CN105505852A - Method for screening high-tolerance cell line - Google Patents
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Abstract
The present application relates to bioengineering technology, and particularly relates to a method for screening a high-tolerance cell line. In one aspect, the method for screening the high-tolerance cell line is provided, and the method is as follows: selecting a growable cell line for in-vitro culture, changing cell culture conditions to force growth and metabolism of cells in poor growth environment, and then screening the high-tolerance cell line adapt to the poor growth environment, wherein the changed cell culture conditions comprise osmotic pressure, lactate concentration, ammonia concentration, pH, dissolved oxygen concentration or stirring speed of a cell culture solution, or any combination of two or more of the cell culture conditions. The high-tolerance cell line screened by the method is particularly applicable to large scale cultivation of a bioreactor.
Description
The divisional application of the Chinese invention patent application of " screening method of high resistant cells strain " that the application is application number is 201210051119.2, the applying date, to be March 1, denomination of invention in 2012 be.
Technical field
The application relates to biotechnology, is particularly suitable for screening and the cultural method of the high resistant cells strain of scale operation.
Background technology
The application of bio-reactor in zooblast pilot scale culture is also the development trend of field of biological pharmacy; a lot of biopharmaceutical company is all devoted to the zymotechnique developing related products, to meet the preparation that Products can carry out product in the extensive situation of low cost.Along with the whole world is to the increase of the demand of biological products, the scale of bio-reactor also constantly improves.Now, zooblast large scale culturing has also just become one of most important gordian technique of field of biological pharmacy.
But in the process that fermentation-scale constantly amplifies, the microenvironment of Growth of Cells has a very large change, and Growth of Cells is subject to remarkably influenced, even causes necrocytosis.When high-density cell growth acquires a certain degree, because himself characteristic is different, its processing parameter tolerance situation is also not quite similar.Along with the growth of cell density, main substrate redox metabolism aggravates, and mass transfer and heat transfer process need to adjust, and strengthening stirring and improving air flow is topmost means, the incident series of physical damage being cell and being inevitably subject to.A large amount of consumption of substrate can cause a large amount of generations of by product, have higher toxicity to various types of cells, and meanwhile, substrate exhaustion can cause Growth of Cells metabolic stasis and then cause a large amount of cell quick death.
Therefore, processing parameter is the major issue of restricted fermentation scale, and polytechnic exploitation depends on the change that cell strain can tolerate microenvironment parameters index, and screening obtains the high resistant cells strain in bio-reactor can seem particularly important.
Adopt high resistant cells strain; cell strain can not only be realized at serum-free, without the growth metabolism in albumen or chemical composition defined medium and Product Expression; simultaneously; significantly can also improve the growth metabolism function of cell strain; strengthen the various severe change of cell strain opposing microenvironment; realize controlling cell proliferation level, extending cell culture period with this, the target such as solve the restriction of mass-producing amplifying parameters, reduce costs, and then improve the usefulness of pilot scale culture.
Summary of the invention
The application relates to preparation and the screening method of the high resistant cells strain being suitable for scale operation.
The one side of the application provides a kind of screening method of high resistant cells strain, comprise: choosing can grown cell strain, carried out vitro culture, change cell culture condition thus force cell growth metabolism in bad growing environment, and then screening obtains the high resistant cells strain being adapted to described bad growing environment, it is characterized in that, the cell culture condition of described change comprises: the osmotic pressure of cell culture fluid, lactic acid concn, ammonia density, pH value, oxyty or stirring velocity, or two or more arbitrary combination of described cell culture condition.
In some embodiments, the screening method of the high resistant cells strain of the application comprises and changes two or more and be selected from the cell culture condition of following group: the osmotic pressure of cell culture fluid, lactic acid concn, ammonia density, pH value, oxyty and stirring velocity.In some embodiments, the cell culture condition described in three kinds or more can be changed.In some embodiments, the cell culture condition described in four or more can be changed.In some embodiments, five kinds or five kinds of above-described cell culture conditions can be changed.In some embodiments, six kinds or six kinds of above-described cell culture conditions can be changed.
