CN104974933B - A kind of extensive continuous several times, which suspend, turns the apparatus and method of expression recombinant protein wink - Google Patents
A kind of extensive continuous several times, which suspend, turns the apparatus and method of expression recombinant protein wink Download PDFInfo
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Abstract
Suspended the invention discloses a kind of extensive continuous several times and turn the apparatus and method of expression recombinant protein wink, the apparatus and method mainly make use of perfusion technique and maintain cell to be stored in a relatively good growth conditions so that the process of transient transfection carries out multiple（≥2）And repeatedly the cell state of transient transfection group motility rate during the transient transfection of continuous several times does not significantly decrease, propagation is not affected, it is thus possible to effectively extend the cycle of whole technique, increase the yield of recombinant protein, reduce the production cost of recombinant protein.
Description
Technical field
The present invention relates to a kind of apparatus and method of bioengineering field, and in particular to a kind of in the reaction of continuous suspended biological
The apparatus and method for expressing recombinant protein for a long time are repeatedly transiently transfected in device.
Background technology
Transfection refers to the process that eukaryotic obtains new genetic marker due to exogenous DNA incorporation.Routine transfection technology can
It is divided into transient transfection and the major class of stable transfection two.Exogenous DNA/RNA unconformity is into host chromosome during transient transfection, because
It may be present multiple copy numbers in this host cell, produce high-caliber expression, but generally only last for several days, after transfection
Harvesting in 24 hours to 96 hours, its specific time depends on many factors such as cell type, vector construction.
In recent years, with the development of transient transfection techniques and cell culture technology, it is already possible to by generally making to some
Cell line（Such as HEK293 and Chinese hamster ovary celI）Suspension culture is carried out, to realize that the extensive recombinant protein of transient transfection is closed
Into.In a suitable case can be with time saving and energy saving, cost-effective from transient expression.
By the retrieval discovery to existing literature, C é line Raymond, Roseanne Tom, Sylvie Perret etc.
People .2011 exists April《Methods》On deliver《A simplified polyethylenimine-mediated
transfection process for large-scale and high-throughput applications》One text report
The large-scale transient transfection process that a kind of technique simplifies has been accused, wink of the suspension cell in orifice plate and reactor is have studied respectively
When transfection expression recombinant protein process.But the transient transfection process in bioreactor has only lasted for one week and has recombinated egg
White yield is very limited.The following defect of presence transiently transfected on a large scale in article：
First, due to this characteristic of plasmid loss during transient transfection, transient transfection techniques are only limitted to laboratory level,
Industrialization promotion has many limitations；
Second, common scale transiently transfects technique（Such as batch）General to continue 3-7 days, the cycle is short, thus causes whole
Individual process costs are high.
In summary, existing gene transiently transfect expression recombinant protein the method cycle it is short, yield poorly, cost it is high, no
It is adapted to the production of industrially scalable, it is difficult to meet demand of the preclinical laboratory to recombinant protein.
The content of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of recombinant protein of continuous, bioreactor scale instantaneously turns
Contaminate the apparatus and method of expression.This method has the cycle long compared with traditional transient transfection method, and yield is high, can automate
Advantage, therefore comparatively large number of recombinant protein can be provided in the shorter time, for new drug development and screening, meet preclinical right
Recombinant protein demand.
A kind of extensive continuous several times for bioreactor scale that the present invention is provided, which suspend, turns expression recombinant protein wink
Device, including device for storing liquid, transfection composite mixing arrangement, cell culture apparatus, central control unit, cell retention device,
Supernatant collection device, cell collection device, some liquid asepsis pushing meanss and it is some can rate controlling liquid flow conduits, it is above-mentioned
The connected mode of each device is：Device for storing liquid by one first can rate controlling liquid flow conduits be connected with cell culture apparatus,
One first liquid asepsis pushing meanss are provided between device for storing liquid and cell culture apparatus；Transfection composite mixing arrangement passes through one
Second can rate controlling liquid flow conduits be connected with cell culture apparatus, transfection composite mixing arrangement and cell culture apparatus it
Between be provided with the sterile pushing meanss of a second liquid；Cell culture apparatus converts output line phase with central control unit by signal
Even；One end of cell culture apparatus can rate controlling liquid flow conduits and the anterior split-phase of retention of cell retention device by one the 3rd
Even, the other end of cell culture apparatus by one the 4th can rate controlling liquid flow conduits with dam after cell retention device phase
Even, one the 3rd liquid asepsis pushing meanss are provided between the cell retention device in cell culture apparatus and after damming；Cell is cut
Stay device to retain preceding one end with supernatant collection device to be connected, supernatant collection device with dam before cell retention device it
Between be provided with one the 4th liquid asepsis pushing meanss；Cell retention device retention after the other end by one the 5th can rate controlling liquid flow move
Pipeline is connected with cell collection device, and one the 5th liquid is provided between the cell retention device in cell collection device and after damming
Sterile pushing meanss.
