CN104845883A - Apparatus and method for producing anti-Her-2 monoclonal antibodies through online industrially robust regulation of cell states - Google Patents
Apparatus and method for producing anti-Her-2 monoclonal antibodies through online industrially robust regulation of cell states Download PDFInfo
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Abstract
The present invention discloses a bioreactor cell culture apparatus capable of onlinely and continuously monitoring physiological and biochemical indexes in a variety of cell culture environments and directly regulating and controlling cell states (including cell cycle and the like), and a method for producing anti-Her-2 monoclonal antibodies through the apparatus. With the apparatus and the method of the present invention, the bacterial pollution risk during the cell culture process can be reduced, the stable cell growth environment can be maintained, the constant cell growth viability can be maintained, and the high-quality, high-efficiency and continuous anti-Her-2 monoclonal antibody production can be achieved.
Description
Technical field
The present invention relates in a kind of bioengineering field can on-line control and control the apparatus and method that cell state produces anti-Her-2 monoclonal antibody, particularly relating to one can every reason biochemical indicator in the cell culture environment of on-line industry Robustness (industrially robust) monitoring continuously, continous pouring bio-reactor and culturing bottle/bag system, and can the cell cultures of direct-on-line regulation and control cell state, amplification and the anti-Her-2 monoclonal antibody of preparation apparatus and method.
Background technology
Mammary cancer is a class disease instead of a kind of disease, it relate to that apoptosis is disorderly with the differentiation of unbalance, the cell of propagation, the exception of the multiple vital movement such as the functional disorder of oncogene and tumor repressive gene and coherent signal conducting system.HER2 has the oncogene of substantial connection as the transfer prognosis with mammary cancer, has become one of current study hotspot.
1987, Slamon etc. were in the research of 189 human breast carcinomas, and the reported first expression of proto-oncogene HER2 in mammary cancer cancer cells, points out that HER2 gene plays an important role in the biological behaviour and pathogenesis of mammary cancer.HER2, also referred to as c-erB2 or HER2, is made up of 922 VITAMIN B4,1382 cytosine(Cyt)s, 1346 guanines and 880 thymus pyrimidines.Proto-oncogene HER2 (i.e. Erb-B2) is positioned at karyomit(e) 17q21, and coding 185kDa has the growth factor receptors of transmembrane tyrosine kinase activity.Be made up of ectodomain (ECD), membrane spaning domain and intracellular domain (ICD) three part, ectodomain is mainly rich in halfcystine district by 2 ligand binding domains (RLD) and 2 and forms.HER2 monomer non-activity, must form dimer and could produce activation signals.HER2 heterodimer reduces the coupling of EGFR and cab1 greatly, decrease the degraded of EGFR in cell endocytic process, promote that EGFR is circulated back to cytolemma, make EGFR in cytolemma overexpression, accelerate the propagation of cell, cause tumour to be formed and increase accelerating.
HER2 participates in signal transduction by molecule in various kinds of cell, and its major downstream material comprises mitrogen-activated protein (MAP) kinases, PI3 kinases, C-src, Shc and Grb2 etc.; The intracellular signalling pathways of HER2 mediation mainly comprises mitogen activated protein kinase (MAPK) approach, phosphatidylinositol 3-kinase (PI-3K) and silk-threonine kinase (AKT) approach.Another signal pathway of HER2 mediation is the PI-3K/AKT approach played a significant role in cell survival.HER2 process LAN can activate AKT and Nuclear factor-кB (NF-к B), AKT and the NF-к B of continuous activation can cause anti-apoptotic cascade reaction, makes the resistance of cancer cells generation to TNF-α, reduces host to the defence capability of tumour.
Tutsi etc. follow up a case by regular visits to discovery to the tracking that 698 routine primary breast cancer patients have carried out continuing 54 months, and be no matter Lymph Node-negative or positive group, the amplification that single factor test or multiplicity all show HER2 gene is main prognostic indicator.Further, Salmon etc. think that HER2 is prognostic indicator more valuable than estrogen receptor, tumor size etc.The research such as Tutsi finds prognosis and the clinical efficacy that can combine both HER2 and ER to judge mammary cancer.HER2 gene is first factor being used for predicting chemotherapeutic efficacy.1994, after the patient that the first time such as Muss reports HER2 gene amplification or protein overexpression is better to the chemotherapy regimen effect containing Zorubicin (Doxorubicin), many research groups all carry out retrospective research in the world, confirm the relation of HER2 and Doxorubicin and have found the biological mechanism of this relation.Have HER2 to increase II, the III phase primary breast cancer patients that the research such as Mazouni 39 is just accepting neoadjuvant finds, to accept chemotherapy especially Trastuzumab treatment patient with breast cancer, the decline level of Serum HER2 ECD is replied relevant with pathology, points out the chemotherapeutic efficacy that Serum HER2 ECD level may be used for the primary breast cancer patients monitoring HER2 process LAN.
Herceptin is the Humanized monoclonal antibodies that a kind of recombinant DNA derives, and optionally acts on the position, extracellular of human epidermal growth factor receptor-2 (HER2).This antibody belongs to IgGl type, containing the framework region of people, and the complementary determining region of the mouse-anti-p185HER2 antibody that can be combined with HER-2.Humanized Anti-HER 2 is produced by outstanding mammalian cell (middle CHO) of supporting in aseptic culture medium, with affinity chromatography and ion exchange methods, comprises the removal program of special inactivation of virus.State's hamster ovary cell HER2 proto-oncogene or C-erbB2 encode a single acceptor sample transmembrane protein, and molecular weight 185kDa is relevant to EGF-R ELISA in its structure.The patient HER2 overexpression of 25%-30% is observed in primary breast cancer patients.The result of HER2 gene amplification is that these tumor cell surfaces HER2 protein expression increases, and causes HER2 receptor activation.Research shows, the tumour patient of HER2 overexpression is short compared with the disease free survival phase without overexpression.The overexpression of HER2 is diagnosed by following methods: evaluation assessment tumor tissue being turned to basis with immune group, the ELISA method of tissue or plasma sample or fluorescence in situ hybridization (FISH).
Herceptin in vitro and in experimentation on animals all display can suppress the propagation of the tumour cell of HER2 overexpression.In addition, Herceptin is the potential medium of the cell-mediated cytotoxic reaction (ADCC) of antibody-dependant.In vitro in research, the ADCC of Herceptin mediation is proved to be in the cancer cells of overexpression more non-than HER2 in the cancer cells of HER2 overexpression and more preferably produces.