If screen high resistant cells strain by changing or adjusting various kinds of cell culture condition, described cell culture condition can change simultaneously or adjust, also can successively change respectively or adjust, also can change by some culture condition, some culture condition changes separately simultaneously.Such as, in some embodiments, the osmotic pressure of cell culture fluid, lactic acid concn, ammonia density, pH value, oxyty and stirring velocity can be changed simultaneously, cell is cultivated, pick out the high resistant cells strain being adapted to the culture condition after changing.In some embodiments, culture condition can be changed successively by predetermined order in cell cultivation process, such as first change osmotic pressure, change lactic acid concn again, then change ammonia density, then change pH value, change oxyty again, change stirring velocity etc. again, such as first change osmotic pressure again, then change stirring velocity, change lactic acid concn and ammonia density etc. again, such as first change osmotic pressure and pH value again, then change stirring velocity, then change lactic acid concn and ammonia density etc.In some embodiments, have at least two kinds to change respectively in cell cultivation process in the cell culture condition changed, be not namely change simultaneously.Such as, in some embodiments, first change initial cell density, osmotic pressure, pH value and stirring velocity, and then change lactic acid concn and ammonia density.
In some embodiments, changing cell culture condition can be progressively improve osmotic pressure to make predetermined Growth of Cells parameter reach preestablished limit value.In some embodiments, with predetermined gradient, such as 50m Ω or 100m Ω, progressively improve osmotic pressure.In some embodiments, initial in nutrient solution osmotic pressure is about 280m Ω or 320m Ω.In some embodiments, the scope that in culture condition, osmotic pressure changes is 300 ~ 600m Ω.In some embodiments, in nutrient solution, osmotic pressure is adjusted to preestablished limit osmotic pressure value by disposable, such as 400m Ω, 500m Ω or 600m Ω, directly filter out high resistant cells strain that can survive under limit osmotic pressure value condition, that growth metabolism is in good condition.
In some embodiments, changing cell culture condition can be progressively reduce oxyty to make predetermined Growth of Cells parameter reach preestablished limit value.In some embodiments, with predetermined gradient, such as 5% or 10%, progressively reduce oxyty.In some embodiments, in nutrient solution, initial oxyty is 80% or 70%.In some embodiments, in nutrient solution the scope of oxyty at 10%-80%, 20% ~ 80% or 30%-80%.In some embodiments, in nutrient solution, oxyty is adjusted to preestablished limit oxyty value, such as 10% or 15% by disposable, directly filters out high resistant cells strain that can survive under limit oxyty condition, that growth metabolism is in good condition.
In some embodiments, changing cell culture condition can be progressively improve lactic acid concn to make predetermined Growth of Cells parameter reach preestablished limit value.In some embodiments, with predetermined gradient, such as 5mM, 10mM, 20mM, 40mM or 80mM, progressively lactic acid concn is improved.In some embodiments, in nutrient solution, initial lactic acid concn is 0mM.In some embodiments, in cell culture condition, the change scope of lactic acid concn is 0 ~ 60mM, 0 ~ 80mM, 0 ~ 100mM or 1 ~ 120mM etc.In some embodiments, in nutrient solution, lactic acid concn is adjusted to preestablished limit lactic acid concn value, such as 110mM or 120mM by disposable, directly filters out high resistant cells strain that can survive under limit lactic acid concn condition, that growth metabolism is in good condition.
In some embodiments, changing cell culture condition can be progressively improve ammonia density to make predetermined Growth of Cells parameter reach preestablished limit value.In some embodiments, with predetermined gradient, such as 2mM, 4mM, 6mM, 8mM, 10mM, progressively ammonia density is improved.In some embodiments, in nutrient solution, initial ammonia density is 0mM.In some embodiments, in cell culture condition, the change scope of ammonia density is 0 ~ 10mM, 0 ~ 15mM, or 0 ~ 20mM etc.In some embodiments, in nutrient solution, ammonia density is adjusted to preestablished limit ammonia density value, such as 10mM or 20mM by disposable, directly filters out high resistant cells strain that can survive under limit ammonia density condition, that growth metabolism is in good condition.
