CN102634476A - Method for screening high-tolerance cell strain - Google Patents
Method for screening high-tolerance cell strain Download PDFInfo
- Publication number
- CN102634476A CN102634476A CN2012100511192A CN201210051119A CN102634476A CN 102634476 A CN102634476 A CN 102634476A CN 2012100511192 A CN2012100511192 A CN 2012100511192A CN 201210051119 A CN201210051119 A CN 201210051119A CN 102634476 A CN102634476 A CN 102634476A
- Authority
- CN
- China
- Prior art keywords
- cell
- culture condition
- strain
- cell strain
- cell culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application relates to the technology of biological engineering, in particular to a method for screening high-tolerance cell strain. On one hand of the application, the application provides a method for screening the high-tolerance cell strain. The method comprises the following steps of: selecting growable cell strain, carrying out cultivation in vitro, and changing the cell culturing condition, so that the cell can grow and metabolize in a bad growth environment, therefore, the high-tolerance cell strain which is suitable for the bad growth environment can be obtained in a screening way, the cell culturing condition comprises the osmotic pressure, the lactic acid concentration, the pH value, the dissolved oxygen concentration or the stirring speed of the cell culture solution, or the cell culturing condition comprises any combination of at least two of the said conditions. The high-tolerance cell strain which is obtained by the application in a screening way is particularly suitable for the large-scale cultivation of the bioreactor.
Description
Technical field
The application relates to biotechnology, particularly is suitable for the screening and the cultural method of the height tolerance cell strain that scale operation uses.
Background technology
The application of bio-reactor in the zooblast mass-producing is cultivated also is the development trend of field of biological pharmacy; A lot of biopharmaceutical companys all are devoted to develop the zymotechnique of related prods, can under the extensive situation of low cost, carry out the preparation of product to satisfy Company products.Along with the increase of the whole world to the demand of biological products, the scale of bio-reactor also constantly improves.At this moment, animal cell large-scale is cultivated and has also just been become one of most important gordian technique of field of biological pharmacy.
But in the process that the fermentation scale is constantly amplified, very big variation has taken place in the microenvironment of cell growth, and the cell growth receives remarkably influenced, even causes necrocytosis.When the cell high-density growth acquires a certain degree, owing to himself characteristic difference, its processing parameter tolerance situation also is not quite similar.Along with the growth of cell density, main substrate redox metabolism aggravation, mass transfer and heat transfer process need be adjusted, and strengthening stirring and improving air flow is topmost means, and incident is a series of physical damnifications that cell inevitably receives.The mass consumption of substrate can cause a large amount of generations of by product, and various types of cells is had higher toxicity, and simultaneously, substrate exhaustion can cause the cell growth metabolism to stagnate and then cause a large amount of cells dead fast.
Therefore, processing parameter is the major issue of restricted fermentation scale, and polytechnic exploitation depends on the variation that cell strain can tolerate microenvironment parameters index, and screening obtains and can seem particularly important by the height tolerance cell strain in bio-reactor.
Adopt high tolerance cell strain; Growth metabolism and the product expression of cell strain in serum-free, no albumen or chemical ingredients defined medium not only can be realized, simultaneously, the growth metabolism function of cell strain can also be significantly improved; Strengthen the various abominable variation of cell strain opposing microenvironment; Realize controlling the cell proliferation level, prolong cell culture period with this, solve the restriction of mass-producing amplifying parameters, target such as reduce cost, and then improve the usefulness that mass-producing is cultivated.
Summary of the invention
The application relates to preparation and the screening method that is suitable for the height tolerance cell strain that scale operation uses.
The application's one side provides a kind of screening method of high tolerance cell strain; Comprise: but the grown cell strain chosen; It is carried out vitro culture; Force cell growth metabolism in bad growing environment thereby change cell culture condition, and then screen the height tolerance cell strain that obtains to be adapted to said bad growing environment, it is characterized in that; The cell culture condition of said change comprises: the osmotic pressure of cell culture fluid, lactic acid concn, ammonia concentration, pH value, oxyty or stirring velocity, or two or more arbitrary combination of said cell culture condition.