The following protection with regard to the relevant patent application of Herceptin product of Genentech company:
(1) a kind of monoclonal antibody that can grow with HER2 receptor extracellular region specific binding, inhibition tumor cell and this its in the application in the cancer patient for the treatment of overexpression HER2 acceptor.
(2) one can with HER2 receptor extracellular region specific binding, make overexpression HER2
The monoclonal antibody that the tumour cell of acceptor is more responsive to cytotoxic factor.
(3) method of the cancer patient of overexpression HER2 acceptor is treated, comprise to above-mentioned patient take significant quantity cytotoxic factor and can with HER2 receptors bind, the antibody that makes the tumour cell of overexpression HER2 acceptor more responsive to cytotoxic factor, to eliminate or to reduce the tumour of patient.
(4) cytotoxic factor and antibody antigen are in conjunction with the application of residue in the claims.
The technology that Genentech company is just relevant to Herceptin product has applied for patent protection in the U.S., Japan, Canada, and concrete patent is: JP3502885, US5677171,
US5720937、US5720954、US5725856、US5770195、US5772997、
JP11255666、JP11335297、JP3040121、CA1341082、US6165464
、US6387371、US6399063、US2002192211。But be directed to the details of antibody technique, too much do not relate to.
With regard to the commercial processes of anti-Her-2 monoclonal antibody, modem animal bioreactor culture expression technology is the key core technology of the said products, and this technical matters is complicated, difficulty is large.The recombinant protein production technology of these advanced persons rests in external minority drugmaker hand, helps these transnational companys to rake in enormous profits.And China differs greatly than world bleeding edge, it is the gap of the globality industry involving a series of technology.Therefore, we not only need to catch up with fast, narrow the gap, and more need to accelerate technical research, obtain great-leap-forward development in association area, form the core key technology of the anti-Her-2 monoclonal antibody production really with independent intellectual property right.
At present, the production expression method of anti-Her-2 monoclonal antibody of recombinating is generally and adopts batch cultivation, Fed batch fementation etc.Although these recombinant protein production technique are amplified to more than 1000L, but technique itself exists following shortcoming: 1) cultivate, it is less to express the state modulator of process, can not completely or can only the physico chemical factor of control culture environment of partial extent as temperature, pH, CO
2and O
2concentration, nutrient concentrations etc.; 2) even if accurately controlled reason, raw, change condition by reactor, but this control method still lacks fine adjustment to cell state itself and control.The cell state be in growth and protein process greatly can affect final recombinant proteins of expressing (as the level of glycosylation of protein, the correct formation of protein secondary, tertiary structure), and cell state is not merely can be determined the impact of cell by parameters such as above-mentioned reason, life, changes, for the albumen that anti-Her-2 monoclonal antibody is such, the protein-active that production obtains is that the protein mass far away than simple is important more; 3) repeatability of the cell of batch cultivation acquisition is lower, and be greatly subject to the impact of different experiments and production technology personnel individual operation, there will be difference (batch to batch variation) between larger batch in test with in producing, this is also one of difficult problem of current medicine quality control and supervision area.Therefore, a kind of (robust) with easy reliability large-scale (scalable) can be applied to the bottleneck that industrialization (industrial) anti-Her-2 monoclonal antibody is current expression process (processdevelopment) research field.
In addition, describing cell for traditional technology cell state " good " and " bad " is a kind of colloquial expression, and " lag period ", " logarithmic phase " and " plateau " etc. speech be also a comparatively general concept, not accurately and accurate.According to the result of study of open report and research department of our company, the state of Growth of Cells and the cell cycle distribution of whole colony have certain dependency.The behavior of the significant difference shown in fission process according to cell, cell cycle can be roughly divided into three phases (Fig. 1 (image credit in: Hwww.newearthbiomed.orgH)): gap 1 phase(G1 phase, the G1 phase) be the initial period of a cell cycle, in this stage, cell interior carries out the behaviors such as the synthesis of related protein, cell volume and genome do not produce any change, subsequently, cell can judge whether across restriction point (restrictionpoint) by the environment residing for self, enter DNA synthesis phase (DNA synthesis phase, S phase, the S phase), in the S phase, the synthesis that cell mainly carries out autogene group DNA copies, after copying, cell becomes 2 times of bodies, namely genome is double, cell enters m period (gap 2/mitotic phase subsequently, G2/M phase, the G2/M phase), phase at this moment, cell completes mitotic division, obtain two identical daughter cells, complete a cell cycle.When environment residing for cell is not good be unfavorable for Growth of Cells time, cell cannot stride across restriction point, thus jumps out the cell cycle, enters an extra quiet period (resting phase, G0 phase), becomes not somatoblast.According to the cell behavior of cell at different cycles, detect nuclear DNA content by nuclei dyeing color reagent, coordinate flow cytometer and the cell cycle residing for distinguishable individual cells and draw the cell cycle distribution figure of whole colony.For the cell in normal culturing process, cells intact is an asynchronous culture system (asynchronous culture), namely in culturing process, cells intact is not in the same position of cell cycle, but is randomly dispersed in each position of cell cycle.The characteristic shown in protein process due to the cell being in cell cycle different times may not be identical, therefore the possibility of result that cells intact presents is in fact by being in the different cell cycle, and the cell cycle distribution of the dynamic change of the proportion of the cell of different states-namely causes.
Summary of the invention
Because cell growth state itself greatly affects final recombinant proteins of expressing, and traditional cell cultures and protein expression are as batch cultivated and lack for the control of cell state is relative in feed-batch culture technique, while cell state be not merely can be determined the impact of cell by above-mentioned physical and chemical parameter.Therefore; realize cell growth state (i.e. Growth of Cells; the design parameters such as cell cycle distribution) accurate control, be set up the Robustness (robust) with easy reliability large-scale (scalable) important process of industrialization (industrial) anti-Her-2 monoclonal antibody expression process (process development) to be applied to.
