CN206843495U - Ezyme bio-reactor and intelligent enzyme reaction work station - Google Patents
Ezyme bio-reactor and intelligent enzyme reaction work station Download PDFInfo
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- CN206843495U CN206843495U CN201720508476.5U CN201720508476U CN206843495U CN 206843495 U CN206843495 U CN 206843495U CN 201720508476 U CN201720508476 U CN 201720508476U CN 206843495 U CN206843495 U CN 206843495U
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Abstract
The utility model discloses a kind of ezyme bio-reactor, it includes closed tank body, retention component and mixing component, the mixing component includes agitating shaft and the agitating paddle being fixedly connected on the agitating shaft, the agitating paddle is distributed in the closed tank inner chamber, the agitating paddle uses elevator belt oar, and the ratio between the diameter of the elevator belt oar and the internal diameter of the sealed shell of tank are 1.0:1.35~1.0：1.95, the blade inclination angle of the elevator belt oar is 5.5~24.5 ° etc..The utility model discloses a kind of intelligent enzyme reaction work station based on the enzyme reactor.The utility model can be used for harvest anchorage dependent animal cell and expression product therein, and by separating cell from the culture medium of saturation in gnotobasis, cell and expression product are collected, and culture medium may be reused.The utility model can be additionally used in the full volumetric culture fluid exchange of anchorage dependent animal cell incubation, and more than 98% can be reached by changing efficiency.
Description
Technical field
It the utility model is related to a kind of bioreactor, and in particular to a kind of intelligent enzyme for being used to release zooblast attaching
React work station, its can apply to the culture of anchorage dependent animal cell in biological products production process, inoculation, amplification,
Change liquid, cleaning and harvest technical field.
Background technology
At present, a large amount of bio-pharmaceuticals are the products of mammalian cell expression.Most of domestic manufacturers are utilizing animal
When cell produces biological products, all realized by cell factory and roller bottle culture technique, but these technologies have batch
Small, the defects of efficiency is low, labor intensity is big.
In general, used and animal vaccines, Recombinant protein medicine and part small-molecule drug for producing people
Zooblast has anchorage dependence type, and this kind of cell can not be directly suspended in nutrient solution, it is necessary to depend on culture in vitro to be situated between
Matter, conventional culture medium have Cytodex1, Cytodex2 of GE companies etc., the DISC carriers of NBS companies, and Taiwan match space
Scraps of paper carrier of company etc..During technical grade bioreactor culture zooblast produces corresponding biological products, it is necessary to first
Reach certain culture scale, generally 500L to 5000 liters, in this course, it is necessary to constantly by institute on the medium of saturation
After the cell contained departs from and is enriched with carrier, enter on new carrier, realize the amplification of cell and cultivate the amplification of scale.
The free attached cell seed for obtaining enrichment is the key of technical grade amplification production.In addition, in process of production, to culture medium
The cleaning of progress full volumetric, replacing nutrient solution are also very crucial.Finally after production terminates, recovery culture medium produces with harvesting
Product also relate to the separation process of cell and medium.
Realizing the method for above-mentioned function at present mainly has directly digestion separation, digestion in site transfer, ball to turn ball migration, off normal
Digest four kinds of transfer method etc..
But no matter use it is any in these separation methods, wherein it is by physical action point that must all have a stage
From zooblast and medium.However, because zooblast is very fragile, so very quick to shearing force caused by physical separation effect
Sense, but shearing force can not enough realize the effect of separation again, how reach the balance of both, be always industry craving solution
Problem certainly.
On the other hand, as what the popularization of process analysis technology (PAT) and GMP standards required gradually steps up, to life
The On-line Control and intelligent automation of production equipment require more and more higher, but existing enzyme reaction equipment is difficult to meet these realities
Border demand.
Utility model content
In view of the shortcomings of the prior art, the purpose of this utility model is to provide a kind of ezyme bio-reactor and intelligent enzyme is anti-
Answer work station.
To realize foregoing purpose of utility model, the technical solution adopted in the utility model includes：
The utility model embodiment provides a kind of ezyme bio-reactor, and it includes closed tank body, mixing component and set
Retention component in the vapor tight tank body, the mixing component include agitating shaft and are fixedly connected on stirring on the agitating shaft
Oar is mixed, the agitating shaft coordinates with the bearing block at the top of the closed tank body, and the agitating paddle is distributed in the vapor tight tank
In intracoelomic cavity, the agitating paddle use with three-dimensional spiral shape structure elevator belt oar, the diameter of the elevator belt oar with it is described
The ratio between internal diameter of sealed shell of tank is 1.0:1.35~1.0：1.95, the blade inclination angle of the elevator belt oar is 5.5~24.5 °.
Among some embodiments, the height of the closed tank body is less than the diameter of the closed tank body, i.e. its Gao Jing
Than less than 1：1.
Among some embodiments, the rotating speed of the agitating shaft is 20~200rpm.
Among some embodiments, the closed tank body mainly by tank body and with tank body upper and lower end be tightly connected it is upper,
Lower sealing cover forms, and material input channel, the material for being also associated with connecting with the tank inner chamber on the upper and lower closure are defeated
Go out pipeline.
Among some embodiments, the retention component includes at least one layer of sieve plate or filter screen, at least one layer of sieve plate
Or filter screen is distributed in the closed tank inner chamber bottom, and the sieve plate or filter screen periphery edge and the sealed shell of tank inwall ring
Around connection, the aperture of the filter opening of the sieve plate or filter screen is from top to bottom sequentially reduced.