In some embodiments, the screening method of the application's height tolerance cell strain comprises that changing two or more is selected from following group cell culture condition: the osmotic pressure of cell culture fluid, lactic acid concn, ammonia concentration, pH value, oxyty and stirring velocity.In some embodiments, can change three kinds or three kinds of above-described cell culture conditions.In some embodiments, can change four or more described cell culture condition.In some embodiments, can change five kinds or five kinds of above-described cell culture conditions.In some embodiments, can change six kinds or six kinds of above-described cell culture conditions.
If through changing or adjusting the various kinds of cell culture condition and screen high tolerance cell strain; Said cell culture condition can change or adjust simultaneously; Also can divide successively to change respectively or adjustment, also can change simultaneously by some culture condition, some culture condition changes separately.For example, in some embodiments, can change osmotic pressure, lactic acid concn, ammonia concentration, pH value, oxyty and the stirring velocity of cell culture fluid simultaneously, cell is cultivated, pick out the height tolerance cell strain of the culture condition after being adapted to change.In some embodiments, in cell cultivation process, can change culture condition successively,, change lactic acid concn again such as changing osmotic pressure earlier by predetermined order; Change ammonia concentration again, change change pH values again, change oxyty again, change stirring velocity etc. again; Such as changing osmotic pressure earlier, change stirring velocity more again, change lactic acid concn and ammonia concentration etc. again; Such as changing osmotic pressure and pH value earlier, change stirring velocity more again, change lactic acid concn and ammonia concentration etc. again.In some embodiments, having two kinds in the cell culture condition that in cell cultivation process, changes at least and change respectively, promptly is not to change simultaneously.For example, in some embodiments, change initial cell density, osmotic pressure, pH value and stirring velocity earlier, and then change lactic acid concn and ammonia concentration.
In some embodiments, changing cell culture condition can be progressively to improve osmotic pressure to make predetermined cell growth parameter(s) reach the preestablished limit value.In some embodiments, the gradient with predetermined such as 50m Ω or 100m Ω, progressively improves osmotic pressure.In some embodiments, initial osmotic pressure is about 280m Ω or 320m Ω in the nutrient solution.In some embodiments, the scope that osmotic pressure changes in the culture condition is 300～600m Ω.In some embodiments; Osmotic pressure is by the disposable preestablished limit osmotic pressure value of adjusting in the nutrient solution; For example 400m Ω, 500m Ω or 600m Ω directly filter out and can under limit osmotic pressure value condition, tolerate cell strain by height that survive, that growth metabolism is in good condition.
In some embodiments, changing cell culture condition can be progressively to reduce oxyty to make predetermined cell growth parameter(s) reach the preestablished limit value.In some embodiments, the gradient with predetermined such as 5% or 10%, progressively reduces oxyty.In some embodiments, initial oxyty is 80% or 70% in the nutrient solution.In some embodiments, in the nutrient solution scope of oxyty at 10%-80%, 20%～80% or 30%-80%.In some embodiments, in the nutrient solution oxyty by the disposable preestablished limit oxyty value of adjusting to, for example 10% or 15%, directly filter out can be under the limit oxyty condition height tolerance cell strain that survive, that growth metabolism is in good condition.
In some embodiments, changing cell culture condition can be progressively to improve lactic acid concn to make predetermined cell growth parameter(s) reach the preestablished limit value.In some embodiments, the gradient with predetermined such as 5mM, 10mM, 20mM, 40mM or 80mM, progressively improves lactic acid concn.In some embodiments, initial lactic acid concn is 0mM in the nutrient solution.In some embodiments, the change scope of lactic acid concn is 0～60mM in the cell culture condition, 0～80mM, 0～100mM or 1～120mM etc.In some embodiments, lactic acid concn is by the disposable preestablished limit lactic acid concn value of adjusting in the nutrient solution, and for example 110mM or 120mM directly filter out and can under limit lactic acid concn condition, tolerate cell strain by height that survive, that growth metabolism is in good condition.
In some embodiments, changing cell culture condition can be progressively to improve ammonia concentration to make predetermined cell growth parameter(s) reach the preestablished limit value.In some embodiments, the gradient with predetermined such as 2mM, 4mM, 6mM, 8mM, 10mM, progressively improves ammonia concentration.In some embodiments, initial ammonia concentration is 0mM in the nutrient solution.In some embodiments, the change scope of ammonia concentration is 0～10mM in the cell culture condition, 0～15mM, or 0～20mM etc.In some embodiments, ammonia concentration is by the disposable preestablished limit ammonia concentration value of adjusting in the nutrient solution, and for example 10mM or 20mM directly filter out and can under limit ammonia concentration conditions, tolerate cell strain by height that survive, that growth metabolism is in good condition.