The device for storing liquid is common cylinder or cube bottle structure or liquid storing bag, and to hold aseptic culture medium, it goes out
Mouthful with can rate controlling pipeline system be tightly connected, and on the apparatus have interface pass through sterilised membrane filter filter connection air；Institute
Device for storing liquid is stated at least provided with three holes, wherein a hole can by can rate controlling liquid flow conduits the nutrient solution after aseptic filtration is noted
Enter the device for storing liquid, another hole can by can rate controlling liquid flow conduits the nutrient solution after aseptic filtration is pushed into cell culture
In device, another hole can be communicated by air filter with external environment condition；The device for storing liquid is can high-temperature sterilization unit.
The transfection composite mixing arrangement at least reagent adds mouth and compound outlet, and with automatic stirring mixing
The function of liquid, is embodied in and possesses stirring mixed function and clocking capability, for example, can adjust stir speed (S.S.) and setting is stirred
Time；Be incubated the transfection composite that terminates can by compound outlet by described second can rate controlling liquid flow conduits enter thin
Born of the same parents' culture apparatus.
The cell culture apparatus is passed back at least provided with nutrient solution entrance, nutrient solution outlet, compound entrance, cell
Mouth, gas（Such as air, oxygen, carbon dioxide, nitrogen）Entrance, cell sampling mouth, alkali entrance, cell inoculation mouth, probe insertion
Mouthful（Such as PH, dissolved oxygen, temperature）, can high-temperature sterilization, enclosed sterile progress cell culture；The cell culture apparatus can pass through center
Various parameters in control device control cell cultivation process（Such as PH, dissolved oxygen, temperature, concentration of glucose）.
The central control unit is that at least can record and adjust the physical-chemical parameters in various incubations（Such as PH,
Dissolved oxygen, temperature, concentration of glucose etc.）Controller, the central control unit can control cell cultivation process various parameters, make
Obtain the growth of optimum cell or obtain purpose product.
The cell retention device（Such as biosep, cell settlement device, Hollow fiber units）It is mixed at least provided with cell
Liquid entrance, cell supernatant outlet, cell bypass outlet, retention outlet are closed, wherein cell mixture entrance is filled with cell culture
Connection is put, for the cell in the cell mixture discharged from cell culture apparatus to be retained, the described 3rd can rate controlling
Liquid flow conduits are arranged between the cell mixture entrance and cell culture apparatus；Cell supernatant outlet is through described the
Four liquid asepsis pushing meanss are connected with supernatant collection device, make the cell supernatant in cell retention device by described
Four liquid asepsis pushing meanss are pushed into supernatant collection device；Cell bypass outlet is promoted through the 3rd liquid asepsis
Device connects cell culture apparatus, and the cell for cell retention device to be retained down passes through the 3rd liquid asepsis pushing meanss
It is pushed into cell culture apparatus；Retention outlet is connected to cell collection device through the 5th liquid asepsis pushing meanss, uses
Cell is pushed into by the 5th liquid asepsis pushing meanss collect dress in the cell for being retained down cell retention device
In putting.
The supernatant collection device is provided with bottle cap, and the bottle cap is provided with liquid inlet, is sterilizing at least two holes
Storage device a, wherein hole is by can rate controlling liquid flow conduits and cell retention device top（Such as cell supernatant outlet）Phase
Even, cell culture supernatant can enter supernatant collection device by the impetus of the 4th liquid asepsis pushing meanss,
Another hole is communicated by air filter with external environment condition.
The cell collection device is the storage device that sterilizes at least two holes, wherein a hole is controllable by the described 5th
Fast liquid flow conduits and cell retention device bottom（Such as retain outlet）It is connected, the cell after retention can pass through the 5th liquid
The impetus of the sterile pushing meanss of body enters cell collection device, and another hole passes through air filter and external environment condition phase
It is logical.
It is described can rate controlling liquid flow conduits for can by slide control valve or clip control cultivate flow velocity pipeline, pipe
Road is, for example, silicone tube.
The liquid asepsis pushing meanss are that can sterilely promote the device that the liquid in pipeline flows, such as peristaltic pump.