In some embodiments, changing cell culture condition can be that progressively raising lactic acid concn and ammonia density make predetermined Growth of Cells parameter reach preestablished limit value simultaneously.In some embodiments, changing cell culture condition can be progressively improve lactic acid concn, ammonia density and osmotic pressure to make predetermined Growth of Cells parameter reach preestablished limit value simultaneously.In some embodiments, changing cell culture condition can be progressively improve lactic acid concn, ammonia density and pH value to make predetermined Growth of Cells parameter reach preestablished limit value simultaneously.
In some embodiments, changing cell culture condition can be progressively improve pH value to make predetermined Growth of Cells parameter reach preestablished limit value.In some embodiments, in nutrient solution, initial pH value is 6.5.In some embodiments, in cell culture condition, the variation range of pH value can be 6.5 ~ 7.8.In some embodiments, in nutrient solution, pH value is adjusted to preestablished limit pH value by disposable, and such as 7.8, directly filter out high resistant cells strain that can survive under limit pH value condition, that growth metabolism is in good condition.
In some embodiments, changing cell culture condition can be progressively improve stirring velocity to make predetermined Growth of Cells parameter reach preestablished limit value.In some embodiments, progressively stirring velocity is improved with predetermined gradient, such as 40rpm, 50rpm, 60rpm, 70rpm, 80rpm, 90rpm, 100rpm, 110rpm, 120rpm, 130rpm, 140rpm, 150rpm.In some embodiments, the initial stirring velocity of nutrient solution is 40rpm, 60rpm, 80rpm or 100rpm.In some embodiments, in cell culture condition, the variation range of stirring velocity can be 40 ~ 150rpm, 40 ~ 200rpm, 40 ~ 250rpm, or 40 ~ 300rpm etc.In some embodiments, stirring velocity is adjusted to preestablished limit stirring velocity by disposable, such as 200rpm or 300rpm, directly filters out high resistant cells strain that can survive under limit stirring velocity condition, that growth metabolism is in good condition.
In some embodiments, high resistant cells strain can be screened under nutrition low consistency conditions.Such as, nutrient concentrations is lower than 50%, 30% or lower concentration of starter nutrient substrate concentration under this cell routine culture condition.Conventional starter nutrient substrate concentration can be such as, fresh
nutrient concentrations contained by 302 serum free mediums (JRHBIOSCIENCE company, article No.: 24326C) is fresh
nutrient concentrations contained by 620 serum free mediums (JRHBIOSCIENCE company, article No.: 14621C).
In some embodiments, high resistant cells strain can be screened under conditions of high cell density.Such as, cell density is higher than 10
7cell/mL, 2 × 10
7cell/mL or 5 × 10
7cell/mL.In some embodiments, the initial culture density of cell can be 0.5 ~ 10 × 10
5cell/mL, or 1 ~ 10 × 10
5cell/mL, or 2 ~ 10 × 10
5cell/mL, or 3 ~ 10 × 10
5cell/mL, or 4 ~ 10 × 10
5cell/mL, or 5 ~ 10 × 10
5cell/mL, or 9 ~ 10 × 10
5cell/mL etc.
In some embodiments, predetermined Growth of Cells metabolizing parameters refers to cytoactive, and preestablished limit value refers to that cytoactive is lower than about 60%, 50%, 40% or 30% etc.
In some embodiments, predetermined Growth of Cells metabolizing parameters refers to cell doubling time, and preestablished limit value cannot keep stable at the phalangeal cell multiplication phase, and such as colony's cell doubling time shortens or extends more than 40%, more than 50% or more than 60%.
In some embodiments, predetermined Growth of Cells metabolizing parameters is the Product Expression amount of phalangeal cell, and preestablished limit value refers to that cellular products expression amount is lower than 90%, 80%, 70% or 60% etc. of the expression amount under conventional culture conditions.