The object of the invention is to, for the above-mentioned shortcoming in current monoclonal antibody conventional preparation techniques, there is provided one both can online, continuously, multiple reason biochemical indicator in monitoring cell culture environment, again can directly regulation and control cell state (comprising the cell cycle etc.) bio-reactor class cell culture apparatus and produced the method for anti-Her-2 monoclonal antibody by this device, it is made to reduce microbiological contamination risk, maintain Growth of Cells ambient stable, keep cell consistent growth vigor, reach high-quality, object that high-efficiency and continuous produces anti-Her-2 monoclonal antibody.
Describing cell for traditional technology cell state " good " and " bad " is a kind of colloquial expression, and " lag period ", " logarithmic phase " and " plateau " etc. speech be also a comparatively general concept, not accurately and accurate.The present invention uses specific cell growth rate (μ) as the kinetic parameter accurately describing Growth of Cells, can reflect cell state to a certain extent.According to the result of study of contriver, this growth velocity equation and cell cycle distribution, cell residence time within the cell cycle are substantial connection, regulated, can realize the target of accuracy controlling cell state by specific device composition linked system.
Foregoing invention object of the present invention is achieved through the following technical solutions:
A kind of on-line industry Robustness regulation and control cell state produces the device of anti-Her-2 monoclonal antibody, in order to taxis regulation and control cell state, this device at least comprises: nutrient solution hold-up vessel, bioreactor can be monitored, harvest product tank, some reason biochemical indicator detectors, online monitoring system, cell state control set for adjusting and linked system, cell retention device, some fluid sterility control speed pushers and some controlled fast pipeline systems, wherein, described nutrient solution hold-up vessel is connected with described bioreactor of monitoring through described fluid sterility control speed pusher by described controlled fast pipeline system, described cell retention device can monitor bioreactor described in connecting, described cell state control set for adjusting and linked system monitor bioreactor through described fluid sterility control speed pusher selectivity with described by described controlled fast pipeline system or described cell retention device is connected, described cell state control set for adjusting and linked system connect described harvest product tank by described controlled fast pipeline system through described fluid sterility control speed pusher, described bioreactor of monitoring connects described online monitoring system by described reason biochemical indicator detector, described online monitoring system is connected with described fluid sterility control speed pusher and sends controls fast instruction to described fluid sterility control speed pusher, above-mentioned all container class devices are all connected by controlled fast pipeline system, wherein, described cell state control set for adjusting and linked system are used for the described regulation and control monitoring different specific cell growth rate and different cell state in bioreactor.
Preferably, described cell retention device comprises concentrating cells outlet and harvested cell outlet, described cell state control set for adjusting and linked system can be selected and one be monitored the tank body of bioreactor with described, the concentrating cells of described cell retention device exports or the harvested cell of described cell retention device exports and is connected, and described cell state control set for adjusting and linked system at least comprise three kinds of work linked manner:
The first, when described cell state control set for adjusting and linked system and the concentrating cells of described cell retention device export be connected time, cell concentrated in described cell retention device can be discharged by the waste discharge cytostome on cell retention device by it, to reach the described regulation and control can monitoring different specific cell growth rate and different cell state in bioreactor;
The second, when described cell state control set for adjusting and linked system are directly connected with the described tank body monitoring bioreactor, the described cell monitored in bioreactor can be discharged by the described waste discharge mouth monitored on bioreactor, to reach the described regulation and control can monitoring different specific cell growth rate and different cell state in bioreactor by it;
The third, when described cell state control set for adjusting and linked system and the harvested cell of described cell retention device export be connected time, the cell of different concns can be discharged by the harvested cell outlet as the cell retention device of waste discharge cytostome when the difference of described cell retention device retains efficiency state by it, to reach the described regulation and control can monitoring different specific cell growth rate and different cell state in bioreactor.
In the preferred embodiment of the present invention, described cell state control set for adjusting and linked system comprise the regulator control system of cellular segregation reflux unit and cell deliverying unit and cell state, described cellular segregation reflux unit is positioned on described cell retention device, and it controls cell return velocity by fluid sterility control speed pusher; Described cell deliverying unit selectivity is positioned at concentrating cells outlet, the harvested cell outlet of described cell retention device or describedly monitors on the cell relief outlet of bioreactor; The regulator control system of described cell state by the set point feedback of described online monitoring system to the sterile pipes speed adjuster of described cell deliverying unit, control the speed of cell discharge and the cell concentration of discharge, when after the discharge cell concentration that the cell concentration regulator control system that reaches described cell state of discharging provides, fluid sterility control speed pusher quits work.
The invention also discloses a kind of method that on-line industry Robustness regulation and control cell state produces anti-Her-2 monoclonal antibody, the method includes the steps of:
1) sterilizing is carried out to cell culture system, then in cell culture system, pass into aseptic PBS damping fluid or aseptic culture fluid, the whole cell culture system of wash cycles;
2) to inoculating cell suspension in cell culture apparatus, the elementary cultivation of cell is carried out;
3) when Growth of Cells increases the cell density 1 ~ 100 × 10 of applicable expression recombinant protein
6during individual cell/ml and cell viability more than 70%, according to the growth characteristics of culturing cell, the parameters of biochemical marker data setting online monitoring system is managed in the cell culture apparatus that the working volume of cell culture apparatus and online monitoring system detect, described online monitoring system is expressed process to the anti-Her-2 monoclonal antibody of perfusion culture and is automatically controlled, indices in cell culture apparatus is maintained the best reason biochemical condition of setting, and regulate the corresponding fluid sterility control speed pusher of cell state control set for adjusting and linked system, it is the optimum cell state of expressing anti-Her-2 monoclonal antibody by cell state regulable control, whole system is made to reach the index of stable state, simultaneously automatic monitoring and record whole cell cultures and anti-Her-2 monoclonal antibody expresses process,
4), after having produced, stop cultivating, product reclaims purifying.
Wherein preferred, described step 2) in, the inoculum density of inoculating cell suspension is 0.5 ~ 6 × 10
6individual cell/ml.
Preferably, described step 2) in, the cell inoculated is one of the following engineering cell for the production of anti-Her-2 monoclonal antibody: CHO, HEK293, BHK or Per are C.6.
Preferably, in described step 3), described reason biochemical marker data comprises cellular metabolism, growth velocity, Product formation balance regulation data, those data are obtained by on-line monitoring glucose, lactic acid, L-glutamic acid, paddy ammonia phthalein amine, oxygen consumption rate and pH value index, monitor specific cell growth rate and protein ratio generating rate simultaneously.