Further, the retention component is mainly formed by stacking by two layers with upper sieve plate or filter screen, and any one layer of sieve
The filter opening aperture of plate or filter screen is less than the distribution filter opening aperture of square sieve plate or filter screen thereon.
Among some embodiments, the enzyme reactor also includes temperature-controlling module, pH control assemblies, air pressure control
Component, online living cells Sensor monitoring component and weigh assembly processed；
The temperature-controlling module includes the temperature sensor located at the tank inner chamber reaction zone, is attached to the tank body
The chuck water system of outer wall, and the electric blanket of the cladding chuck water system；
The pH control assemblies include be arranged at the tank inner chamber reaction zone pH sensors and with the tank body
The acid ＆ alkali liquid of chamber connection adds pipeline, and the acid ＆ alkali liquid, which is added, is distributed with the pump housing on pipeline；
The pressure control component includes the respirator that is installed on the tank body and connected with the tank inner chamber
Discharge duct, the discharge duct are provided with pressure-regulating valve；
The online living cells Sensor monitoring component includes the electrode located at the tank inner chamber reaction zone, the electrode
Press close to the retention component.
Among some more specific embodiment, the weigh assembly located at the closed tank base, can not
The opening position being in contact with any material.
The utility model additionally provides a kind of intelligent enzyme reaction work station, and it includes described enzyme reactor and center control
Cabinet processed, the central control cabinet include respectively with the mixing component, temperature-controlling module, pH control assemblies, pressure control group
The central control unit that part, online living cells Sensor monitoring component connect with weigh assembly.
Among some embodiments, described intelligent enzyme reaction work station is also included such as at least one of lower component：
Quick liquid in-out module, for the liquid in the enzyme reactor quickly to be exported, and meanwhile required liquid is fast
Speed imports the enzyme reactor；
Circulating water temperature control module, to chuck water system cooperation and to the temperature in the ezyme bio-reactor
Regulated and controled；
Spectral analysis module, for being monitored to the response situation in the enzyme reactor；
Tank pressure provides module, for regulating and controlling the air pressure in the enzyme reactor；
The quick liquid in-out module, circulating water temperature control module, spectral analysis module, tank pressure provide module also and institute
State central control unit connection.
Among some more specific embodiment, described intelligent enzyme reaction work station also includes：With the center
The parallel arrangement of independent monitoring module of control unit, and the independent monitoring module also with the mixing component, temperature control
Component, pH control assemblies, pressure control component, online living cells Sensor monitoring component, weigh assembly, quick liquid in-out mould
Block, circulating water temperature control module, spectral analysis module, tank pressure provide members or all components connection in module.
Among some more specific embodiment, the central control unit includes PLC, the PLC and touch screen
Connection.
Compared with prior art, include the advantages of the utility model：
1. the utility model, which releases the intelligent enzyme reaction work station that zooblast attaches, can be used for anchorage-dependent animal
The seed of cell prepares and expands culture, by gnotobasis that above-mentioned cell is (such as micro- from the culture medium of saturation
Carrier, porous carrier or scraps of paper carrier etc.) on separate, fresh seed cell is formed after being enriched with, is inoculated in new blank
Culture medium or the culture that directly suspends, the amplification formed in culture scale, it is empty that another aspect seed cell obtains new growth
Between, continue amplification and improve cell total amount.
It can be used for harvesting anchorage dependence 2. the utility model releases the intelligent enzyme reaction work station that zooblast attaches
Zooblast and expression product therein, by gnotobasis that above-mentioned cell is (such as micro- from the culture medium of saturation
Carrier, porous carrier or scraps of paper carrier etc.) on separate, cell and expression product are collected, and culture medium may be reused
Or carry out nuisanceless disposal.
3. the utility model, which releases the intelligent enzyme reaction work station that zooblast attaches, can be used for anchorage-dependent animal
The full volumetric culture fluid exchange of cell cultivation process, more than 98% can be reached by changing efficiency.
Brief description of the drawings
Fig. 1 a are a kind of structure of the central control cabinet of intelligent enzyme reaction work station in the exemplary embodiments of the utility model one
Schematic diagram；
Fig. 1 b are a kind of knot of the ezyme bio-reactor of intelligent enzyme reaction work station in the exemplary embodiments of the utility model one
Structure schematic diagram；
Fig. 1 c are a kind of auxiliary control of intelligent enzyme reaction work station in the exemplary embodiments of the utility model the utility model one
The structural representation of molding block；
Fig. 2 is a kind of workflow diagram of intelligent enzyme reaction work station in the exemplary embodiments of the utility model one；
Fig. 3 is a kind of implementing procedure figure of intelligent enzyme reaction work station in another exemplary embodiments of the utility model；
Fig. 4 a are the picture before PK-15 (Pig testicular cell) cells depart from medium (Cytodex-1)；
Fig. 4 b are the utility model after applied to PK-15 (Pig testicular cell) cells and medium (Cytodex-1) is departed from
Design sketch；
Description of reference numerals：A- ezyme bio-reactors, 1-glass tank body, 2-gas distributor, 3-agitating paddle, 4-mark
Quasi- screw socket, 5-fixed bottle cap, 6-standard electric in highest terms, 7-air inlet, 8-sealing ring, 9-magnet fluid sealing, 10-servo electricity
Machine frame, 11-servomotor, 12- cleaning fluid tanks, 13- enzyme reaction flow containers, 14- culture tanks, the sterile acid solution tanks of 15-, 16- without
Bacterium alkali liquid tank, 17-90L bioreactors, 18-300L bioreactors, 19- microcarrier collecting tanks, 20- collect feed supplement bottle, 21-
Virus protection flow container, 23-Weighing module, 24-central control module, 25-loop temperature-control module, 26-man machine operation interface,
27-tank pressure provides module, 28-quick liquid in-out module.