The above-mentioned extensive continuous several times that the present invention is provided, which suspend, turns the device of expression recombinant protein wink, and its principle is as follows：
A kind of cell is after transient transfection, and only part cell can receive external source recombinant plasmid and carry out exogenous plasmid transcription, expression
The process of recombinant protein, the metabolic rate of the cell because successfully receiving plasmid（Such as specific growth rate, than glucose consumption rate, than thin
Born of the same parents' yield etc.）Will be less than not receiving the cell of plasmid, thus do not receive under identical condition of culture the growth of the cell of plasmid
Situation is better than the cell for receiving plasmid.After cell in cell culture apparatus is by cell retention device, active cell retention
The sterile pushing meanss of device（Such as peristaltic pump）, the different cell of speed will be grown and be uniformly pushed into cell retention device and root
Need, be partly refluxed in cell culture apparatus according to cell growth state, while collecting the detection that supernatant carries out recombinant protein
And purifying.Due to the difference of the cell for not receiving plasmid and the specific cell growth rate for receiving plasmid, compare in cell culture apparatus
The big cell of growth rate（Do not receive recombinant plasmid）Quantity can be more and more, meanwhile, received the cell of exogenous plasmid because
To transiently transfect the characteristic of plasmid loss, with the growth of incubation time, it can also turn into the cell for not receiving exogenous plasmid at any time,
Thus after a period of time, the cell in cell culture apparatus is provided with the ability for receiving recombinant plasmid transient transfection once more, this
When can then carry out second of transient transfection.This technique of the present invention also uses perfusion technique（perfusion）Remain thin
Born of the same parents are stored in a relatively good growth conditions so that the process of transient transfection carries out multiple（≥2）, it is thus possible to effective extension
In the cycle of whole technique, increase the yield of recombinant protein, reduce the production cost of recombinant protein.
When the extensive continuous several times suspension wink of the present invention turns the device work of expression recombinant protein, the training in device for storing liquid
Nutrient solution the first liquid asepsis pushing meanss effect under, by described first can rate controlling liquid flow conduits enter cell training
Support in device, and central control unit controls the various parameters of cell culture（Such as, but not limited to PH, dissolved oxygen, temperature etc.）, carefully
Born of the same parents grow under the conditions of the cell culture Optimal Parameters that central control unit is controlled.When completing to transiently transfect for the first time, liquid storage
Nutrient solution in device, can rate controlling liquid flow conduits by described first under the first liquid asepsis pushing meanss effect
Into in cell culture apparatus, and central control unit controls the various parameters of cell culture（Such as, but not limited to PH, dissolved oxygen,
Temperature etc.）, cell grows under the conditions of the cell culture Optimal Parameters that central control unit is controlled；Meanwhile, central control unit
The setting weight claimed by continuous pouring regulates and controls the speed of the 4th liquid asepsis pushing meanss at supernatant collection device,
So that the nutrient solution cumulative volume of cell culture apparatus keeps constant.The cell bypass outlet of cell retention device is filled with cell culture
The 3rd liquid asepsis pushing meanss between putting（Such as peristaltic pump）The speed of service according to cell cultivation requirement sets itself,
It is unsuitable excessive or too small（For example in cell retention device, the excessive then backflow cellular damage of speed is excessive, and speed is too small, retains
Effect is not good, causes all to enter in supernatant compared with many cells）.When filling of the cell density in cell culture apparatus higher than setting
Note cell density when, central control unit then active cell device for trapping retention outlet cell collection device between the 5th
Liquid asepsis pushing meanss（Such as peristaltic pump）, the part cell of retention is pushed into cell collection device, works as cell culture apparatus
In cell density less than setting perfusion cell density when, then retention outlet and the cell for closing cell retention device are collected and filled
The 5th liquid asepsis pushing meanss between putting（Such as peristaltic pump）So that it is constant that cell density maintains setting density.By this
The control process of one intelligence, cell can maintain a relatively good cell state.It can now mix and fill to transfection composite
Put and add recombinant plasmid, transfection mediation reagent and cell transfecting liquid again, start timing agitation function, prepare fresh transfection
Compound, under the sterile pushing meanss effect of the second liquid, transfection composite is pushed into cell culture apparatus, carried out
Secondary transfection.Secondary Transfected cells enter the state similar to after once transfection again, can be regulated and controled again by central control unit
Filling process is to maintain the good growth of cell, expression status.The collection cell training that supernatant collection device can continue simultaneously
The recombinant protein in supernatant, purified pool supernatant is supported to be analyzed, detected and the detection of other preclinical pharmacologies, toxicity.