Preferably, in described step 3), the method detecting the reason biochemical marker data in cell culture apparatus is: make the nutrient solution in cell culture apparatus not have reason biochemical indicator detector set to detect, and the signal data after reason biochemical indicator detector is detected and changed returns to online monitoring system.
Preferably, in described step 3), the index that reason biochemical indicator detector set can detect at least comprises: temperature, pH value, dissolved oxygen, molten carbonic acid gas, glucose, lactic acid, cell density, ammonia index, liquid level.
Preferably, in described step 3), online monitoring system is expressed process to the anti-Her-2 monoclonal antibody of perfusion culture and is carried out automatically controlling to comprise: online monitoring system receives reason biochemical marker data and processes, then send instruction and automatically regulate the interpolation speed of fresh pre-treatment nutrient solution and the useless cell velocity of discharge, the reason biochemical indicator maintaining cell culture environment is the best reason biochemical condition of setting, wherein, the interpolation speed of fresh pre-treatment nutrient solution is the velocity of discharge and the waste discharge cytostome flow velocity sum of cell retention device upper liquid outlet.
Preferably, in described step 3), described the best reason biochemical condition at least comprises: nutrient solution adds the serum (v/v) of 10%-20%, if use serum-free medium, does not then add serum; Culture-liquid temp 34.5 DEG C ± 5.5 DEG C, pH value 7.0 ± 2.0, dissolved oxygen 10% ~ 65%, molten carbonic acid gas 5% ± 8%.
Preferably, in described step 3), the cell state index of online monitoring system regulable control at least comprises: specific cell growth rate, cell cycle distribution, cell cycle mean residence time.
Preferably, in described step 3), the optimum cell state of expressing anti-Her-2 monoclonal antibody at least comprises: G0/G1 cell cycle, cell phase accounts for 35% ~ 85%, S phase cell accounts for 15% ~ 70%, G2/M phase cell accounts for 1% ~ 40% and its summation is 100%, cell G0/G1 phase mean residence time 1 hour ~ 80 hours, S phase mean residence time 2 hours ~ 40 hours, G2/M phase mean residence time 4 hours ~ 20 hours.
Preferably, in described step 3), the index of the stable state of described whole system mainly refers to lactic acid generating rate, glucose consumption rate ratio, and the value of specific cell growth rate keeps basicly stable.
Preferably, in described step 3), automatic monitoring and record whole cell cultures and expression of recombinant proteins process is: setting online monitoring system records each index probe monitors data and equipment operating parameter automatically, and the self registering timed interval is 5 ~ 300 seconds, and stores data.
Preferably, in described step 4), judge that the standard of having produced is: Cell viability declines, or total cellular score declines obviously, or protein expression obviously reduces, the preliminary experiment before amplifying by technique is determined in conjunction with the service data of whole system.
Compared with prior art, beneficial effect of the present invention is as follows:
One provided by the invention both can online, continuously, multiple reason biochemical indicator in monitoring cell culture environment, again can directly the bio-reactor class cell culture apparatus of regulation and control cell state (comprising the cell cycle etc.) and the method for being produced by this device, anti-Her-2 monoclonal antibody production process is made to reduce microbiological contamination risk, maintain Growth of Cells ambient stable, keep cell consistent growth vigor, reach high-quality, object that high-efficiency and continuous produces anti-Her-2 monoclonal antibody.
Accompanying drawing explanation
Fig. 1 is cell cycle schematic diagram;
Fig. 2 is that the present invention realizes to produce the schematic diagram of a kind of embodiment of the device of anti-Her-2 monoclonal antibody by on-line industry Robustness (industrially robust) regulation and control cell state;
Fig. 3 is the elementary cultivation of the cell of example 1 of the present invention and secondary culturing process growth kinetics of cells figure;
Fig. 4 is the elementary cultivation of the cell of example 1 of the present invention and secondary culturing process cell cycle distribution histogram;
Fig. 5 is the secondary culturing process anti-Her-2 monoclonal antibody expression figure of example 1 of the present invention.
Embodiment
Above description general description content of the present invention; several specific embodiments below will more directly embody situation of the present invention; but it must be noted that; the object be illustrated is only used at this example enumerated; the present invention is not limited thereto; the changes that any person skilled in the art can think of, all should drop in protection scope of the present invention.That uses in following examples can produce the device of anti-Her-2 monoclonal antibody see Fig. 2 with control Chinese hamster ovary celI state by on-line control, it mainly comprises: nutrient solution hold-up vessel, bioreactor can be monitored, harvest product tank (in corresponding diagram supernatant liquor results tank), connect and be arranged on the some reason biochemical indicator detectors can monitored on bioreactor, online monitoring system (in corresponding diagram on-line monitoring terminal), (mode of operation comprises linked manner one for cell state control set for adjusting and linked system, linked manner two and linked manner three), cell retention device, some fluid sterility control speed pushers and some controlled fast pipeline systems, wherein, described nutrient solution hold-up vessel is connected with described bioreactor of monitoring through described fluid sterility control speed pusher by described controlled fast pipeline system, described cell retention device can monitor bioreactor described in connecting, described cell state control set for adjusting and linked system monitor bioreactor through described fluid sterility control speed pusher selectivity with described by described controlled fast pipeline system or described cell retention device is connected, described cell state control set for adjusting and linked system connect described harvest product tank by described controlled fast pipeline system through described fluid sterility control speed pusher, described bioreactor of monitoring connects described online monitoring system by described reason biochemical indicator detector, described online monitoring system is connected with described fluid sterility control speed pusher and sends controls fast instruction to described fluid sterility control speed pusher, above-mentioned all container class devices are all connected by controlled fast pipeline system, wherein, described cell state control set for adjusting and linked system are used for the described regulation and control monitoring different specific cell growth rate and different cell state in bioreactor.It should be noted that the object of this schematic diagram 2 helps better to understand the present invention, and not show the form that apparatus and method of the present invention are only confined to this accompanying drawing and show.