In some embodiments, changing cell culture condition can be progressively to improve lactic acid concn simultaneously to make the cell growth parameter(s) of being scheduled to reach the preestablished limit value with ammonia concentration.In some embodiments, changing cell culture condition can be progressively to improve lactic acid concn, ammonia concentration and osmotic pressure simultaneously to make predetermined cell growth parameter(s) reach the preestablished limit value.In some embodiments, changing cell culture condition can be progressively to improve lactic acid concn, ammonia concentration and pH value simultaneously to make predetermined cell growth parameter(s) reach the preestablished limit value.
In some embodiments, changing cell culture condition can be progressively to improve the pH value to make predetermined cell growth parameter(s) reach the preestablished limit value.In some embodiments, original ph is 6.5 in the nutrient solution.In some embodiments, the variation range of pH value can be 6.5～7.8 in the cell culture condition.In some embodiments, in the nutrient solution pH value by the disposable preestablished limit pH value of adjusting to, for example 7.8, directly filter out can be under the limit pH value condition height tolerance cell strain that survive, that growth metabolism is in good condition.
In some embodiments, changing cell culture condition can be progressively to improve stirring velocity to make predetermined cell growth parameter(s) reach the preestablished limit value.In some embodiments, progressively improve stirring velocity 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, the variation range of stirring velocity can be 40～150rpm in the cell culture condition, 40～200rpm, and 40～250rpm, or 40～300rpm etc.In some embodiments, stirring velocity is by the disposable preestablished limit stirring velocity of adjusting to, and for example 200rpm or 300rpm directly filter out and can under limit stirring velocity condition, tolerate cell strain by height that survive, that growth metabolism is in good condition.
In some embodiments, can under the nutrition low consistency conditions, screen high tolerance cell strain.Such as, nutrient concentrations is lower than 50%, 30% or lower concentration of initial nutrient concentrations under this cell routine culture condition.Conventional initial nutrient concentrations for example can be; Fresh EX-
302 serum free mediums (JRH BIOSCIENCE company; Article No.: 24326C) contained nutrient concentrations; Fresh EX-
620 serum free mediums (JRH BIOSCIENCE company, article No.: 14621C) contained nutrient concentrations.
In some embodiments, can under the high-cell density condition, screen high tolerance cell strain.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 cell growth metabolism parameter is meant cytoactive, and the preestablished limit value is meant that cytoactive is lower than about 60%, 50%, 40% or 30% etc.
In some embodiments, predetermined cell growth metabolism parameter is meant cell doubling time, and the preestablished limit value is the phalangeal cell multiplication phase can't keep stable, for example colony's cell doubling time shorten or prolong 40% or more, more than 50%, perhaps more than 60%.
In some embodiments, predetermined cell growth metabolism parameter is the product expression amount of phalangeal cell, and the preestablished limit value is meant that the cellular products expression amount is lower than 90%, 80%, 70% or 60% etc. of expression amount under the conventional culture condition.
In some embodiments, further screen, obtain the stable height tolerance cell strain of growth metabolism changing the cell that obtains behind the culture condition with serum-free cloning method.In some embodiments; After changing one or more cell culture conditions, carry out serum-free culture and screening, and then further change cell culture condition; Carry out serum-free culture and screening once more, hocketing like this changes the cultivation and the screening of culture condition.Can be about the typical method of serum-free cloning referring to people's such as Mi Li and Qu Ying Chinese patent CN200810150056.X number, its open day is on December 31st, 2008, the disclosed full content of this patent mode by reference is incorporated among the application.
In some embodiments, said cell strain is the zooblast strain.In some embodiments, said cell strain is a mammalian cell strain, encloses hamster ovary cell (CHO) in for example.In some embodiments, said cell strain has effable reorganization foreign gene.In some embodiments, said reorganization foreign gene encoding exogenous albumen antibody protein for example.In some embodiments, but high tolerance cell strain express recombinant protein.In some embodiments, the quality of the high tolerance cell strain recombinant protein of expressing and the cell strain expression of not advancing screening is consistent.In some embodiments, the purity of the high tolerance cell strain recombinant protein of expressing is with the consistent of the cell strain expression of not advancing screening or differ and be no more than 5%, 10% or 20%.