The device that the extensive continuous several times suspension wink of the present invention turns expression recombinant protein can be transfected repeatedly, and specifically be transfected secondary
Number can flexibly be determined according to cell state, the actual conditions such as plasmid property and product.
The method that the extensive continuous several times suspension wink of the present invention turns expression recombinant protein is mainly that make use of perfusion
（perfusion）Technology maintains cell to be stored in a relatively good growth conditions so that the process of transient transfection carries out multiple（≥
2 times）, specifically, this method for the device for turning expression recombinant protein using above-mentioned extensive continuous several times suspension wink can be led
Comprise the following steps：
（1）Install, connect package unit and the progress for turning expression recombinant protein in above-mentioned extensive continuous several times suspension wink
High-temperature sterilization；High-temperature sterilization condition high-temperature sterilization can be 121 DEG C known to technical staff, 30min；
（2）A certain amount of aseptic culture fluid is pumped under aseptic condition into cell culture apparatus, the amount of nutrient solution at least can
Detection probe such as detection temperature, dissolved oxygen, pH probe are not crossed, Sterility testing is carried out, for example, sterile inspection is carried out under the conditions of 37 DEG C
Survey up to 48 hours；
（3）Aseptically, it is cultivating in advance, in good condition to being inoculated with certain cell density in cell culture apparatus
Cell to be transfected, and according to the optimal transfection conditions of different cells, different plasmid（Plasmid concentration, transfection reagent and plasmid
Concentration ratio, incubation time）, by DNA, transfection mediation reagent, cell transfecting liquid is mixed simultaneously in transfection composite mixing arrangement
The transfection composite obtained after incubation is added in cell culture apparatus；Generally, the cell density to be transfected of inoculation can be skill
Common cell density during recombinant plasmid cell transfecting known to art personnel, such as 0.05~1 × 107Individual cell/mL（According to
Volume is inoculated with to calculate）；
（4）According to the growth characteristics of culture cell and the working volume of cell culture apparatus, by the anti-of cell culture apparatus
Every reason of device is answered, gives birth to, change target setting in cell culture and the optimum condition of protein expression, while passing through central control unit
Perfusion parameters are controlled, perfusion parameters, back-flow velocity, useless cell velocity of discharge of the reactor etc. is set；
（5）According to the change of specific protein expression level, after perfusion culture certain time, appropriate turn is added again
Compound is contaminated, according to above-mentioned steps（3）In transfection conditions transfect again, this process may be repeated several times；
（6）Each work period for example collects detection and subsequent recombination albumen that cell culture fluid carries out protein content daily
Purification process, is collected after enough albumen, terminates cell culture and protein expression process.
The optimal transfection conditions（Plasmid concentration, transfection reagent concentration, incubation time）Can be：Plasmid DNA concentration is
0.5-3 μ g/mL, DNA:Transfection mediation ratio of reagents is 1:2-1:10, incubation time is 5-30 minutes, to form stable DNA-
Transfection mediation reagent complex.Transfection mediation reagent can select various reagents commonly used in the trade, such as Lipofectamine2000,
25KD PEI, PEI MAX etc., but it is not limited to only mentioned reagent.
Described step（4）In, the optimum condition of cell culture and protein expression can be each known to technical staff
Temperature set by bioreactor cell culture, pH value, oxyty value etc..For example can be：Nutrient solution uses T39
（CHO）Or Free Style（293）, pretreatment to 37 DEG C ± 0.2 DEG C, pH value 7.1 ± 0.1, DO25%~55%, with 50~150
The index of the velocity interpolation nutrient solution turned is uniform.
Described step（4）In, control perfusion parameters are tested according to cell growth condition and protein expression process
Determine, its concrete numerical value can be determined by persons skilled in the art through overtesting, for example, can be 0.05 reactor training
Support volume/day to 20 bioreactor culture volume/days.
The step（5）In, the determination for repeating the number of repetition of transient transfection process is according to different cell, different
Protein expression level under the conditions of recombinant plasmid is different, is determined for the purpose of reaching and finally harvesting enough destination proteins, can
To be more than 2 times or more frequently carry out transfection procedure.
Compared with prior art, beneficial effects of the present invention are as follows：
The extensive continuous several times that the present invention is provided suspend the apparatus and method for turning expression recombinant protein wink and traditional wink
When transfection method compare and have the cycle long, yield is high, the advantage such as can automate, therefore can be provided in the shorter time than relatively large
Recombinant protein, for new drug development with screening, meet preclinical to recombinant protein demand.
