CN103255238A - Method for controlling generation of mammalian cell lactic acid - Google Patents
Method for controlling generation of mammalian cell lactic acid Download PDFInfo
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- CN103255238A CN103255238A CN2013101824752A CN201310182475A CN103255238A CN 103255238 A CN103255238 A CN 103255238A CN 2013101824752 A CN2013101824752 A CN 2013101824752A CN 201310182475 A CN201310182475 A CN 201310182475A CN 103255238 A CN103255238 A CN 103255238A
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Abstract
The invention relates to the biotechnical field, and in particular relates to a fed-batch cultivation method for controlling generation of mammalian cell lactic acid. The method comprises the following steps: adding CuSO4.5H2O into a basal culture medium; inoculating mammalian cells in logarithmic phase in a reactor to be cultivated for 12-18 days; controlling the pH to 6.6-7.0 in previous 4-8 days; feeding a culture medium and glucose concentrated liquor in the cultivation process; and controlling the glucose concentration to 1-3g/L. The fed-batch cultivation method for controlling of CHO cell lactic acid on a large scale and pilot amplified production are suitable for mammalian cell cultivation of recombinant proteins and further suitable for mammalian cell cultivation for producing anti-TNF-alpah (Tumor Necrosis Factor) antibodies.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of method that mammalian cell lactic acid generates of controlling.
Background technology
The antibody class medicine is because its good active and high advantage such as selectivity extensively is used to treatment of diseases such as tumour, immunity, infection.In recent years, along with the listing in succession of some " cookle " medicine Enbrel, Avastin, Humira, Herceptin, Rituxan etc., the antibody class medicine has started " sensational effect " in whole field of biological pharmacy, and its market share is soaring year by year.Mammalian cell is owing to can make the external source recombinant protein carry out a series of posttranslational modification, comprise correct folding, the formation of disulfide linkage, phosphorylation, especially glycosylation modified, these rhetorical functions have great importance for the biologic activity of recombinant protein, therefore, become recombinant protein gradually, especially the important host cell of antibody class medicine.
Continuous increase along with market antagonist class pharmaceutical requirements, feeding culture becomes the main flow training method of mammalian cell high-cell-density cultivation, but, in the feeding culture process because the mass consumption of nutritive substance glucose, amino acid etc., a large amount of accumulations of metabolic by-prods, limited the accumulation of further optimization, the especially lactic acid of feeding culture process, for the growth of cell and the expression of product the obvious suppression effect has been arranged.Therefore, in the feeding culture process, for being controlled to for culturing process optimization one of successful key whether of lactic acid.People (Biotechnol Bioeng72:55-61,2001) such as Keqin Chen have reported the Chinese hamster ovary celI system that obtains the lactic dehydrogenase enzyme defect by screening after the homologous recombination, and the result has reduced the generation of lactic acid, and the antibody expression level is 5 times of maternal clone.But in the pharmacy research process, changing clone is a thing that wastes time and energy, and can waste a large amount of R﹠D work amounts and time before antibody class medicine clinical, and this method is real in being a kind of very unwise move for the control that lactic acid generates.
Summary of the invention
The shortcoming of prior art in view of the above, the invention provides a kind of method that control lactic acid generates in the feeding culture process of simple possible, and then amplify production step by step, and adopted a kind of new seed amplification and cell culture processes, be used for solving the problems of the prior art.
First aspect present invention provides a kind of method that mammalian cell lactic acid generates of controlling, and comprises the steps: to add in basic medium CuSO
45H
2O, the mammalian cell that will be in logarithmic phase cultivated 12-18 days after being inoculated in reactor, wherein before 4-8 days control pH=6.6-7.0, fed-batch medium and glucose concentrated solution in the culturing process, and the control glucose concn is at 1-3g/L.
Preferably, add CuSO in the basic medium
45H
2The amount of O is 30-70 μ mol/L.
