture method |
| CN113846017A (en) * | 2021-10-13 | 2021-12-28 | 无锡药明生物技术股份有限公司 | Selective entrapment device and method of reducing entrapment efficiency |
| CN113846017B (en) * | 2021-10-13 | 2024-04-26 | 无锡药明生物技术股份有限公司 | Selective entrapment device and method for reducing entrapment rate |
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Address after: 510000 No.1, jinfengyuan Road, Guangzhou Economic and Technological Development Zone, Guangdong Province Patentee after: Shijiazhuang Pharmaceutical Group mingfule Pharmaceutical (Guangzhou) Co.,Ltd. Address before: 510000 No.1, jinfengyuan Road, Guangzhou Economic and Technological Development Zone, Guangdong Province Patentee before: GUANGZHOU RECOMGEN BIOTECH CO.,LTD. |