Brief description of the drawings
Fig. 1 suspends for the extensive continuous several times of an example of the invention turns the schematic diagram for expressing the device of recombinant protein wink；
Fig. 2 for an example of the invention transfection process in cell density with transfection after cultivated days variation diagram；
Fig. 3 for an example of the invention transfection process in transfection efficiency with transfection after cultivated days variation diagram；
Fig. 4 for an example of the invention one transfection process cycle during accumulation cell express the blood coagulation II factors it is total
Amount.
Embodiment
Certain specific embodiments of the invention are described in detail below in conjunction with accompanying drawing, it is of the invention to help understanding, but
Content described further below is not intended to limit the scope of the invention.Description above generally illustrates that the present invention's is interior
Hold, specific embodiment below will more directly embody the situation of the present invention, but it must be noted that the example enumerated herein
Just for the sake of the purpose being illustrated, the present invention is not limited to this.
Fig. 1 is referred to, is suspended for a kind of extensive continuous several times that the present invention is provided and turns the device of expression recombinant protein wink
Concrete structure schematic diagram.As illustrated, the device includes device for storing liquid 1, transfection composite mixing arrangement 2, cell culture dress
3, central control unit 4, cell retention device 5, supernatant collection device 6, cell collection device 7, five liquid asepsis are put to push away
Dynamic device（8、9、10、11、12）, it is multiple can rate controlling liquid flow conduits；Its connected mode is：Device for storing liquid 1 passes through one first
Can rate controlling liquid flow conduits（Such as silicone tube）It is connected with cell culture apparatus 3, is set between device for storing liquid and cell culture apparatus
There are one first liquid asepsis pushing meanss 9；Transfection composite mixing arrangement 2 by one second can rate controlling liquid flow conduits with it is thin
Born of the same parents' culture apparatus 3 is connected, and the rwo is middle provided with the sterile pushing meanss 8 of a second liquid；Cell culture apparatus 3 is controlled with center
Device 4 converts output line by signal and is connected；The one end of cell culture apparatus 3 by one the 3rd can rate controlling liquid flow conduits with it is thin
The retention forward part of born of the same parents' device for trapping 5 is connected, the other end by one the 4th can rate controlling liquid flow conduits and the cell after damming cut
Stay device 5 to be connected, promote and fill provided with one the 3rd liquid asepsis between cell culture apparatus 3 and cell retention device 5 after damming
Put 12；Cell retention device 5 retains preceding one end and is connected with supernatant collection device 6, supernatant collection device 6 and the cell before damming
One the 4th liquid asepsis pushing meanss 10 are provided between device for trapping 5；The other end passes through one the 4th after cell retention device 5 is retained
Can rate controlling liquid flow conduits be connected with cell collection device 7, cell collection device 7 and cell retention device 5 after damming it
Between be provided with one the 5th liquid asepsis pushing meanss 11.
Wherein, cell retention device 5 is provided with cell mixture entrance, cell supernatant outlet, cell bypass outlet, retention
Outlet, wherein cell mixture entrance by the described 3rd can rate controlling liquid flow conduits be connected with cell culture apparatus 3, be used for
Cell in the cell mixture that will be discharged from cell culture apparatus 3 is retained；Cell supernatant is exported through the 4th liquid
Sterile pushing meanss 10 are connected with supernatant collection device 6, the cell supernatant in cell retention device is passed through the 4th liquid
Sterile pushing meanss 10 are pushed into supernatant collection device 6；Cell bypass outlet is through the 3rd liquid asepsis pushing meanss 12
Cell culture apparatus 3 is connected, the 3rd liquid is passed through through cell bypass outlet for the cell that is retained down cell retention device 5
Sterile pushing meanss 12 are pushed into cell culture apparatus 3；Retention outlet is connected to carefully through the 5th liquid asepsis pushing meanss 11
Born of the same parents' collection device 7, the cell for cell retention device 5 to be retained down is promoted by retaining the 5th liquid asepsis in exit
Device 11 is pushed into cell collection device 7.
In a following specific embodiment of the invention, liquid asepsis pushing meanss use peristaltic pump.
Below exemplified by expression people's restructuring blood coagulation II factors are transiently transfected using the method for the present invention, the present invention is described in detail
Extensive continuous several times suspend wink turn expression recombinant protein method.