In some embodiments, further the cell obtained after change culture condition is screened by serum-free cloning method, obtain the high resistant cells strain that growth metabolism is stable.In some embodiments, after one or more cell culture conditions of change, carry out serum-free culture and screening, and then change cell culture condition further, again carry out serum-free culture and screening, hocket cultivation and screening like this that change culture condition.About the typical method of serum-free cloning can see No. CN200810150056.Xth, the Chinese patent of the people such as meter Li and Qu Ying, its publication date is on December 31st, 2008, and disclosed in this patent, full content is incorporated in the application by reference.
In some embodiments, described cell strain is cell lines.In some embodiments, described cell strain is mammalian cell strain, such as Chinese hamster ovary cell (CHO).In some embodiments, described cell strain is with effable restructuring foreign gene.In some embodiments, described restructuring exogene encodes foreign protein such as antibody protein.In some embodiments, high resistant cells strain can express recombinant protein.In some embodiments, the quality of recombinant protein expressed of high resistant cells strain with do not enter that the cell strain that screens expresses consistent.In some embodiments, the purity of recombinant protein expressed of high resistant cells strain and do not enter that the cell strain that screens expresses consistent or differ and be no more than 5%, 10% or 20%.
In some embodiments, the initiator cell strain that described screening method adopts is the cell strain that growth conditions is good.In some embodiments, the initiator cell strain that described screening method adopts is the cell being in logarithmic phase.In some embodiments, the initiator cell that described screening method adopts can be normally without the cell of height tolerance screening, also can be through the cell strain of one or many height tolerance screening.
In some embodiments, the cycle of screening can be 10 ~ 40 days, 20 ~ 40 days, 30 ~ 40 days etc.
Accompanying drawing explanation
Fig. 1 show embodiment 1 screen obtain high resistant cells strain with initiating cell strain under same culture conditions in substratum L-glutaminate concentration along with the changing conditions of time.
Fig. 2 show embodiment 1 screen obtain high resistant cells strain with initiating cell strain under same culture conditions in substratum glucose concn along with the changing conditions of time.
Fig. 3 show embodiment 1 screen obtain high resistant cells strain with initiating cell strain under same culture conditions cell density along with the changing conditions of time.
Fig. 4 show embodiment 1 screen obtain high resistant cells strain with initiating cell strain under same culture conditions cytoactive along with the changing conditions of time.
Embodiment
Below in conjunction with accompanying drawing, the application is described in further detail.
Embodiment described below is for the purpose of illustration and also not intended to be restriction only.When not departing from the spirit or scope of theme of the application, can adopt other embodiments, and can make other changes, all these is within the application's scope, and forms a part for teachings herein.
Embodiment 1: by the method for the high resistant cells strains of screening such as adjustment osmotic pressure, stirring velocity, pH, high-cell density and nutrient concentrations
1) cell strain and substratum
Initiator cell strain is the Chinese hamster ovary cell with target recombinant gene, this cell strain called after CERC-10 (the Fourth Military Medical University of P.L.A's cell engineering center construction).
Used fresh in the present embodiment
302 serum free mediums are purchased from JRHBIOSCIENCE company (article No.: 24326C).
Conditioned medium used in the present embodiment is fresh
302 serum free mediums and gained medium supernatant centrifugal after cell cultures mix with certain proportion and obtain.Such as, conditioned medium obtains by following steps: fresh by treating that culturing cell is inoculated into
in 302 serum free mediums, at 5%CO
2, saturated humidity incubator in be cultured to time or the cell density of expection, then obtain culture supernatant with 1200rpm centrifugal 5 minutes removing cells; By culture supernatant according to a certain percentage with fresh
302 serum free mediums are mixed to form conditioned medium.