Embodiment
In view of deficiency of the prior art, inventor is able to propose that this practicality is new through studying for a long period of time and largely putting into practice
The technical scheme of type, it relates generally to a kind of intelligent enzyme reaction work station for being used to release zooblast attaching.
Embodiment of the present utility model provides a kind of intelligent enzyme reaction work station for being used to release zooblast attaching, should
Equipment includes two cores：Ezyme bio-reactor and central control cabinet.In addition, the work station also includes auxiliary control mould
Block：Such as quick liquid in-out module, loop control module and spectral analysis module.
The various pieces of the work station have communication line to be connected with module, and wherein enzyme reactor power supply is controlled by center
Cabinet is provided, and in addition, central control cabinet is also powered to spectral analysis module, and quick liquid in-out module and loop control module
Individually connect power supply.
The main body of the enzyme reactor is a closed reaction zone, by closed tank body, retention component and mixing component group
Into.Closed tank body typically uses glass or 316L stainless steels, and it is generally cylindrical in shape, and ratio of height to diameter is less than 1:1.
Retort upper end is provided with closure, there is material access way on closure, and tank body side is provided with water leg, and
Chuck is by being connected to the temperature control electric blanket of central control cabinet.Tank base is again provided with closure, has material to enter on closure
Go out passage.
The bottom closure support of the enzyme reactor the component that dams.The component that dams is multi-layer circular stainless steel filtering net (2-10
Layer), use 20-200 mesh as needed.The stainless steel filtering net mounting means of retention component is with conventional art on the contrary, front
(above) is rough filter opening, and reverse side is then thin filter opening processed, the purpose for the arrangement is that reducing the congestion of cell.
The mixing component of the enzyme reactor uses upper magnetic agitation, and agitating shaft is entered by top cover labyrinth by bearing block
Tank body.Fluid in tank is calculated by CFD technologies, with reference to the physiological property of single-cell suspension state, stirring system in tank
Conventional flat leaf oar (blade tilt 30-60 degree) and three leaf high inclination-angle oars (blade tilt 30-60 degree) can be typically used, these
Blade can play a part of mixing for the less carrier of microcarrier isodensity, and then difficult for the higher mounting medium of density
With equilibrium shear power, the shearing force of carrier movement is promoted to frequently can lead to cellular damage.So enzyme reactor of the present utility model
Use lifting belt stirring system.Used elevator belt oar is preferably three-dimensional spiral structure, its blade inclination angle be 5.5~
24.5 °, preferably 15~22 °, the diameter of oar and the ratio of tank diameter are 1.0:1.35~1.0：1.95, preferably 1：1.6
~1.95, the mandatory lifting of low-density or high density carrier is arrived by mid blade, then the work for passing through centrifugal force by castering action
With throwing away oar area.Calculated by CFD and it was verified that this process only produces extremely low shearing force.Because it can be with Large Copacity
High density mounting medium is transported, more foregoing blade preferably serves the effect that mixing separates with cell medium.Stirring system
Driving is realized by servomotor, servomotor speed setting scope 20-200rpm, by power line and data wire with
Entreat switch board connection.
Temperature-controlling module involved by the enzyme reactor includes：(1) temperature of tank body is entered by tank body top cover labyrinth
Sensor；(2) chuck water system；(3) three parts of electric blanket heating system.Specifically, temperature sensor and central control cabinet
It is connected；Chuck water system also includes a water inlet and a delivery port, and corresponding disengaging taps and pipeline；Electric blanket
It is connected with central control cabinet, is provided power supply by central control cabinet and controlled.In temperature controlled processes, temperature electrode can be to center
Control system sends real time temperature detected value, if detected value is less than setting value, electric blanket can calculate according to PID principles to be added
Thermal power and heating jacket, jacket water (J.W.) is heated to retort internal heat transfer, and inside heating response tank, because retort is using
During internal liquid capacity regular can change, so in the guarantee retort that the heating system can be continually and steadily
Liquids and gases temperature stabilization is in setting value.
The enzyme reactor contains pH control assemblies, and it includes pH sensors and soda acid adds two parts of control, wherein
PH sensors enter tank interior by the pg-13.5 normal electrodes hole of top cover labyrinth, and press close to lower retention component, pass through
Data wire is connected with central control cabinet, the pH numerical value into central control system real-time Transmission retort.Soda acid is added by upper
Androgynous binary channels on closure is respectively enterd in retort.Acid solution is stored in external reagent bottle, can use high salt concentration
Acid, nitric acid, boric acid etc..Alkali lye is also stored in external reagent bottle, can use high-concentration sodium hydroxide, sodium acid carbonate, carbonic acid
Sodium etc..Mentioned reagent bottle is connected by silicone tube with androgynous binary channels respectively, and the small peristaltic pump for passing through central control cabinet
Control enters and flow velocity.