Wherein, described cell retention device comprises concentrating cells outlet and harvested cell outlet, described cell state control set for adjusting and linked system can be selected and one be monitored the tank body of bioreactor with described, the concentrating cells of described cell retention device exports or the harvested cell of described cell retention device exports and is connected, described cell state control set for adjusting and linked system at least comprise three kinds of work linked manner, incorporated by reference to Fig. 2:
The first (in corresponding diagram 2 linked manner one), when described cell state control set for adjusting and linked system and the concentrating cells of described cell retention device export be connected time, cell concentrated in described cell retention device can be discharged by the waste discharge cytostome on cell retention device by it, to reach the described regulation and control can monitoring different specific cell growth rate and different cell state in bioreactor;
The second (in corresponding diagram 2 linked manner two), when described cell state control set for adjusting and linked system are directly connected with the described tank body monitoring bioreactor, the described cell monitored in bioreactor can be discharged by the described waste discharge mouth monitored on bioreactor, to reach the described regulation and control can monitoring different specific cell growth rate and different cell state in bioreactor by it;
The third (in corresponding diagram 2 linked manner three), when described cell state control set for adjusting and linked system and the harvested cell of described cell retention device export be connected time, the cell of different concns can be discharged by the harvested cell outlet as the cell retention device of waste discharge cytostome when the difference of described cell retention device retains efficiency state by it, to reach the described regulation and control can monitoring different specific cell growth rate and different cell state in bioreactor.
Described cell state control set for adjusting and linked system comprise the regulator control system of cellular segregation reflux unit and cell deliverying unit and cell state, described cellular segregation reflux unit can be cell refluxing opening, it is positioned on described cell retention device, and controls cell return velocity by fluid sterility control speed pusher; Described cell deliverying unit can be useless cell relief outlet, and its selectivity is positioned at the concentrating cells outlet of described cell retention device, harvested cell outlet or describedly monitors on the cell relief outlet of bioreactor; The regulator control system of described cell state by the set point feedback of described online monitoring system to the fluid sterility control speed pusher of described cell deliverying unit, control the speed of cell discharge and the cell concentration of discharge, when after the discharge cell concentration that the cell concentration regulator control system that reaches described cell state of discharging provides, fluid sterility control speed pusher quits work.
Wherein, specifically in the examples below, nutrient solution hold-up vessel is common cylinder or square body bottle structure, in order to hold aseptic culture medium, its outlet is tightly connected with described controlled fast pipeline system, and described nutrient solution hold-up vessel is communicated with air by the sterilised membrane filter strainer that interface that its tank body is arranged and described interface are arranged.
Can monitor bioreactor is general commercial bioreactor, possess multiple reason biochemical indicator probe interface, multiple liquid entrance and multiple gas inlet and outlet, can aseptically use or change fluidic interface or the gas interface of different number, enter to carry different liquid stream or gas or leave and described can monitor bioreactor, and different reason biochemical indicator probe groups can be selected.In addition, bioreactor can be monitored and also the quantity of liquid entrance or gas inlet and outlet according to actual needs can be set to one.In addition the self-control animal bioreactor possessing above-mentioned said function can also be used.
Reason biochemical indicator detector is all selected from commercial reason biochemical indicator detector, as detecting electrode, it possesses capacity switching signal, the reason biochemical indicator numbers translate that reason biochemical indicator sensor detects can be signal and transfer to described online monitoring system, each reason biochemical indicator detector is connected with described online monitoring system by electrode cable, wherein manages biochemical indicator detector and at least comprises pH value, temperature, dissolved oxygen, molten CO
2, osmotic pressure, cell density, glucose, lactic acid, ammonia index, liquid level detector one or more.In addition the self-control reason biochemical indicator detector possessing above-mentioned said function can also be used.
Fluid sterility control speed pusher is selected from commodity peristaltic pump, liquid-flow can be promoted with the speed of setting, flow velocity signal can be transferred to described online monitoring system, automatically regulate operating power to reach the object controlling different in flow rate or stop flowing by manual or described online monitoring system.In addition the controlled fast fluid forces device of the self-control possessing above-mentioned said function can also be used.
Online monitoring system is selected from commercial control tower, the function of the signal that it possesses the signal receiving described reason biochemical indicator sensor transmissions, described fluid control speed pusher transmits, and signal conversion processes function, reason biochemical indicator and fluid control speed pusher steering order send function, data logging memory function, and there is complete human-computer interaction interface.In addition the self-control online monitoring system possessing above-mentioned said function can also be used.
Controlled fast pipeline system forms pipeline system body by the ordinary silicon sebific duct meeting recombinant protein Production requirement and pipeline system body between any two container class devices is all with slides control speed folder or controls fast valve, described slip control speed folder or control fast valve and be located at related device exit.In addition, ordinary silicon sebific duct can also replace with high-molecular polymerization membrane material pipe.
In following examples, the on-line industry Robustness regulation and control cell state using said apparatus to carry out cell cultures produces the method for anti-Her-2 monoclonal antibody, comprises following steps:
1) sterilizing is carried out to cell culture system, then in cell culture system, pass into aseptic PBS damping fluid or aseptic culture fluid, the whole cell culture system of wash cycles;
2) to inoculating cell suspension in cell culture apparatus, the elementary cultivation of cell is carried out;
3) when Growth of Cells increases the cell density 1 ~ 100 × 10 of applicable expression recombinant protein
6during individual cell/ml and cell viability more than 70%, according to the growth characteristics of culturing cell, the parameters of biochemical marker data setting online monitoring system is managed in the cell culture apparatus that the working volume of cell culture apparatus and online monitoring system detect, described online monitoring system is expressed process to the anti-Her-2 monoclonal antibody of perfusion culture and is automatically controlled, indices in cell culture apparatus is maintained the best reason biochemical condition of setting, and regulate the corresponding fluid sterility control speed pusher of cell state control set for adjusting and linked system, it is the optimum cell state of expressing anti-Her-2 monoclonal antibody by cell state regulable control, whole system is made to reach the index of stable state, simultaneously automatic monitoring and record whole cell cultures and anti-Her-2 monoclonal antibody expresses process,
4), after having produced, stop cultivating, product reclaims purifying.
Wherein, described step 2) in, the cell inoculated is one of the following engineering cell for the production of anti-Her-2 monoclonal antibody: CHO, HEK293, BHK or Per are C.6; The inoculum density of inoculating cell suspension is 0.5 ~ 5 × 10
6individual cell/ml.