The non-blood serum low density culture medium that in the present embodiment, serum-free cloning step is used is obtained by following steps: prepare
substratum based on 302 serum free mediums; Add Regular Insulin to 15mg/L, add Transferrins,iron complexes to 5mg/L, add insulin-like growth factor-i to 100ug/L, add thanomin to 10mol/L and add Sodium Selenite to 60nmol/L and prepare non-blood serum low density culture medium (described concentration is all the final concentration in the medium of these additives).
2) detection method and instrument
Cell density and cytoactive adopt trypan exclusion stain to detect, wherein, from substratum, cell is taken out at Different periods, by the cell suspension of equivalent and trypan blue dye liquor Homogeneous phase mixing, after 1 minute, the cell suspension dyeed is added in blood counting chamber, under an optical microscope viable cell and dead cell is counted respectively.Cytoactive=viable count/(viable count+dead cell number).
Adopt sandwich enzyme linked immunosorbant assay (ELISA) to detect expression product in supernatant liquor, wherein, adopt two section (purchased from Corning company, article No.: the XH329) bag of Fab by 96 orifice plates, concentration is 100 μ g/mL; Two anti-employing horseradish peroxidases (HRP) mark goat-anti people two anti-(purchased from PIERCE company, article No.: HC002), and concentration is 1:8000.Microplate reader is adopted to carry out reading value after nitrite ion colour developing.
Glucose concn and L-glutaminate concentration adopt Biochemical Analyzer (YSI Inc., YSI2700 model) to detect.
Bio-reactor adopts the B5L biological stirring aeration type bio-reactor of German Bei Lang company (B.BraunBiotech).Expression product in bio-reactor adopts AKTA protein purification system (GEHealthcareLifeSciences, AKTAexplorer model) to carry out recovery purifying by ProteinA affinity column.The purity of expression product uses high performance liquid chromatograph HPLC (Waters600) to detect, and the expression product purity be recovered to is about 95%.
3) following steps are adopted to set up high resistant cells strain:
1. 20 milliliters of CERC-10 cells are inoculated in 80 milliliters fresh
302 serum free mediums, cell initial density is 5 × 10
5about cells/ml, with sodium chloride solution, osmotic pressure is adjusted to 400m Ω, regulates pH to 7.0, be placed in 5%CO
2, cultivate in saturated humidity incubator, and rotating speed is set to 80rpm/min.
After 2.3 days, by previous step gained cell centrifugation, and collecting cell is diluted in conditioned medium (fresh
302 serum free mediums: the culture supernatant=1:1 of previous step gained is obtained by mixing), make cell density be adjusted to about 2 × 10
6cells/ml, adopts sodium-chlor that osmotic pressure is adjusted to 450m Ω, adopts 5mM sodium hydroxide to be 7.5 by pH regulator, be placed in 5%CO
2, cultivate in saturated humidity incubator, and rotating speed is set to 90rpm/min.
After 3.6 days, by previous step gained cell centrifugation, and collecting cell is diluted in conditioned medium (fresh
302 serum free mediums: the culture supernatant=1:3 of previous step gained is obtained by mixing), make cell density be adjusted to about 5 × 10
6cells/ml, adopts sodium-chlor that osmotic pressure is adjusted to 500m Ω, is placed in 5%CO
2, cultivate in saturated humidity incubator, and rotating speed is set to 100rpm/min.
After 4.9 days, by previous step gained cell centrifugation, and collecting cell is diluted in conditioned medium (fresh
302 serum free mediums: the culture supernatant=1:5 mixing of previous step gained), make cell density be adjusted to about 10 × 10
6cells/ml, adopts sodium-chlor that osmotic pressure is adjusted to 550m Ω, is placed in 5%CO
2, cultivate in saturated humidity incubator, and rotating speed is set to 110rpm/min.
After 5.12 days, by previous step gained cell centrifugation, and collecting cell is diluted in conditioned medium (fresh
302 serum free mediums: the culture supernatant=1:9 mixing of previous step gained), make cell density be adjusted to about 20 × 10
6cells/ml, adopts sodium-chlor that osmotic pressure is adjusted to 500m Ω, with 5mM hydrochloric acid, pH is adjusted to 6.8, is placed in 5%CO
2, cultivate in saturated humidity incubator, and rotating speed is set to 120rpm/min.