The enzyme reactor also contains pressure control component, and top cover labyrinth is equipped with respirator, entrance and row for gas
Go out.Other top cover labyrinth also contains a discharge duct, and the pipeline is equipped with pressure-regulating valve, can adjust gas according to pressure inside the tank
The rate of discharge of body.In the case where lacking quick liquid in-out module, user usually can to realize the purpose of quick fluid-discharge
The mode of pressure inside the tank is improved using compressed air increases discharge opeing speed, and the pressure-regulating valve may insure the pressure in retort
In safe range, and it is constant.
The enzyme reactor also contains online living cells Sensor monitoring component.The electrode passes through the pg- on top cover labyrinth
13.5 normal electrode interfaces are entered inside retort, and press close to retain component.Electrode is connected by data wire and central control cabinet
Connect.The component can be close to characterize free living cells by the voltage on at-once monitor retort inner cell yardstick and capacitance variations
Degree, and can on reaction medium cell detachment degree change, its data-signal fed back is by central control system conversion
Afterwards, it can be used as adjusting the most important index of every control parameter.
The enzyme reactor also contains weigh assembly, and it is located at the bottom of retort, is not in contact with any material, and
Connected by data wire with central control cabinet.The gross weight of weighing system Real-time Feedback reacting tank body, it is mainly used in measurement reaction
The disengaging liquid measure of tank body, and feedback regulation liquid in-out is opened and closed.
If the upper-lower seal lid of the enzyme reactor also contains dry material inlet and outlet piping.
The central control cabinet is the electrical control cabinet with industrial computer and execution component, and it is individual small-sized to be provided with 2-8
Peristaltic pump.The core component of the equipment is programmable logic controller (PLC) (PLC), and it is connected with host computer (touch-screen), control
The electrically-controlled component of whole.In addition, central control cabinet also includes such as lower component：
(1) motor transmitter, by being controlled in PLC, the operation of servomotor, the change of speed of agitator are controlled；
(2) 2-8 independent constant speed peristaltic pumps, by being controlled in PLC, flow velocity and the direction of silica gel liquid in pipe are controlled；
(3) electric blanket transmitter, by being controlled in PLC, heating power and the time of electric blanket are controlled with PID mode；
(4) pH electrodes transmitter, by being controlled in PLC, real-time Transmission pH electrode detection values；
(5) temperature electrode transmitter, by being controlled in PLC, real-time Transmission temperature electrode detected value；
(6) living cells electrode transmitter, by being controlled in PLC, real-time Transmission detected value；
(7) electronic-weighing transmitter, by being controlled in PLC, real-time Transmission detected value；
(8) transmitter being connected with each supplementary module etc., by being controlled in PLC, each module operation and parameter are controlled.
The host computer operating system that above-mentioned central control cabinet is developed by specific aim, realizes human-computer interaction.What can be implemented answers
Miscellaneous linkage function is as follows：
(1) mixing control：Change the rotating speed of mixing component in enzyme reactor according to setting value, and adjusted according to program setting
Section.
(2) pH is controlled：According to setting value, by the data of pH sensor feedbacks, peristaltic pump is controlled to adjust by PID mode
The addition of acid solution and alkali lye, pH value is rapidly achieved setting value, and reduce fluctuation, alarming value can be set.
(3) temperature control：According to setting value, by the data of temperature sensor feedback, electric blanket is controlled by PID mode
Heating power and the cycle, heating jacket, and further control enzyme reactor in temperature.
(4) enzyme reaction Process flowchart：According to the numerical value of living cells electrode-feedback, the process of simulation enzyme reaction progress, and journey
Corresponding assembly is closed in the startup of sequence, ensures the stability and accuracy of enzyme reaction process.
(5) liquid in-out controls：The numerical value fed back by electronic-weighing, peristaltic pump is controlled, makes liquid inlet volume and liquid outlet quantity with setting
Definite value is coincide.
Programming automation controls：By the way that parameter is previously set, the automatic running of each component is controlled, and monitors the stabilization of operation
Property, there is provided warning function.
The intelligent workstation can also strengthen function by supplementary module, and supplementary module has independent equipment and matched somebody with somebody
Part, is connected with central control cabinet or independent operating, conventional module have following several：
Quick liquid in-out module：It is made up of the regular collocation of the peristaltic pump of polytype and specification, for enzyme is biological
Liquid in reactor is quickly exported, while required liquid is quickly introduced wherein；The module is connected with central control cabinet
Connect, controlled by operation software.Because during enzyme reactor, the reaction time is very short, so the quick disengaging of liquid is closed very much
Key, the specification of peristaltic pump contained by the module are much larger than the peristaltic pump of central control cabinet, strengthen its function, ensure in not damaging cells
On the premise of activity, completion liquid relief as fast as possible, the process of input and output material are longer to cytoactive to reduce usual pipetting processes
The adverse effect brought.The time of liquid relief is normally controlled in 1~3min, and the time of input and output material is normally controlled in 2~5min.
(1) circulating water temperature control module：It is made up of circulation constant temperature case, governor impeller and pipeline, is transported independently of central control cabinet
OK, for ezyme bio-reactor more accurately temperature control, and reduce the temperature adjustment time；Because its can circulate offer from
16-40 DEG C of thermostatted water, the electric blanket mode of heating that compares can change the temperature in enzyme reactor faster, in addition, when needs
During fast cooling, the module can play a great role, and be adapted to work in Non-Air-Conditioned Buildings.
(2) spectral analysis module：It is made up of small-sized Raman spectrum detector and accessory, for being reacted in ezyme bio-reactor
The analysis of situation and feedback control；The module is connected with central control cabinet, can with the cell number in Real-time Feedback enzyme reactor,
The parameters such as state, the concentration of key substance, and do not influenceed by complex environment in tank body, data are more reliable and more stable, can be with auxiliary
The detection of living cells electrode is helped, strengthens the enzyme reactor process feedback control function of control system.