In described step 3), described reason biochemical marker data comprises cellular metabolism, cell growth rate, Product formation balance regulation data, those data are obtained by on-line monitoring glucose, lactic acid, L-glutamic acid, paddy ammonia phthalein amine, oxygen consumption rate and pH value index, monitor specific cell growth rate and protein ratio generating rate simultaneously; The method detecting the reason biochemical marker data in cell culture apparatus is: make the nutrient solution in cell culture apparatus not have reason biochemical indicator detector set to detect, and the signal data after reason biochemical indicator detector is detected and changed returns to online monitoring system; The index that reason biochemical indicator detector set can detect at least comprises: temperature, pH value, dissolved oxygen, molten carbonic acid gas, glucose, lactic acid, cell density, ammonia index.
In described step 3), online monitoring system is expressed process to the anti-Her-2 monoclonal antibody of perfusion culture and is carried out automatically controlling to comprise: online monitoring system receives reason biochemical marker data and processes, then send instruction and automatically regulate the interpolation speed of fresh pre-treatment nutrient solution and the useless cell velocity of discharge, the reason biochemical indicator maintaining cell culture environment is the best reason biochemical condition of setting, wherein, the interpolation speed of fresh pre-treatment nutrient solution is the velocity of discharge and the waste discharge cytostome flow velocity sum of cell retention device upper liquid outlet.
In described step 3), described the best reason biochemical condition at least comprises: nutrient solution adds the serum (v/v) of 10%-20%, if use serum-free medium, does not then add serum; Culture-liquid temp 34.5 DEG C ± 5.5 DEG C, pH value 7.0 ± 2.0, dissolved oxygen 10% ~ 65%, molten carbonic acid gas 5% ± 8%.
In described step 3), the cell state index of online monitoring system regulable control at least comprises: specific cell growth rate, cell cycle distribution, cell cycle mean residence time.
In described step 3), the optimum cell state of expressing anti-Her-2 monoclonal antibody at least comprises: G0/G1 cell cycle, cell phase accounts for 35% ~ 85%, S phase cell accounts for 15% ~ 70%, G2/M phase cell accounts for 1% ~ 40% and its summation is 100%, cell was G0/G1 phase mean residence time 1 hour ~ 80 hours, S phase mean residence time 2 hours ~ 40 hours, G2/M phase mean residence time 4 hours ~ 20 hours.
In described step 3), the index of the stable state of described whole system mainly refers to lactic acid generating rate, glucose consumption rate ratio, and the value of specific cell growth rate keeps basicly stable.
In described step 3), automatic monitoring and record whole cell cultures and expression of recombinant proteins process is: setting online monitoring system records each index probe monitors data and equipment operating parameter automatically, the self registering timed interval is 5 ~ 300 seconds, and stores data.
In described step 4), judge that the standard of having produced is: Cell viability declines, or total cellular score declines obviously, or protein expression obviously reduces.
Following examples some specific exampless for using on-line industry Robustness of the present invention (industrially robust) regulation and control Chinese hamster ovary celI state to produce the method for anti-Her-2 monoclonal antibody.
Example 1, can the apparatus and method of the anti-Her-2 monoclonal antibody of on-line industry Robustness (industrially robust) regulation and control Chinese hamster ovary celI state bio-reactor Restruction
The reagent used in this example and instrument as follows:
Nutrient solution: U.S. Sigma produces Excell CD CHO serum-free medium;
Cell: the Chinese hamster ovary celI of expressing the anti-Her-2 monoclonal antibody of restructuring stablizes strain;
Bioreactor can be monitored: the 7L zooblast reactor that Dutch Applicon company produces;
Online monitoring system: the control tower that Dutch Applicon company produces.
The method of producing anti-Her-2 monoclonal antibody in this example is as follows:
1) design temperature 37 DEG C on the control tower of Dutch Applicon company production, pH value 7.1, oxyty value 45%, molten gas concentration lwevel 5%, by heating jacket control temperature, the flow valve on control tower is control CO automatically
2, N
2, O
2the method of AIR tetra-tunnel air inlet bubbling oxygen supply controls oxyty and molten gas concentration lwevel, control tower is connected with the peristaltic pump control PH of 1mol/L NaOH and 1mol/LHCL, adds nutrient solution to the speed of peristaltic pump of cell culture apparatus can monitoring bioreactor according to the signal control of level electrode (control liquid level), the tank body can monitoring bioreactor is connected with the liquid inlet of supplementary fresh medium, this liquid inlet connects nutrient solution hold-up vessel, bioreactor can be monitored be connected with the cell state setting device of external placed type simultaneously, this cell state control set for adjusting comprises cellular segregation reflux unit and cell deliverying unit, the regulator control system of this cell state control set for adjusting and cell state constitutes cell state control set for adjusting and linked system jointly, cell state control set for adjusting is also connected to be expelled to by the supernatant liquor of results in this harvest product tank with harvest product tank (herein for tank gathered in the crops by supernatant liquor), complete assembly has been connected and installed latter 121 DEG C, and high pressure steam sterilization 20min is placed in sterilisable chamber, passes into aseptic PBS damping fluid, wash cycles whole system in system.Subsequently, discharge PBS, add fresh medium, circulation 24h, check whether microbiological contamination and system run all right.Check aseptic after, by nutrient solution process to set(ting)value, even with the index of the velocity interpolation nutrient solution of 80RPM;
2) the elementary cultivation of cell
With 1 × 10
6the density of individual cell/ml prepares cell suspension, and in 7L bio-reactor (working volume 5L), inoculating cell suspension is to final concentration 3 × 10
5individual cell/ml, cultivates 96 hours when not carrying out perfusion cultivation, treats that cell amplification is to 5 × 10
6open perfusion during the density of individual cell/ml, perfusion rate is set to 0.5 reactor tank body volume/sky (i.e. 2.5L/ days), continuation culturing cell 72 hours, culturing cell density to 9 × 10
6individual cell/ml, cell viability more than 95%, this result can see Fig. 3;
3) the secondary cultivation of cell and expressing protein
After elementary cultivation completes, design temperature 35 DEG C on control tower, pH value 7.0, oxyty value 35%, molten gas concentration lwevel 5%, carry out perfusion to cultivate and Restruction protein process, perfusion initial rate is set to 2 reactor tank body volume/skies (i.e. 10L/ days), data measured by glucose electrode carry out dynamic adjustments, glucose concn in nutrient solution is maintained 2g/L, the simultaneously online monitoring system fast pusher of fluid sterility control that regulates the cell refluxing opening (being one of embodiment of cellular segregation reflux unit) of cell state control set for adjusting and linked system to be connected with useless cell relief outlet (being one of embodiment of cell deliverying unit), useless cell relief outlet flow velocity is set to 1.5L/ days, be specific cell growth rate by cell state regulable control be 0.34d
-1s phase cell in cell cycle, G0/G1 phase cell and G2/M phase cell proportion be respectively 18.76%, 72.15% and 9.09%(refer to Fig. 4), cell is in the G0/G1 phase, the residence time of S phase and G2/M phase is respectively 25.91 hours, 12.75 hours and 5.7 hours, and cell density reaches 1.2 × 10
7individual cell/ml, cell viability more than 90% (referring to Fig. 4), harvest product tank cultivates with phase same rate results the supernatant liquor obtained accordingly, every day samples, to cell density, cell viability and expressing quantity detect, carry out secondary cultivation and the protein expression process of cell, simultaneously automatic monitoring and record whole cell cultures and protein expression production process, arranges control tower every 30 seconds record one secondary data and stores,
4) produced
According to the data of control tower record, after starting to pour into about 48 hours, the peristaltic pump rotating speed substantially constant of perfusion nutrient solution fluid inlet and liquid outlet, remain on 2 reactor tank body volume/skies, cell density maintains 1.2 × 10
7individual cell/ml, cell viability more than 90%, continuously perfused culture, after 10 days, after obtaining enough albumen, gathers in the crops whole supernatant liquor, stops the process of cell cultures and expressing protein.Carry out next step protein purification, quality test and preparation set-up procedure.And close online monitoring system, cleaning culture apparatus etc. simultaneously.Judge in this step that the standard of having produced is: Cell viability declines, or total cellular score declines obviously, or protein expression obviously reduces.