Example：Bioreactor continuous several times transiently transfect expression people's restructuring blood coagulation II factors
Cell：Serum free suspension tames HEK293 cell lines；
Nutrient solution：The Excell293 serum-free mediums of U.S. Sigma productions；
Cell culture apparatus：The 3L zooblast reactors of Dutch Applicon companies production；
1) the aseptic detection of the system before being inoculated with and Detection of Stability
37 DEG C of the design temperature on the control tower that Dutch Applicon companies produce, pH value 7.2, oxyty value 50%, by
Thermostatic water-jacket controls the flow valve on temperature, control tower to automatically control CO2、N2、O2Three tunnel air inlets are controlled with the method for bubbling oxygen supply
1mol/L NaOH and 1mol/L HCL peristaltic pump is connected with oxyty and molten gas concentration lwevel, control tower to control
PH, according to the weighing system signal of perfusion control addition nutrient solution to setting value, is connected with simultaneously on the tank body of bioreactor
The liquid inlet of fresh medium, and the cell retention device flowed back for cell are supplemented, package unit connection installs rear 121
DEG C, high pressure steam sterilization 30min.After cooling, it is placed in desinfection chamber, sterile PBS, circulation cleaning is passed through into system
Wash whole system.Then, PBS is discharged, fresh medium is added, starts temperature control and the stirring of control tower, continues 24h, inspection
Look into whether microbiological contamination and system run all right.It is uniform with the index of 80rpm velocity interpolation nutrient solution after inspection is sterile.
2）Culture and transient transfection for the first time before the transfection of cell
With 1 × 106Individual cell/mL density prepares cell suspension, to above-mentioned 3L（Working volume 2L）In bioreactor
Inoculating cell suspension is to final concentration 2.5 × 105Individual cell/mL, after criticizing culture 48 hours, treats cell amplification to 1 × 106Individual cell/
First time transient transfection is carried out during mL density, this example selects direct transfection method, to transfection composite mixing arrangement（In this reality
It is cylindrical appliance in example, there is an inlet and a liquid outlet, sealed with expanded rubber plug, inserts controllable on rubber consent
Speed, can timing agitation device）Middle addition recombinant plasmid pGMAX-FII and cell transfecting liquid, 120rpm stirring 5min, are then added
Transfection mediation reagent PEI MAX, 120rpm stirring 5min, stirring terminates rear stationary incubation 10min.Wherein plasmid pGMAX-FII
Measure as 2 μ g/106Individual cell, DNA:PEI=1:2.5.Transfection composite is pushed into cell culture apparatus by peristaltic pump, carefully
Born of the same parents are transfected.Open perfusion within 48 hours after transfection, it is respectively 37 DEG C of temperature, pH value 7.0, oxyty value to set each desired value
40%, concentration of glucose 1.5g/L, control to automatically adjust the inlet and liquid outlet of cell culture apparatus by central control system
Wriggling revolution speed, with maintain setting cell growth environment, while central control system is according to bioreactor level electrode
And temperature, PH, dissolved oxygen, the peristaltic pump that nutrient solution is added in the signal auto-adjustment control device for storing liquid of molten carbon dioxide electrode
Rotating speed and control device, to ensure the supply of nutrient solution.Irrigation rate is set to 1 reactor tank body volume/day, cell discharge of giving up
Speed is set to 1/3 reactor tank body volume/day.Supernatant is collected daily and in -80 DEG C of storages, for carrying out the solidifying of next step
The purifying of blood II factor proteins, quality inspection and preparation set-up procedure.
3）Cell is repeatedly transiently transfected
The 6th day after transiently transfecting for the first time, stop perfusion culture, abandon a part of cell and add fresh medium, adjust
Ganglion cell's density is to 1 × 106Individual cell/mL, incubated overnight 24 hours adjusts cell to 1 × 10 again6Individual cell/mL carries out the
It is secondary to transiently transfect.To transfection composite mixing arrangement（It is in this example cylindrical appliance, has an inlet and one goes out
Liquid mouthful, is sealed with expanded rubber plug, on rubber consent insertion can rate controlling, can timing agitation device）Middle addition recombinant plasmid
Then pGMAX-FII and cell transfecting liquid, 120rpm stirring 5min add transfection mediation reagent PEI MAX, 120rpm stirrings
5min, stirring terminates rear stationary incubation 10min.Wherein plasmid pGMAX-FII amounts are 1.5 μ g/106Individual cell, DNA:PEI=1:
2.5.Transfection composite is pushed into cell culture apparatus by peristaltic pump, cell is transfected.Open within 48 hours after transfection
Perfusion, it is consistent that perfusion culture transiently transfects incubation with first time.