In some embodiments, the initiator cell strain that said screening method adopted is the strain of growth conditions good cell.In some embodiments, the initiator cell strain that said screening method adopted is the cell that is in logarithmic phase.In some embodiments, the initiator cell that said screening method adopted can be the cell that screens without the height tolerance normally, also can be the cell strain through the high tolerance screening of one or many.
In some embodiments, the cycle of screening can be 10～40 days, 20～40 days, and 30～40 days etc.
Description of drawings
Fig. 1 show height tolerance cell strain that embodiment 1 screening obtains with the initiating cell strain under same culture conditions in the substratum L-glutaminate concentration along with the change of time situation.
Fig. 2 show height tolerance cell strain that embodiment 1 screening obtains with the initiating cell strain under same culture conditions in the substratum glucose concn along with the change of time situation.
Fig. 3 show height tolerance cell strain that embodiment 1 screening obtains with the initiating cell strain under same culture conditions cell density along with the change of time situation.
Fig. 4 show height tolerance cell strain that embodiment 1 screening obtains with the initiating cell strain under same culture conditions cytoactive along with the change of time situation.
Embodiment
Below in conjunction with accompanying drawing the application is done further detailed description.
The embodiment of below describing is merely illustration purpose and is not to be intended to limit.Under the situation of the spirit of the theme that does not depart from the application or scope, other embodiments can be adopted, and other variations can be made, all these and constitutes the part of teachings herein within the application's scope.
Embodiment 1: through the method for the high tolerance of screening cell strains such as adjustment osmotic pressure, stirring velocity, pH, high-cell density and nutrient concentrations
1) cell strain and substratum
The initiator cell strain is the Chinese hamster ovary cell that has the target recombinant gene, this cell strain called after CERC-10 (The Fourth Military Medical University of P.L.A's cell engineering center construction).
Fresh EX-
302 serum free mediums used in the present embodiment are available from JRH BIOSCIENCE company (article No.: 24326C).
In the present embodiment used conditioned medium be fresh EX-
302 serum free mediums with cell cultures after centrifugal gained medium supernatant mix acquisition with certain proportion.For example, conditioned medium can obtain through following steps: will treat that culturing cell is inoculated into fresh EX-
In 302 serum free mediums, at 5%CO
2, saturated humidity incubator in be cultured to the time or the cell density of expection, removed cell in centrifugal 5 minutes with 1200rpm then and obtain culture supernatant liquid; Culture supernatant liquid is followed fresh EX-according to a certain percentage
302 serum free mediums mix the formation condition substratum.
The used non-blood serum low density culture medium of serum-free cloning step makes through following steps in the present embodiment: prepare EX-
302 serum free mediums as basic medium; Thereby 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 and prepare non-blood serum low density culture medium (said concentration all is the finally concentration in substratum of these additives) to 60nmol/L.
2) detection method and instrument
Cell density and cytoactive adopt the trypan blue exclusion method to detect; Wherein, From substratum, take out cell in the different periods, with the cell suspension and the trypan blue dye liquor uniform mixing of equivalent, after 1 minute; Painted cell suspension is added in the blood counting chamber, under opticmicroscope, viable cell and dead cell are counted respectively.Cytoactive=viable count/(viable count+dead cell number).
Adopt sandwich enzyme linked immunosorbant assay (ELISA) to detect the expression product in the supernatant, wherein, (available from Corning company, article No.: XH329) encapsulate 96 orifice plates, concentration is 100 μ g/mL to adopt the two sections of Fab; Two anti-adopt horseradish peroxidase (HRP) mark goat-anti people two anti-(available from PIERCE company, article No.: HC002), concentration are 1:8000.The ELIASA value of reading is adopted in colour developing liquid colour developing back.
Glucose concn and L-glutaminate concentration adopt Biochemical Analyzer (YSI Inc., YSI 2700 models) to detect.
Bio-reactor adopts moral to enclose the biological aeration type bio-reactor that stirs of B5L of Bei Lang company (B.Braun Biotech).Expression product in the bio-reactor adopts AKTA protein purification system (GE Healthcare Life Sciences, AKTAexplorer model) to reclaim purifying through Protein A affinity column.The purity of expression product uses high performance liquid chromatograph HPLC (Waters 600) to detect, and the expression product purity that is recovered to is about 95%.