Find according to the physico-chemical property detected result of results albumen, gather in the crops the purity of albumen and activity is all cultivated with conventional batch or Fed batch fementation is similar, but because cell density and expression amount are better, so finally gathered in the crops more high reactivity high purity protein, protein yield has significantly improved.Refer to Fig. 5, use culture systems and the technique of this embodiment of the invention, regulate the irrigation rate of cell cultures, cell keeps good growth conditions, expressing protein from the 5th day, is efficiently maintained to the 30th day always, cytoactive is good, expression amount maintains higher state, and every system control parameters is good, belongs to higher level in the technique of report at present.
Apparatus and method provided by the invention not only can be used for the anti-Her-2 monoclonal antibody of the bioreactor processes Restruction based on stable recombinant cell strain as above-mentioned example 1, also may be used for various astable recombinant cell strain bioreactor processes (biological example reactor gene transfection transient expression process) the anti-Her-2 monoclonal antibody of Restruction simultaneously.Under the instruction of the present invention and above-described embodiment, those skilled in the art are easy to predict, cited or each raw material that exemplifies of the present invention or its equivalent alterations, each working method or its equivalent alterations can realize the present invention, and the parameter bound value of each raw material and working method, interval value can realize the present invention, do not enumerate embodiment at this.
Claims (15)
1. an on-line industry Robustness regulation and control cell state produces the device of anti-Her-2 monoclonal antibody, in order to taxis regulation and control cell state, it is characterized in that, this device at least comprises: nutrient solution hold-up vessel, bioreactor can be monitored, harvest product tank, some reason biochemical indicator detectors, online monitoring system, cell state control set for adjusting and linked system, cell retention device, some fluid sterility control speed pushers and some controlled fast pipeline systems, wherein, described nutrient solution hold-up vessel is connected with described bioreactor of monitoring through described fluid sterility control speed pusher by described controlled fast pipeline system, described cell retention device can monitor bioreactor described in connecting, described cell state control set for adjusting and linked system monitor bioreactor through described fluid sterility control speed pusher selectivity with described by described controlled fast pipeline system or described cell retention device is connected, described cell state control set for adjusting and linked system connect described harvest product tank by described controlled fast pipeline system through described fluid sterility control speed pusher, described bioreactor of monitoring connects described online monitoring system by described reason biochemical indicator detector, described online monitoring system is connected with described fluid sterility control speed pusher and sends controls fast instruction to described fluid sterility control speed pusher, above-mentioned all container class devices are all connected by controlled fast pipeline system, wherein, described cell state control set for adjusting and linked system are used for the described regulation and control monitoring different specific cell growth rate and different cell state in bioreactor.
2. on-line industry Robustness regulation and control cell state as claimed in claim 1 produces the device of anti-Her-2 monoclonal antibody, it is characterized in that, described cell retention device comprises concentrating cells outlet and harvested cell outlet, described cell state control set for adjusting and linked system can be selected and one be monitored the tank body of bioreactor with described, the concentrating cells of described cell retention device exports or the harvested cell of described cell retention device exports and is connected, and described cell state control set for adjusting and linked system at least comprise three kinds of work linked manner:
The first, when described cell state control set for adjusting and linked system and the concentrating cells of described cell retention device export be connected time, cell concentrated in described cell retention device can be discharged by the waste discharge cytostome on cell retention device by it, to reach the described regulation and control can monitoring different specific cell growth rate and different cell state in bioreactor;
The second, when described cell state control set for adjusting and linked system are directly connected with the described tank body monitoring bioreactor, the described cell monitored in bioreactor can be discharged by the described waste discharge mouth monitored on bioreactor, to reach the described regulation and control can monitoring different specific cell growth rate and different cell state in bioreactor by it;
The third, when described cell state control set for adjusting and linked system and the harvested cell of described cell retention device export be connected time, the cell of different concns can be discharged by the harvested cell outlet as the cell retention device of waste discharge cytostome when the difference of described cell retention device retains efficiency state by it, to reach the described regulation and control can monitoring different specific cell growth rate and different cell state in bioreactor.
3. on-line industry Robustness regulation and control cell state as claimed in claim 1 or 2 produces the device of anti-Her-2 monoclonal antibody, it is characterized in that, described cell state control set for adjusting and linked system comprise the regulator control system of cellular segregation reflux unit and cell deliverying unit and cell state, described cellular segregation reflux unit is positioned on described cell retention device, and it controls cell return velocity by fluid sterility control speed pusher; Described cell deliverying unit selectivity is positioned at concentrating cells outlet, the harvested cell outlet of described cell retention device or describedly monitors on the cell relief outlet of bioreactor; The regulator control system of described cell state by the set point feedback of described online monitoring system to the sterile pipes speed adjuster of described cell deliverying unit, control the speed of cell discharge and the cell concentration of discharge, when after the discharge cell concentration that the cell concentration regulator control system that reaches described cell state of discharging provides, fluid sterility control speed pusher quits work.