The 13rd day after transiently transfecting for the first time, 3 are repeated the above steps）The processing procedure of second described of transient transfection,
Carry out within the 14th day after being transiently transfected in first time condition of culture and second of wink after third time transient transfection, transfection conditions and transfection
When transfection process it is completely the same.
Daily supernatant of collecting is placed in -80 DEG C of preservations in cell cultivation process, the blood coagulation II factors for carrying out next step
Protein purification, quality inspection and preparation set-up procedure.
4）Production is completed
After culture terminates, reactor and cleaning experiment equipment are closed.
The incubation parameter of the present embodiment refers to Fig. 2 and Fig. 3.Wherein Fig. 2 show the transfection process of examples detailed above
The variation diagram of middle cell density and cultivated days after transfection.As seen from Figure 2, multiple transient transfection group in this example is thin
Born of the same parents' state Cell viability during the transient transfection of continuous three times is substantially steady, and later stage appearance slightly declines, and Cell viability exists
Do not significantly decreased in whole transfection period, propagation is not affected.Cell keeps good life in whole incubation
Long status, Cell viability is maintained at more than 90%.
It is shown in Figure 3, be examples detailed above transfection process in transfection efficiency with transfection after cultivated days variation diagram.
Visible in figure, in this example, the transfection efficiency and blood coagulation II factor proteins expression quantity of cell are presented after transiently transfecting for the first time
The transfection efficiency and the blood coagulation II factors of cell after downward trend after first rising, second of transient transfection and third time are transiently transfected
There is obvious rise in the expression quantity of albumen.Whole continuous several times transiently transfect process cycle 21 days, the technique week of transient transfection
Phase, more traditional batch of culture was obviously prolonged.
Fig. 4 is referred to, the total of the blood coagulation II factors that cell is expressed is calculated it illustrates accumulation during whole process cycle
Amount, the transient transfection technique of the example of the invention obtains blood coagulation II factors total amount and adds 97% than tradition transient transfection technique.
Illustrated above is only example of the present invention, under the teaching of the present invention and above-described embodiment, this area
Technical staff be easy to it is envisioned that the present invention it is cited or enumerate each raw material or its equivalent alterations, each processing method or its
Equivalent alterations can realize the present invention, and the parameter bound value of each raw material and processing method, interval value can be realized
The present invention, embodiment numerous to list herein.
Claims (6)
1. a kind of extensive continuous several times, which suspend, turns the method for expression recombinant protein wink, it is characterised in that main to include following step
Suddenly：
（1）One extensive continuous several times are provided and suspended and turn the device of expression recombinant protein wink, installed, connected package unit and carry out
High-temperature sterilization；The device that the extensive continuous several times suspension wink turns expression recombinant protein includes：Device for storing liquid, transfection composite
Mixing arrangement, cell culture apparatus, central control unit, cell retention device, supernatant collection device, cell collection device,
Some liquid asepsis pushing meanss and it is some can rate controlling liquid flow conduits, the connected mode of above-mentioned each device is：Device for storing liquid
By one first can rate controlling liquid flow conduits be connected with cell culture apparatus, set between device for storing liquid and cell culture apparatus
There are one first liquid asepsis pushing meanss；Transfection composite mixing arrangement can rate controlling liquid flow conduits and cell by one second
Culture apparatus is connected, and is filled between transfection composite mixing arrangement and cell culture apparatus provided with sterile promote of a second liquid
Put；Cell culture apparatus converts output line by signal with central control unit and is connected；One end of cell culture apparatus passes through one
3rd can rate controlling liquid flow conduits be connected with the retention forward part of cell retention device, the other end of cell culture apparatus passes through
One the 4th can rate controlling liquid flow conduits be connected with the cell retention device after damming, cell culture apparatus with dam after it is thin
One the 3rd liquid asepsis pushing meanss are provided between born of the same parents' device for trapping；Cell retention device retains preceding one end and filled with supernatant collection
Put connected, one the 4th liquid asepsis pushing meanss are provided between the cell retention device in supernatant collection device and before damming；
Cell retention device retention after the other end by one the 5th can rate controlling liquid flow conduits be connected with cell collection device, in cell
One the 5th liquid asepsis pushing meanss are provided between collection device and cell retention device after damming；Wherein, the cell is cut