(3) tank pressure provides module：It is made up of small-sized air compressor and pressure-regulating valve and pipe fitting, for keeping in tank
The liquid in-out efficiency of malleation and enhancing enzyme reactor.The module independent operating, can be by into enzyme reactor using the module
The mode fast evacuation liquid of pressurization, can also quickly squeeze into liquid by way of being pressurizeed to fluid reservoir.Pressure-regulating valve is adjusted
Pressure is saved, and ensures pressure that tank body is born in zone of reasonableness.Used when the module and typically different quick liquid in-out module.
Independent computer monitoring module：Refer to computer system is separately configured, substitute work station host computer operating system, improve control
The convenience of system is, it is necessary to install control software.
In addition, also other modules, can be selected according to being actually needed.
Above-mentioned intelligent enzyme reactor work station, following several stages are mainly included using process：
(1) preparatory stage：Work station installation, debugging connect with pipeline.Prepare buffer solution, enzyme liquid and acid solution, alkali lye, culture
Base, each fluid reservoir, reagent bottle and the sterilizing of related ducts.
(2) the sterilization stage：Needing glass enzyme reactor is carried out offing normal sterilizing before use (can for stainless steel
To use online sterilization method), need to remove on servomotor, ducts valve and electrode data line etc. before sterilizing of offing normal,
And the pipe sealing that will be fixed on tank body, reacting tank body add 20%-50% ultra-pure waters, chuck needs to add 20%-50%
Pure water, the 121 DEG C of sterilizings of steam sterilization pan or steam sterilization cabinet are sent into, are reinstalled after taking-up, liquids and gases pipeline need to lead to
The sterile connection of snap joint is crossed, electrode needs to connect with central control cabinet by data wire, and liquid in-out silicone tube is installed to wriggling
Pump, and connect soda acid bottle.
(3) sterile erection stage：It is depressed into slightly just in use, first passing through the tank that tank pressure is provided in the constant enzyme reactor of module
Pressure, temperature in tank is on the one hand reduced, on the other hand checks tank body sealing.Start circulating water temperature control system, according to temperature electricity
Temperature is set in suitable numerical value by the numerical value of pole, is such as higher than setting value, then starts electric blanket heat temperature raising as needed, such as low
In setting value, then cooled using circulation water dispenser with water at low temperature.Adjusted after the water temperature in enzyme reactor reaches setting value in chuck
Circulating water temperature now only passes through temperature control electric blanket to setting value.
(4) first stage is cleaned：Liquid in-out control module is quickly discharged the liquid in tank body by big flow peristaltic pump,
And it is net using cleaning fluid flushing tank body heel row, it need not control temperature during being somebody's turn to do.Culture suspension (is contained in culture medium and covered with carefully
The mounting medium of born of the same parents) it is imported into by inlet in tank body, the data fed back according to weigh assembly are compared by system, are reached
Stop peristaltic pump after setting value, feed liquor stops, and system will start the discharge gate peristaltic pump of closure bottom, fast evacuation tank afterwards
Internal culture medium, and culture carrier will be trapped interception component in tank body.After liquid accompanies sky, system is again started up cleaning
Liquid peristaltic pump, tank body is entered by charging aperture, after amount to be weighed reaches setting value, system will start stirring group according to setting value
Part.Stir after opening, the numerical value that system will be fed back according to temperature electrode, start electric blanket, heating jacket water, make liquid in tank body
Temperature is rapidly achieved design temperature, generally cell culture temperature.After the completion of of short duration cleaning, stop stirring and temperature control, and quickly
Empty cleaning fluid.Above-mentioned steps repeat 1-3 times.
(5) the enzyme reaction stage：After stage to be cleaned terminates, system will open enzyme liquid and enter step, quick by peristaltic pump
Enzyme liquid is imported into tank body by single enzyme liquid import, weigh assembly control enters volume, stirring system low-speed running.With
This simultaneously, system simultaneously start two control, one side system by according to survey and setting pH value difference, pass through PID control
Pattern adds acid solution or alkali lye, quick regulation pH value to setting value；Another aspect system will be surveyed according to temperature and setting value
Gap, by PID control pattern heat electric blanket (temperature of enzyme liquid almost be respectively less than design temperature, such as need to cool, need to shift to an earlier date
Circulating temperature control module is opened and sets relatively low chuck water temperature).Said process should be completed in a short period of time.It is living thin
Born of the same parents' sensor or spectral analysis module start after starting at this stage, immediately monitoring enzyme reaction overall process, and dissociating, it is living thin to suspend
Born of the same parents' number initial value is extremely low and a period of time keeps stable, then gradually rises, and present and accelerate elevated situation.System will
Above-mentioned numerical value is analyzed, and according to the parameter of setting, starts the peristaltic pump of enzyme liquid passing away a moment, closes stirring, pH
Control and temperature control, enzyme liquid will be by quick emptying, and enters next stage.
(6) second stage is cleaned：Essentially identical with the first stage, system opens the peristaltic pump of cleaning fluid passage, cleaning fluid
Into appropriate volume and start stirring at low speed, temperature control is in cultivation temperature.The process is repeatable 1-3 times, living cells sensor
Or spectral analysis module complete monitoring loss cell situation.