4. on-line industry Robustness regulation and control cell state produces a method for anti-Her-2 monoclonal antibody, it is characterized in that, comprises following steps:
1) sterilizing is carried out to cell culture system, then in cell culture system, pass into aseptic PBS damping fluid or aseptic culture fluid, the whole cell culture system of wash cycles;
2) to inoculating cell suspension in cell culture apparatus, the elementary cultivation of cell is carried out;
3) when Growth of Cells increases the cell density 1 ~ 100 × 10 of applicable expression recombinant protein
6during individual cell/ml and cell viability more than 70%, according to the growth characteristics of culturing cell, the parameters of biochemical marker data setting online monitoring system is managed in the cell culture apparatus that the working volume of cell culture apparatus and online monitoring system detect, described online monitoring system is expressed process to the anti-Her-2 monoclonal antibody of perfusion culture and is automatically controlled, indices in cell culture apparatus is maintained the best reason biochemical condition of setting, and regulate the corresponding fluid sterility control speed pusher of cell state control set for adjusting and linked system, it is the optimum cell state of expressing anti-Her-2 monoclonal antibody by cell state regulable control, whole system is made to reach the index of stable state, simultaneously automatic monitoring and record whole cell cultures and anti-Her-2 monoclonal antibody expresses process,
4), after having produced, stop cultivating, product reclaims purifying.
5. on-line industry Robustness regulation and control cell state according to claim 4 produces the method for anti-Her-2 monoclonal antibody, it is characterized in that, described step 2) in, the inoculum density of inoculating cell suspension is 0.5 ~ 5 × 10
6individual cell/ml.
6. on-line industry Robustness regulation and control cell state according to claim 4 produces the method for anti-Her-2 monoclonal antibody, it is characterized in that, in described step 3), described reason biochemical marker data comprises cellular metabolism, cell growth rate, Product formation balance regulation data, those data are obtained by on-line monitoring glucose, lactic acid, L-glutamic acid, paddy ammonia phthalein amine, oxygen consumption rate and pH value index, monitor specific cell growth rate and protein ratio generating rate simultaneously.
7. the on-line industry Robustness regulation and control cell state according to claim 4 or 6 produces the method for anti-Her-2 monoclonal antibody, it is characterized in that, in described step 3), the method detecting the reason biochemical marker data in cell culture apparatus is: make the nutrient solution in cell culture apparatus not have reason biochemical indicator detector set to detect, and the signal data after reason biochemical indicator detector is detected and changed returns to online monitoring system.
8. on-line industry Robustness regulation and control cell state according to claim 7 produces the method for anti-Her-2 monoclonal antibody, it is characterized in that, in described step 3), the index that reason biochemical indicator detector set can detect at least comprises: temperature, pH value, dissolved oxygen, molten carbonic acid gas, glucose, lactic acid, cell density, ammonia index.
9. on-line industry Robustness regulation and control cell state according to claim 4 produces the method for anti-Her-2 monoclonal antibody, it is characterized in that, in described step 3), online monitoring system is expressed process to the anti-Her-2 monoclonal antibody of perfusion culture and is carried out automatically controlling to comprise: online monitoring system receives reason biochemical marker data and processes, then send instruction and automatically regulate the interpolation speed of fresh pre-treatment nutrient solution and the useless cell velocity of discharge, the reason biochemical indicator maintaining cell culture environment is the best reason biochemical condition of setting, wherein, the interpolation speed of fresh pre-treatment nutrient solution is the velocity of discharge and the waste discharge cytostome flow velocity sum of cell retention device upper liquid outlet.
10. the on-line industry Robustness regulation and control cell state according to claim 4 or 9 produces the method for anti-Her-2 monoclonal antibody, it is characterized in that, in described step 3), described the best reason biochemical condition at least comprises: nutrient solution adds the serum (v/v) of 10%-20%, if use serum-free medium, then do not add serum; Culture-liquid temp 34.5 DEG C ± 5.5 DEG C, pH value 7.0 ± 2.0, dissolved oxygen 10% ~ 65%, molten carbonic acid gas 5% ± 8%.
11. on-line industry Robustness regulation and control cell states according to claim 4 produce the method for anti-Her-2 monoclonal antibody, it is characterized in that, in described step 3), the cell state index of online monitoring system regulable control at least comprises: specific cell growth rate, cell cycle distribution, cell cycle mean residence time.
12. on-line industry Robustness regulation and control cell states according to claim 4 or 11 produce the method for anti-Her-2 monoclonal antibody, it is characterized in that, in described step 3), the optimum cell state of expressing anti-Her-2 monoclonal antibody at least comprises: G0/G1 cell cycle, cell phase accounts for 35% ~ 85%, S phase cell accounts for 15% ~ 70%, G2/M phase cell accounts for 1% ~ 40% and its summation is 100%, cell was G0/G1 phase mean residence time 1 hour ~ 80 hours, S phase mean residence time 2 hours ~ 40 hours, G2/M phase mean residence time 4 hours ~ 20 hours.
13. on-line industry Robustness regulation and control cell states according to claim 4 produce the method for anti-Her-2 monoclonal antibody, it is characterized in that, in described step 3), the index of the stable state of described whole system mainly refers to lactic acid generating rate, glucose consumption rate ratio, the value of specific cell growth rate keeps basicly stable.
14. on-line industry Robustness regulation and control cell states according to claim 4 produce the method for anti-Her-2 monoclonal antibody, it is characterized in that, in described step 3), automatic monitoring and record whole cell cultures and expression of recombinant proteins process is: setting online monitoring system records each index probe monitors data and equipment operating parameter automatically, the self registering timed interval is 5 ~ 300 seconds, and stores data.
15. on-line industry Robustness regulation and control cell states according to claim 4 produce the method for anti-Her-2 monoclonal antibody, it is characterized in that, in described step 4), judge that the standard of having produced is: Cell viability declines, or total cellular score declines obviously, or protein expression obviously reduces.
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