3) adopt following steps to set up high tolerance cell strain:
With 20 milliliters of CERC-10 cell inoculations in 80 milliliters of fresh EX-
302 serum free mediums, cell initial density are 5 * 10
5About cells/ml, osmotic pressure is adjusted to 400m Ω, regulates pH to 7.0, place 5%C0 with sodium chloride solution
2, cultivate in the saturated humidity incubator, and rotating speed is set to 80rpm/min.
2.3 after it, will go up a step gained cell centrifugation, and collecting cell is diluted in (fresh EX-in the conditioned medium
302 serum free mediums: the culture supernatant liquid=1: 1 of a last step gained is mixed and is got), make cell density be adjusted to about 2 * 10
6Cells/ml adopts sodium-chlor that osmotic pressure is adjusted to 450m Ω, and adopting 5mM sodium hydroxide is 7.5 with pH regulator, places 5%CO
2, cultivate in the saturated humidity incubator, and rotating speed is set to 90rpm/min.
3.6 after it, will go up a step gained cell centrifugation, and collecting cell is diluted in (fresh EX-in the conditioned medium
302 serum free mediums: the culture supernatant liquid=1: 3 of a last step gained is mixed and is got), make cell density be adjusted to about 5 * 10
6Cells/ml adopts sodium-chlor that osmotic pressure is adjusted to 500m Ω, places 5%CO
2, cultivate in the saturated humidity incubator, and rotating speed is set to 100rpm/min.
4.9 after it, will go up a step gained cell centrifugation, and collecting cell is diluted in (fresh EX-in the conditioned medium
302 serum free mediums: the culture supernatant liquid of a last step gained=mix at 1: 5), make cell density be adjusted to about 10 * 10
6Cells/ml adopts sodium-chlor that osmotic pressure is adjusted to 550m Ω, places 5%CO
2, cultivate in the saturated humidity incubator, and rotating speed is set to 110rpm/min.
5.12 after it, will go up a step gained cell centrifugation, and collecting cell is diluted in (fresh EX-in the conditioned medium
302 serum free mediums: the culture supernatant liquid of a last step gained=mix at 1: 9), 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 into 6.8, places 5%CO
2, cultivate in the saturated humidity incubator, and rotating speed is set to 120rpm/min.
6.18 after it, adopt limiting dilution assay to carry out the serum-free cloning, cultivated 7-14 days.This method may further comprise the steps: use the D-poly-lysine of sterile pure water preparation 100mg/L, room temperature encapsulates 96 orifice plate 2h, uses aseptic water washing to encapsulate the hole 3 times; Is 50 cells/ml according to 1: 100 ratio serial dilution until cell density with the cell suspension of step 5 and non-blood serum low density culture medium, with 24 holes of ratio inoculation in 100 μ L/ holes; Continuing diluting cells suspension to cell density is 20 cells/ml, with 24 holes of ratio inoculation in 100 μ L/ holes; Continuing diluting cells suspension to cell density is 10 cells/ml, with 48 holes of ratio inoculation in 100 μ L/ holes; After cultivating through 7-14 days at last, filter out growth and product and express equal ideal clone strain.Growth and product are expressed equal ideal clone strain and should be satisfied growth metabolism and stablize, the standard that the title product of expression is superior in quality, and with the growth metabolism basically identical of initiating cell strain.
7. examine under a microscope the mono-clonal growing state, detect the expression of title product in the supernatant simultaneously with sandwich ELISA method, take all factors into consideration the result who grows and express two aspects, screening growth and all comparatively ideal tolerance clone strain of expression.
8. prepare 5L bio-reactor and fresh EX-
302 serum free mediums, the substratum that 3L is fresh joins in the reactor drum.
9. with about 5 * 10
5The tolerance cell inoculation that the initial cell density of cells/ml obtains step 7 screening is in above-mentioned 5L bio-reactor, and the starting rotating speed is 80rpm, and pH is 7.5.