(7) termination phase：After second stage to be cleaned is completed, system opens the peristaltic pump that culture medium enters passage, training
Support base to enter appropriate volume and start high-speed stirred, temperature control is in cultivation temperature.The process can be with last longer, typically
It is not further added by according to the free suspension cell number that living cells sensor or spectral analysis module monitor, or under cytoactive starts
It is reduced to termination.
(8) cell transition phase：After treating that cell separates with mounting medium, system opens the wriggling of cell transfering channel
Pump, culture medium carries cell and entered in new container, and mounting medium is stayed in enzyme reactor, realizes both sterile
Separation.
The technical scheme, its implementation process and principle etc. will further be solved with reference to accompanying drawing and some embodiments as follows
Release explanation.
Embodiment 1：The embodiment realizes PK-15 cells from 90 liters of biological respinses using the intelligent enzyme reactor work station
Device culture is transferred to the mode (referring to Fig. 2) of 300 liters of bioreactor cultures.The intelligent enzyme reactor work station such as Fig. 1 used
Shown, the working volume of enzyme reactor is 12 liters, and provisioned supplementary module includes quick liquid in-out module, circulation temperature control module
Control module is pressed with tank.Enzyme reactor uses conveyer belt oar, and diameter of propeller blade is 1.0 with tank diameter ratio:1.8.
90 liters of bioreactor culture PK-15 cells, volume of culture are 60 liters, and mounting medium is produced using GE companies
Cytodex-1,3 g/l, cell density is 2.5 × 106cells/mL.300 liters of 180 liters of bioreactor culture volumes, equally
Use 3 g/l, it is contemplated that inoculating cell density is 0.6 × 106cells/mL。
First stage：Related preparation, 100 liters of cleaning fluid is prepared, is contained in 100 liters of fluid reservoirs.Prepare enzyme liquid
10 liters of (0.25% pancreatin), 10 liters of asepsis feeding bottles are contained in, 30 liters of fresh culture, are contained in 100 liters of fluid reservoirs, culture medium
In contain 3% NBCS.Remaining sterile liquid storage pot, feed supplement bottle, silica gel pipeline and joint are some, sterile acid solution (1N HCl) and
Each 1 liter of alkali lye (3N NaOH).
Second stage：Enzyme reactor includes 3 liters of 18 megaohms of ultra-pure waters, and chuck contains 1 liter of 2 megaohms of deionized water, retains component
Using 3 layers of 150 mesh, 316 stainless steel filtering net.It is placed in 1 cubic metre of steam sterilization cabinet 121 DEG C to sterilize 45 minutes, pacifies again after taking-up
Dress, liquids and gases pipeline are connected by the sterile connection of snap joint, each electrode by data wire with central control cabinet, liquid in-out
Silicone tube is installed to peristaltic pump, and connects acid, alkali bottle.
Phase III：After installing, press offer module that pressure in tank body is increased into 0.1bar by tank.Start circulation
Water temperature control system, leaving water temperature is set as 36 DEG C, tank body temperature is set as 37 DEG C, and system is given by electric blanket heating jacket
Pot liquid heats up, and waits 10 minutes, treats that system is stable.
Fourth stage：The first stage is cleaned, liquid in-out control module is by big flow peristaltic pump quickly by the liquid in tank body
Body is discharged, and adds 5 liters of cleaning fluid, and it is net to rinse tank body heel row.The nutrient solution cultivated in 60 liters of bioreactors is settled first,
Supernatant is taken out, now the sterile connection of remaining about 10 liters of suspensions is transferred in tank body by bottom valve, the transfer process uses
Big flow wriggles this, after tank weight is not further added by, stops peristaltic pump, system wriggles the discharge gate for starting closure bottom
Pump, the culture medium in fast evacuation tank body, and microcarrier is intercepted in tank body.System is again started up cleaning fluid peristaltic pump afterwards,
Add 8 liters of cleaning fluids.Speed of agitator 60rpm is opened, temperature is set as 37 DEG C, and cleaning continues 5 minutes, stops stirring and temperature control,
And fast evacuation cleaning fluid.Above-mentioned steps are repeated twice.
5th stage：Enzyme reaction, after the stage to be cleaned terminates, system is quickly passed through 6 liters of enzyme liquids by peristaltic pump independent
Enzyme liquid import imported into tank body, speed of agitator 20rpm, set pH as 7.2, temperature be 37 DEG C.Living cells sensor is shown
Cell density is in low level, and after about 15 minutes, numerical value gradually rises, when living cells sensor values reach 0.6 through conversion ×
106After cells/mL, system starts the peristaltic pump of enzyme liquid passing away, closes stirring, pH is controlled and temperature control, enzyme liquid will be quick
Emptying.
6th stage：Cleaning second stage, the peristaltic pump of system unlatching cleaning fluid passage, 8 liters of cleaning fluids of addition, 20rpm,
Discharged after 37 DEG C, 5 minutes, the stage living cells sensor detects cell density such as more than 2.0 × 106Cells/mL, then clean
Terminate immediately, stop stirring and emptying immediately cleaning fluid.
7th stage：Enzyme reaction terminates, and after second stage to be cleaned is completed, system opens culture medium and enters the compacted of passage
Dynamic pump adds 10 liters of culture mediums, speed of agitator 100rpm, and temperature is set as 37 DEG C, and stirring lasts about 10 minutes, treats living cells
Sensor detection cell density reaches 11 × 106More than cells/mL is stable, or cytoactive begins to decline stopping.