After 6.18 days, adopt limiting dilution assay to carry out serum-free cloning, cultivate 7-14 days.The method comprises the following steps: the D-poly-lysine using sterile pure water preparation 100mg/L, and room temperature bag, by 96 orifice plate 2h, uses aseptic water washing bag by hole 3 times; By the cell suspension of step 5 and non-blood serum low density culture medium according to the ratio serial dilution of 1:100 until cell density is 50 cells/ml, inoculate 24 holes with the ratio in 100 μ L/ holes; Continuing diluting cells suspension is 20 cells/ml to cell density, inoculates 24 holes with the ratio in 100 μ L/ holes; Continuing diluting cells suspension is 10 cells/ml to cell density, inoculates 48 holes with the ratio in 100 μ L/ holes; After eventually passing 7-14 days and cultivating, filter out growth and all desirable clone strain of Product Expression.Growth and all desirable clone strain of Product Expression should meet growth metabolism and stablize, the standard that the target product of expression is superior in quality, and basically identical with the growth metabolism of initiating cell strain.
7. examine under a microscope mono-clonal growing state, detect the expression of target product in supernatant liquor simultaneously with sandwich ELISA method, consider the result of growth and expression two aspects, screening grows and expresses all comparatively ideal tolerance clone strain.
8. prepare 5L bio-reactor and fresh
302 serum free mediums, join substratum fresh for 3L in reactor.
9. with about 5 × 10
5step 7 is screened the resistant cells obtained and is inoculated in above-mentioned 5L bio-reactor by the initial cell density of cells/ml, and starting rotating speed is 80rpm, pH is 7.5.
10. by rotating speed according to 80,90,100,110,120,130,140, the gradient of 150rpm constantly raises, and controls the speed stirred with different cell densities.Initial cell density is 5 × 10
5cells/ml to 1 × 10
6cells/ml, stirs and starts with 80rpm; Cell density reaches 5 × 10
6during cells/ml, stirring can be adjusted to 120rpm; Cell density reaches 1 × 10
7time more than cells/ml, stirring velocity can be adjusted to 150rpm.Stirring velocity between this scope can according to the adhesion situation (adhesion between cell and cell of cell, and cell is with the adhesion between bio-reactor inwall) adjust, such as when cell adhesion aggravation (such as occurred conglomeration, assemble or adhered to bio-reactor inwall), can consider to heighten stirring velocity.
11. reach 5 × 10 at cell density
6after cells/ml, progressively improve the concentration of lactic acid and ammonia every day, the concentration gradient of lactic acid is: 5,10,20,40,80mM, and the concentration gradient of ammonia is: 2,4,6,8,10mM.Lactic acid concn can be adjusted by 200mM lactic acid, and the concentration of ammonia can be adjusted by 200mM ammonium chloride.
12. continue cultivation makes cell density constantly raise, until cell density reaches about 2 × 10
7cells/ml.
13. constantly detect the cytoactive in 5L bio-reactor, when cytoactive drops to about 50%, and sterile sampling.
14. adopt limiting dilution assay to carry out serum-free cloning screening according to the identical mode of step 6, therefrom filter out growth and all desirable clone strain of Product Expression.
4) result:
Adopt the method for serum-free cloning to carry out the screening of high resistant cells strain subclone, its cloning efficiency is 33.7%, and positive rate 100%.As can be seen from accompanying drawing 1 and accompanying drawing 2, the height tolerance subcloned cells strain (being called for short " high resistant cells strain ") that the present embodiment screening obtains and initiating cell strain are basically identical on the accretion rate of L-glutaminate and glucose and trend under same culture conditions.In whole process of growth, substrate L-glutaminate and glucose maintain in setting interval range substantially, and glutamine concentration maintains about 1-4mM, and glucose maintains about 2-6g/L.Stream adds concentration in L-glutaminate and glucose 24 hours and all meets the ultimate value of setting, and it is 1mM that its GLN arranges lower bound, and it is 1g/L that glucose arranges lower bound.