With rotating speed according to 80,90,100,110,120,130,140, the gradient of 150rpm constantly raises, and controls the speed of stirring with different cells density.Initial cell density is 5 * 10
5Cells/ml to 1 * 10
6Cells/ml, stirring begins with 80rpm; Cell density reaches 5 * 10
6During cells/ml, stirring can be adjusted to 120rpm; Cell density reaches 1 * 10
7When cells/ml was above, stirring velocity can be adjusted to 150rpm.Can be in the stirring velocity between this scope according to the adhesion situation (adhesion between cell and the cell of cell; And cell is with the adhesion between the bio-reactor inwall) adjust; For example when cell adhesion aggravates (such as conglomeration having occurred, assemble or adhered to the bio-reactor inwall), can consider to heighten stirring velocity.
11. reach 5 * 10 at cell density
6After the cells/ml, progressively improve the concentration of lactic acid and ammonia every day, the concentration of lactic acid gradient is: 5,10,20,40, and 80mM, the concentration gradient of ammonia is: 2,4,6,8,10mM.Lactic acid concn can be adjusted through 200mM lactic acid, and the concentration of ammonia can be adjusted through 200mM ammonium chloride.
12. continue to cultivate cell density is constantly raise, reach about 2 * 10 until cell density
7Cells/ml.
13. constantly detect the cytoactive in the 5L bio-reactor, when cytoactive drops to 50% left and right sides, sterile sampling.
14., therefrom filter out growth and product and express equal ideal clone strain according to adopt limiting dilution assay to carry out serum-free cloning screening with the identical mode of step 6.
4) result:
Adopt the method for serum-free cloning to carry out the screening of high tolerance cell strain subclone, its cloning efficiency is 33.7%, and positive rate 100%.Can find out by accompanying drawing 1 and accompanying drawing 2, the height tolerance subclone cell strain that the present embodiment screening obtains (being called for short " high tolerance cell strain ") and initiating cell strain under same culture conditions on the accretion rate of L-glutaminate and glucose and trend basically identical.In whole growth process, substrate L-glutaminate and glucose maintain basically to be set in the interval range, and glutamine concentration maintains about 1-4mM, and glucose maintains about 2-6g/L.The concentration in L-glutaminate and the glucose 24 hours of adding stream all satisfies the ultimate value of setting, and wherein lower bound is set is 1mM to Stimulina, and it is 1g/L that glucose is provided with lower bound.
Can find out that by accompanying drawing 3 the high-cell density of high tolerance cell strain and initiating cell strain is respectively about 9.3 * 10
6Cells/ml and about 8.5 * 10
6Cells/ml (all cultivate to reach high-cell density in about the 5th day), both do not have significant difference.It can also be seen that from accompanying drawing 3 in logarithmic phase (about 1-4 days), high tolerance cell strain and initiating cell strain do not have significant difference aspect cell density; The plateau (about 4-6 days) of high tolerance cell strain, prolonged one day than initiating cell strain (about 4-5 days).
Can find out that by accompanying drawing 4 decline phase (rising in the 5th day or the 6th day), the cytoactive lowering speed of high tolerance cell strain is slower than the cytoactive lowering speed of initiating cell strain.In addition, the culture cycle of high tolerance cell strain prolongs two days than the culture cycle of initiating cell strain, extends to 10 days from 8 days.According to the ELISA detected result, high tolerance cell strain reaches mxm. the 10th day expression product concentration, is about 280.2mg/L.
Claims (17)
1. the screening method of a high tolerance cell strain; Comprise: but the grown cell strain chosen; It is carried out vitro culture; Force cell growth metabolism in bad growing environment thereby change cell culture condition, and then screen the height tolerance cell strain that obtains to be adapted to said bad growing environment, it is characterized in that; The cell culture condition of said change comprises: the osmotic pressure of cell culture fluid, lactic acid concn, ammonia concentration, pH value, oxyty or stirring velocity, perhaps two or more arbitrary combination of said cell culture condition.
2. the method for claim 1 is characterized in that, changes two or more described cell culture condition.
3. method as claimed in claim 2 is characterized in that, has 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 to improve osmotic pressure to make predetermined cell growth parameter(s) reach the preestablished limit value.
5. the method for claim 1 is characterized in that, changing cell culture condition is progressively to improve lactic acid concn to make predetermined cell growth parameter(s) reach the preestablished limit value.
6. the method for claim 1 is characterized in that, changing cell culture condition is progressively to improve ammonia concentration to make predetermined cell growth parameter(s) reach the preestablished limit value.