8th stage：Cell transition phase, system open the peristaltic pump of cell transfering channel, and culture medium carries cell and led to
Cross the component that dams to enter in 300 liters of bioreactors, after supplementing culture medium, inoculating cell density is about 0.6 × 106cells/
ML, cell has been attached on new microcarrier after 12 hours.
The embodiment of embodiment 2 realizes production Porcine epidemic diarrhea virus using the intelligent enzyme reactor work station
Vero cells depart from from 300 liters of bioreactors with Cytodex-1 microcarriers, the mistake that the cell containing virion is collected
Journey (refers to Fig. 3).The intelligent enzyme reactor work station that uses is provisioned as shown in figure 1, the working volume of enzyme reactor is 18 liters
Supplementary module include quick liquid in-out module and tank pressure control module.Enzyme reactor uses three Ye Pingye oars, diameter of propeller blade with
Tank diameter ratio is 1.0:1.1.
200 liters of bioreactor culture Vero cells, volume of culture are 150 liters, and incubation time is to connect after poison 96 hours, are carried
Body medium uses Cytodex-1, and 3 g/l, total cell density is 2.0 × 106cells/mL.It is expected that collection TCS 2.0 ×
1011Cells, reclaim 430 grams of microcarrier.
First stage：Related preparation, 100 liters of cleaning fluid is prepared, is contained in 100 liters of fluid reservoirs.Prepare enzyme liquid
20 liters of (0.25% pancreatin), 20 liters of asepsis feeding bottles are contained in, virus 20 liters of liquid of protection, are contained in 20 liters of feed supplement bottles.Remaining
Sterile liquid storage pot, feed supplement bottle, silica gel pipeline and joint are some, sterile acid solution (1N HCl) and each 1 liter of alkali lye (3N NaOH).
Second stage：Enzyme reactor includes 5 liters of 18 megaohms of ultra-pure waters, and chuck contains 2 liters of 2 megaohms of deionized waters, retains component
Using 2 layers of 200 mesh, 316 stainless steel filtering net.It is placed in 1 cubic metre of steam sterilization cabinet 121 DEG C to sterilize 50 minutes, pacifies again after taking-up
Dress, liquids and gases pipeline are connected by the sterile connection of snap joint, each electrode by data wire with central control cabinet, liquid in-out
Silicone tube is installed to peristaltic pump, and connects soda acid bottle.
Phase III：After installing, press offer module that pressure in tank body is increased into 0.05bar by tank.In tank body
Temperature is set as 37 DEG C, and system is heated up by electric blanket heating jacket to pot liquid, is waited 10 minutes, treats that system is stable.
Fourth stage：Feed liquor is cleaned, liquid in-out control module is quickly arranged the liquid in tank body by big flow peristaltic pump
Go out, and add 5 liters of cleaning fluid, it is net to rinse tank body heel row.The nutrient solution cultivated in 200 liters of bioreactors is settled first, taken out
Fall supernatant, now about 35 liters of remaining suspension in tank body, by the way that in the sterile connection tank body of bottom valve, the transfer process uses big flow
This is wriggled, shifts 18 liters of suspensions for the first time, starts the discharge gate peristaltic pump of closure bottom, the culture in fast evacuation tank body
Base, and microcarrier is intercepted in tank body, is transferred to 17 liters of tank body again afterwards, is operated before repeating, by whole microcarriers and
Cell is transferred in enzyme reactor.System is again started up cleaning fluid peristaltic pump afterwards, adds 16 liters of cleaning fluids.Stirring is opened to turn
Fast 80rpm, cleaning continue 5 minutes, stop stirring and temperature control, and fast evacuation cleaning fluid, the process are repeated 2 times.
5th stage：Enzyme reaction, after the stage to be cleaned terminates, system is quickly passed through 15 liters of enzyme liquids by peristaltic pump independent
Enzyme liquid import imported into tank body, speed of agitator 40rpm, set pH as 7.0, temperature be 36.5 DEG C.Living cells sensor shows
Show that cell density is in low level, after about 5 minutes, numerical value gradually rises, when living cells sensor values reach 2.0 through conversion ×
106After cells/mL, system starts the peristaltic pump of enzyme liquid passing away, closes stirring, pH is controlled and temperature control, enzyme liquid will be quick
Emptying.
6th stage：Enzyme reaction terminates, and system opens 18 liters of virus protection liquid of peristaltic pump addition that culture medium enters passage,
Speed of agitator is 160rpm, and stirring lasts about 15 minutes, or treat living cells sensor detection cell density reach 10 ×
106More than cells/mL stably terminates.
7th stage：Collect, system opens the peristaltic pump of cell transfering channel, and virus protection liquid carries cell by cutting
Sterile the entering of stream component is collected in feed supplement bottle.Then enzyme reactor is opened, collects microcarrier.Complete separation.
It should be appreciated that above-described embodiment is only to illustrate technical concepts and features of the present utility model, it is ripe its object is to allow
Content of the present utility model can be understood and implement according to this by knowing the personage of technique, can not be limited with this of the present utility model
Protection domain.All equivalent change or modifications made according to the utility model Spirit Essence, should all cover of the present utility model
Within protection domain.