As can be seen from accompanying drawing 3, the most high-cell density of high resistant cells strain and initiating cell strain is respectively about 9.3 × 10
6cells/ml and about 8.5 × 10
6cells/ml (all reaching most high-cell density for about the 5th day in cultivation), both are without significant difference.It can also be seen that from accompanying drawing 3, in logarithmic phase (about 1-4 days), high resistant cells strain and initiating cell strain in cell density without significant difference; The plateau (about 4-6 days) of high resistant cells strain, extends one day than initiating cell strain (about 4-5 days).
As can be seen from accompanying drawing 4, decline phase (within the 5th day or the 6th day, rising), the cytoactive lowering speed of high resistant cells strain is slower than the cytoactive lowering speed of initiating cell strain.In addition, the culture cycle of high resistant cells strain extends two days than the culture cycle of initiating cell strain, extends to 10 days from 8 days.According to ELISA detected result, high resistant cells strain reaches maximum the 10th day expression product concentration, is about 280.2mg/L.
Claims (17)
1. the screening method of one kind high resistant cells strain, comprise: choosing can grown cell strain, carried out vitro culture, change cell culture condition thus force cell growth metabolism in bad growing environment, and then screening obtains the high resistant cells strain being adapted to described bad growing environment, it is characterized in that, the cell culture condition of described change comprises: the osmotic pressure of cell culture fluid, lactic acid concn, ammonia density, pH value, oxyty or stirring velocity, or two or more arbitrary combination of described cell culture condition.
2. the method for claim 1, is characterized in that, changes the cell culture condition described in two or more.
3. method as claimed in claim 2, is characterized in that having at least two kinds to change respectively in the cell culture condition of described change.
4. the method for claim 1, is characterized in that, changing cell culture condition is progressively improve osmotic pressure to make predetermined Growth of Cells parameter reach preestablished limit value.
5. the method for claim 1, is characterized in that, changing cell culture condition is progressively improve lactic acid concn to make predetermined Growth of Cells parameter reach preestablished limit value.
6. the method for claim 1, is characterized in that, changing cell culture condition is progressively improve ammonia density to make predetermined Growth of Cells parameter reach preestablished limit value.
7. the method for claim 1, is characterized in that, changing cell culture condition is that progressively raising lactic acid concn and ammonia density make predetermined Growth of Cells parameter reach preestablished limit value simultaneously.
8. the method for claim 1, is characterized in that, changing cell culture condition is progressively improve pH value to make predetermined Growth of Cells parameter reach preestablished limit value.
9. the method for claim 1, is characterized in that, changing cell culture condition is progressively improve stirring velocity to make predetermined Growth of Cells parameter reach preestablished limit value.
10. the method for claim 1, is characterized in that, described cell culture condition is directly adjusted to predetermined value.
11. as claim 4 to 9 any one as described in method, it is characterized in that, described predetermined Growth of Cells parameter is cytoactive, and preestablished limit value refers to that cytoactive is lower than 50%.
12. as claim 4 to 9 any one as described in method, it is characterized in that, described predetermined Growth of Cells parameter be cell multiplication the phase, preestablished limit value be phalangeal cell multiplication the phase cannot keep stable.
13. the method for claim 1, is characterized in that, described cell strain is mammalian cell strain.
14. the method for claim 1, is characterized in that, screen further, obtain the high resistant cells strain that growth metabolism is stable by serum-free cloning method to the cell obtained.
15. methods as claimed in claim 14, is characterized in that, the product quality expressed by described high resistant cells strain is qualified.
16. the method for claim 1, is characterized in that, screen under the condition of low nutrition substrate concentration to described cell strain.
17. the method for claim 1, is characterized in that, screen under the condition of high-cell density to described cell strain.
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