7. the method for claim 1 is characterized in that, changing cell culture condition is progressively to improve lactic acid concn simultaneously to make the cell growth parameter(s) of being scheduled to reach the preestablished limit value with ammonia concentration.
8. the method for claim 1 is characterized in that, changing cell culture condition is progressively to improve the pH value to make predetermined cell growth parameter(s) reach the preestablished limit value.
9. the method for claim 1 is characterized in that, changing cell culture condition is progressively to improve stirring velocity to make predetermined cell growth parameter(s) reach the preestablished limit value.
10. the method for claim 1 is characterized in that, directly adjusts to predetermined value to described cell culture condition.
11. any described method like claim 4 to 9 is characterized in that said predetermined cell growth parameter(s) is a cytoactive, the preestablished limit value is meant that cytoactive is lower than 50%.
12. any described method like claim 4 to 9 is characterized in that, said predetermined cell growth parameter(s) is the cell multiplication phase, and the preestablished limit value is the phalangeal cell multiplication phase can't keep stable.
13. the method for claim 1 is characterized in that, said cell strain is a mammalian cell strain.
14. the method for claim 1 is characterized in that, further with serum-free cloning method the cell that obtains is screened, and obtains the stable height tolerance cell strain of growth metabolism.
15. method as claimed in claim 14 is characterized in that, it is qualified that said height tolerates the expressed product quality of cell strain.
16. the method for claim 1 is characterized in that, under the condition of low nutrient concentrations, said cell strain is screened.
17. the method for claim 1 is characterized in that, under the condition of high-cell density, said cell strain is screened.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100511192A CN102634476A (en) | 2012-03-01 | 2012-03-01 | Method for screening high-tolerance cell strain |
| CN201510725233.2A CN105505852A (en) | 2012-03-01 | 2012-03-01 | Method for screening high-tolerance cell line |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100511192A CN102634476A (en) | 2012-03-01 | 2012-03-01 | Method for screening high-tolerance cell strain |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510725233.2A Division CN105505852A (en) | 2012-03-01 | 2012-03-01 | Method for screening high-tolerance cell line |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102634476A true CN102634476A (en) | 2012-08-15 |
Family
ID=46619055
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012100511192A Pending CN102634476A (en) | 2012-03-01 | 2012-03-01 | Method for screening high-tolerance cell strain |
| CN201510725233.2A Pending CN105505852A (en) | 2012-03-01 | 2012-03-01 | Method for screening high-tolerance cell line |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510725233.2A Pending CN105505852A (en) | 2012-03-01 | 2012-03-01 | Method for screening high-tolerance cell line |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN102634476A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104862280A (en) * | 2015-05-29 | 2015-08-26 | 上海大学 | Method for screening target protein CHO expressing cell strain applied to large-scale suspension culture |
| EP2970916B1 (en) | 2013-03-13 | 2021-04-14 | Merck Sharp & Dohme Corp. | Adapted lepidopteran insect cells for the production of recombinant proteins |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102080058A (en) * | 2010-12-09 | 2011-06-01 | 江南大学 | Lactobacillus casei and breeding method thereof |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1453948A2 (en) * | 2001-11-28 | 2004-09-08 | Sandoz Gmbh | Cell culture process |
| CN101857851A (en) * | 2002-12-23 | 2010-10-13 | 布里斯托尔-迈尔斯斯奎布公司 | Mammalian cell culture processes for protein production |
| CN1238498C (en) * | 2004-02-12 | 2006-01-25 | 陈志南 | Method for parameter control of the process for culturing serum-suspension free animal cell |
| WO2010017338A1 (en) * | 2008-08-06 | 2010-02-11 | Praxair Technology, Inc. | METHOD FOR CONTROLLING pH, OSMOLALITY AND DISSOLVED CARBON DIOXIDE LEVELS IN A MAMMALIAN CELL CULTURE PROCESS TO ENHANCE CELL VIABILITY AND BIOLOGIC PRODUCT YIELD |