Device is stayed to be exported at least provided with cell mixture entrance, cell supernatant outlet, cell bypass outlet, retention, wherein cell is mixed
Close liquid entrance to be connected with cell culture apparatus, for the cell in the cell mixture discharged from cell culture apparatus to be carried out
Retention, the described 3rd can rate controlling liquid flow conduits be arranged between the cell mixture entrance and cell culture apparatus；Carefully
Born of the same parents' supernatant outlet is connected through the 4th liquid asepsis pushing meanss with supernatant collection device, is made in cell retention device
Cell supernatant is pushed into supernatant collection device by the 4th liquid asepsis pushing meanss；Cell bypass outlet is passed through
The 3rd liquid asepsis pushing meanss connect cell culture apparatus, and the cell for cell retention device to be retained down passes through
3rd liquid asepsis pushing meanss are pushed into cell culture apparatus；Retention outlet connects through the 5th liquid asepsis pushing meanss
Cell collection device is connected to, the cell for cell retention device to be retained down passes through the 5th liquid asepsis pushing meanss
It is pushed into cell collection device；Wherein, it is described can rate controlling liquid flow conduits for can be by sliding control valve or clip control
The pipeline of system culture flow velocity；The liquid asepsis pushing meanss are peristaltic pump；
（2）Be pumped into aseptic culture fluid under aseptic condition into cell culture apparatus, the amount of nutrient solution can at least not have detection temperature,
The probe of dissolved oxygen, pH, then carries out Sterility testing；
（3）Aseptically, cell to be transfected cultivating in advance, in good condition, root are inoculated with into cell culture apparatus
Optimal transfection conditions are determined according to different cells, different plasmid, by DNA, transfection mediation reagent, thin under the optimal transfection conditions
Dysuria with lower abdominal colic dye liquor is incubated in transfection composite mixing arrangement and obtained transfection composite is added in cell culture apparatus；Its
In, the optimal transfection conditions include plasmid concentration, transfection reagent and plasmid concentration ratio, incubation time；
（4）According to the growth characteristics of culture cell and the working volume of cell culture apparatus, by the reactor of cell culture apparatus
Items reason, raw, change target setting are in cell culture and the optimum condition of protein expression, while being by central control unit control
System perfusion parameters, perfusion of the systemic perfusion parameter including reactor, back-flow velocity, the useless cell velocity of discharge；Cell culture
After cell in device is by cell retention device, the sterile pushing meanss of active cell device for trapping, will growth speed it is different
Cell be uniformly pushed into cell retention device and according to cell growth state needs, be partly refluxed to cell culture apparatus
In, while collecting detection and purifying that supernatant carries out recombinant protein；
（5）According to the change of specific protein expression level, after perfusion culture certain time, appropriate transfection is added again and is answered
Compound, according to above-mentioned steps（3）In transfection conditions transfect again, this process is repeated at least once more；
（6）Each work period collects the detection and the processing of subsequent recombination protein purification that cell culture fluid carries out protein content, receives
After the enough albumen of collection, cell culture and protein expression process are terminated.
2. extensive continuous several times according to claim 1, which suspend, turns the method for expression recombinant protein wink, it is characterised in that
Described step（3）In, the inoculating cell density of cell to be transfected is 0.05~1 × 107Individual cell/mL.
3. extensive continuous several times according to claim 1, which suspend, turns the method for expression recombinant protein wink, it is characterised in that
Described step（3）In, optimal transfection conditions include：Plasmid DNA concentration is 0.5-3 μ g/mL, DNA:Transfection reagent ratio
For 1:2-10, incubation time is 5-30 minutes, to form stable DNA- transfection mediation reagent complex.
4. extensive continuous several times according to claim 1, which suspend, turns the method for expression recombinant protein wink, it is characterised in that
Described step（4）In, the optimum condition of cell culture and protein expression includes：Temperature is 37 °C ± 0.2 °C, and pH value is
7.1 ± 0.1, dissolved oxygen is 25%~55%, and rotating speed is 50 ~ 150 revs/min.
5. extensive continuous several times according to claim 1, which suspend, turns the method for expression recombinant protein wink, it is characterised in that
Described step（4）In, the perfusion of the reactor, back-flow velocity, the useless cell velocity of discharge are 0.05-20 bioreactor culture
Volume/day.
6. extensive continuous several times according to claim 1, which suspend, turns the method for expression recombinant protein wink, it is characterised in that
The number of repetition of the transfection process is more than 2 times.
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| US20230313232A1 (en) | 2022-02-28 | 2023-10-05 | Corning Incorporated | Methods and systems for real-time, continuous production of non-viral carrier nucleic acid particles |
| CN116769832B (en) * | 2023-05-23 | 2024-01-26 | 皓阳生物科技(上海)有限公司 | Transient transfection method of mammalian cells |
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