Claims (10)
1. a kind of ezyme bio-reactor, it is characterised in that including closed tank body, mixing component and in the vapor tight tank body
Retention component, the mixing component includes agitating shaft and the agitating paddle that is fixedly connected on the agitating shaft, the agitating shaft
Coordinate with the bearing block at the top of the closed tank body, the agitating paddle is distributed in the closed tank inner chamber, described to stir
Mix oar and use the elevator belt oar with three-dimensional spiral shape structure, the height of the closed tank body is less than the straight of the closed tank body
The ratio between internal diameter of footpath, the diameter of the elevator belt oar and the closed tank body is 1.0:1.35~1.0：1.95, the elevator belt
The blade inclination angle of oar is 5.5~24.5 °.
2. ezyme bio-reactor according to claim 1, it is characterised in that：The closed tank body is mainly by tank body and difference
Formed with the upper and lower closure that tank body upper and lower end is tightly connected, be also associated with the upper and lower closure with the tank body
Material input channel, the material output channel of chamber connection.
3. ezyme bio-reactor according to claim 1, it is characterised in that：The retention component includes at least one layer of sieve plate
Or filter screen, at least one layer of sieve plate or filter screen are distributed in the closed tank inner chamber bottom, and the sieve plate or filter screen outer peripheral edge
With the closed inner tank wall around connection, the aperture of the filter opening of the sieve plate or filter screen is from top to bottom sequentially reduced in portion.
4. ezyme bio-reactor according to claim 3, it is characterised in that：The retention component is mainly sieved by more than two layers
Plate or filter screen are formed by stacking, and the filter opening aperture of any one layer of sieve plate or filter screen is less than distribution square sieve plate or filter screen thereon
Filter opening aperture.
5. ezyme bio-reactor according to claim 1, it is characterised in that：The ezyme bio-reactor also includes temperature
Control assembly, pH control assemblies, pressure control component, online living cells Sensor monitoring component and weigh assembly；
The temperature-controlling module includes the temperature sensor located at the tank inner chamber reaction zone, is attached to the tank wall
Chuck water system, and the electric blanket of the cladding chuck water system；
The pH control assemblies include the pH sensors for being arranged at the tank inner chamber reaction zone and connected with the tank inner chamber
Logical acid ＆ alkali liquid adds pipeline, and the acid ＆ alkali liquid, which is added, is distributed with the pump housing on pipeline；
The pressure control component includes the respirator being installed on the tank body and the exhaust connected with the tank inner chamber
Pipeline, the discharge duct are provided with pressure-regulating valve；
The online living cells Sensor monitoring component includes the electrode located at the tank inner chamber reaction zone, and the electrode is pressed close to
The retention component.
6. ezyme bio-reactor according to claim 5, it is characterised in that：The weigh assembly is located at the closed tank body
Bottom, the opening position that can not be in contact with any material.
A kind of 7. intelligent enzyme reaction work station, it is characterised in that including：Ezyme bio-reactor described in claim 5；In and
Entreat switch board, the central control cabinet include respectively with the mixing component, temperature-controlling module, pH control assemblies, air pressure control
The central control unit that component processed, online living cells Sensor monitoring component connect with weigh assembly.
8. intelligent enzyme reaction work station according to claim 7, it is characterised in that also include such as at least one in lower component
Kind：
Quick liquid in-out module, for the liquid in the enzyme reactor quickly to be exported, while required liquid is quickly led
Enter the enzyme reactor；
Circulating water temperature control module, the temperature in the ezyme bio-reactor is carried out to coordinate with the chuck water system
Regulation and control；
Spectral analysis module, for being monitored to the response situation in the enzyme reactor；
Tank pressure provides module, for regulating and controlling the air pressure in the enzyme reactor；
The quick liquid in-out module, circulating water temperature control module, spectral analysis module, tank pressure provide module also with it is described in
Entreat control unit connection.
9. intelligent enzyme reaction work station according to claim 8, it is characterised in that also include and the central control unit
Parallel arrangement of independent monitoring module, and the independent monitoring module is also controlled with the mixing component, temperature-controlling module, pH
Component processed, pressure control component, online living cells Sensor monitoring component, weigh assembly, quick liquid in-out module, circulating water temperature
Control module, spectral analysis module and tank pressure provide members or all components connection in module.
10. intelligent enzyme reaction work station according to claim 7, it is characterised in that the central control unit includes PLC,
The PLC is connected with touch screen.
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| CN201720508476.5U CN206843495U (en) | 2017-05-09 | 2017-05-09 | Ezyme bio-reactor and intelligent enzyme reaction work station |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609378A (en) * | 2018-12-20 | 2019-04-12 | 杭州洪扬生物工程有限公司 | A kind of bioreactor |
| CN109679850A (en) * | 2019-02-20 | 2019-04-26 | 美闺(长沙)医疗美容有限公司 | A kind of self living cells breeding apparatus |
| CN110079446A (en) * | 2019-02-28 | 2019-08-02 | 保定蕴生生物科技有限公司 | Microbial fermentation product monitors fermentor on-line |
-
2017
- 2017-05-09 CN CN201720508476.5U patent/CN206843495U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609378A (en) * | 2018-12-20 | 2019-04-12 | 杭州洪扬生物工程有限公司 | A kind of bioreactor |
| CN109679850A (en) * | 2019-02-20 | 2019-04-26 | 美闺(长沙)医疗美容有限公司 | A kind of self living cells breeding apparatus |
| CN110079446A (en) * | 2019-02-28 | 2019-08-02 | 保定蕴生生物科技有限公司 | Microbial fermentation product monitors fermentor on-line |
| CN110079446B (en) * | 2019-02-28 | 2022-05-17 | 保定蕴生生物科技有限公司 | Fermentation tank for on-line monitoring of microbial fermentation products |
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