| US8178318B2 (en) * | 2008-08-06 | 2012-05-15 | Praxair Technology, Inc. | Method for controlling pH, osmolality and dissolved carbon dioxide levels in a mammalian cell culture process to enhance cell viability and biologic product yield |
| CN102277380B (en) * | 2010-06-08 | 2013-03-27 | 齐鲁制药有限公司 | A dhfr complementarily expressed cotransfection eukaryotic expression vector and preparation method and application thereof |
| CN102181530B (en) * | 2011-03-18 | 2013-07-17 | 上海复宏汉霖生物技术有限公司 | Kit for detecting DNA residues of CHO cell and using method thereof |
| CN102994547B (en) * | 2011-09-08 | 2015-05-13 | 哈药集团技术中心 | Recombinant human erythropoietin-CTP fusion protein production process and application |
-
2012
- 2012-03-01 CN CN2012100511192A patent/CN102634476A/en active Pending
- 2012-03-01 CN CN201510725233.2A patent/CN105505852A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102080058A (en) * | 2010-12-09 | 2011-06-01 | 江南大学 | Lactobacillus casei and breeding method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 严以兰: "自然发酵乳中优良乳酸菌株的筛选及发酵条件研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》, no. 3, 15 March 2008 (2008-03-15), pages 024 - 57 * |
| 李莉莉等: "酿酒酵母乙醇发酵性能与胁迫耐性的相关性", 《华南理工大学学报（自然科学版）》, vol. 39, no. 9, 30 September 2011 (2011-09-30), pages 134 - 139 * |
| 杨汝德等: "耐高渗压乳酸菌在酱油酿造中的应用研究", 《现代食品科技》, vol. 21, no. 4, 30 April 2005 (2005-04-30), pages 37 - 40 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2970916B1 (en) | 2013-03-13 | 2021-04-14 | Merck Sharp & Dohme Corp. | Adapted lepidopteran insect cells for the production of recombinant proteins |
| CN104862280A (en) * | 2015-05-29 | 2015-08-26 | 上海大学 | Method for screening target protein CHO expressing cell strain applied to large-scale suspension culture |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105505852A (en) | 2016-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103193315A (en) | Method for preparing bioactive filler through immobilization of anaerobic ammonia oxidizing bacterium | |
| CN106011005A (en) | Bacillus amyloliquefaciens T600 and preparation method and application of microbial agent | |
| CN102443565B (en) | Medium suitable for cultivating CHO cell and cultivation technology thereof | |
| CN106434457B (en) | One plant of alkali protease superior strain and its produced alkali protease | |
| CN101831414A (en) | Process for extracellularly producing recombinant alpha-cyclodextrin glucosyltransferase | |
| CN116496950B (en) | Lysine production strain and application thereof, and lysine production method | |
| CN110079481A (en) | One bacillus amyloliquefaciens SL-7 and its application | |
| CN103773741A (en) | Method for preparing influenza vaccine by using cage type ventilating and stirring bioreactor | |
| CN102690781B (en) | Method for replacing culture medium in perfusion-type cell culture | |
| CN105861380A (en) | Escherichia coli JLTrp and application thereof to fermentation production of L-tryptophan | |
| CN108642041B (en) | Method for improving L-alanine fermentation production capacity of recombinant escherichia coli | |
| CN105671115B (en) | A method of building microbial co culture system produces bacteria cellulose | |
| CN104312963A (en) | Method for separating purified bdellovibrio from active sludge | |
| CN116731933B (en) | Corynebacterium glutamicum and application thereof in valine production | |
| CN102634476A (en) | Method for screening high-tolerance cell strain | |
| CN107604026B (en) | Method for increasing cordycepin yield of cordyceps militaris liquid fermentation | |
| CN102634447B (en) | Micro-array dialysis chamber and enrichment culture method using same | |
| CN109266578B (en) | Escherichia coli ACThr1032 and application thereof in fermentation production of L-threonine | |
| CN104263749B (en) | A kind of expression of scorpion derived antimicrobial peptide IsCT in Pichia pastoris and its clinically application for the treatment of infectious trauma | |
| CN107904200B (en) | It is a kind of expression adalimumab Combined culture base and its application | |
| CN102604884B (en) | Fermentation culture method of cells | |
| CN103966110B (en) | A kind of penicillium oxalicum Host Strains for improving filamentous fungus protein expression | |
| Sim et al. | Shikonin production by hairy roots of Lithospermum erythrorhizon in bioreactors with in situ separation | |
| CN109251914A (en) | A kind of Bacillus cereus and its application in production cellulase | |
| CN103255238A (en) | Method for controlling generation of mammalian cell lactic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120815 |