Preferably, described mammalian cell is Chinese hamster ovary celI.
Preferably, the mode of control pH is for passing through CO
2Gas and sodium bicarbonate control.
Second aspect present invention provides a kind of feeding culture process of producing the Chinese hamster ovary celI of anti-TNF-Alpha antibodies, comprises the steps: the CHO basic medium is added in the reactor, and add CuSO in described basic medium
45H
2O, the CHO-TNF-α cell inoculation that will be in logarithmic phase was cultivated behind reactor 12-18 days, wherein controlled pH=6.6-7.0 in preceding 4-8 days, fed-batch medium and glucose concentrated solution in the culturing process, and the control glucose concn is at 1-3g/L.
The construction process of described CHO-TNF-α cell is with reference to Flora Forouzesh, Samira Shakeri Tabarian, Shaghayegh Emami, et al.Construction and Stable Expression of a Truncated Human Receptor Tyrosine Kinase Ror1 (Ror1-ECD), Avicenna J Med Biotech2012; 4 (1): 41-45.
Preferably, add CuSO in the basic medium
45H
2The amount of O is 30-70 μ mol/L.
Preferably, cell inoculation density is 0.58-0.64 * 10
6Cells/ml.
Preferably, in the described step 1, the cumulative volume of inoculation back nutrient solution is 2.5-3.5L, and the volume of reactor is 6.5-8.5L.
Preferably, the mode of control pH is for passing through CO
2Gas and sodium bicarbonate control.
Preferably, the control condition of reactor is as follows: temperature is 36.3-37.3 ° of C; Stirring velocity is 80-120rpm; DO is 20-60%.
Preferred, the deep ventilation of reactor is set to Air/CO
2/ O
2Three gas patterns, flow velocity are 0.1L/min, CO
2And O
2Ratio arranges automatic adjusting according to pH and DO.
When preferably, the add-on of fed-batch medium is cell inoculation 30% of the cumulative volume of nutrient solution.
The feeding culture process that control Chinese hamster ovary celI lactic acid provided by the present invention generates, not only can in the reactor of small volume, have good effect, reach very high final antibody concentration, can also amplify step by step, carried out the middle trial production of extensive (the above reactor of 250L).
Preferred, described fed-batch medium is CD EfficientFeed
TMC AGT
TM(being called for short EFC, Gibco company).
Third aspect present invention provides the method for described control mammalian cell lactic acid generation in the application that improves cell cultures antibody expression field.
Fourth aspect present invention provides the feeding culture process of the Chinese hamster ovary celI of the anti-TNF-Alpha antibodies of described production to express the application in field at the anti-TNF-Alpha antibodies of raising.
Production is amplified in the feeding culture process that control Chinese hamster ovary celI large scale culturing lactic acid involved in the present invention generates and pilot scale, and the mammalian cell that is suitable for recombinant protein is cultivated, and is more suitable for cultivating in the mammalian cell of producing anti-TNF-Alpha antibodies.The invention discloses the comprehensive means that adds copper sulfate and conditioned reaction device pH by being combined in the substratum, the method that lactic acid generates when controlling the extensive feeding culture of mammalian cell has formed the feeding culture technology of high antibody expression level according to this.And carry out seed amplification and the cultivation of disposable stirred reactor by disposable WAVE reactor, and further this feeding culture technology is carried out the reactor scale and amplify production step by step, reached the scale of pilot scale.
Description of drawings
Fig. 1: do not control the feeding culture process that lactic acid generates in the 7.5L reactor, X-coordinate is represented incubation time, and left side ordinate zou is represented viable cell concentrations, the right ordinate zou 1 expression antibody concentration, and ordinate zou 2 expression lactic acid in the right generate concentration.
Fig. 2: the feeding culture process that control lactic acid generates in the 7.5L reactor, X-coordinate is represented incubation time, left side ordinate zou is represented viable cell concentrations, the right ordinate zou 1 expression antibody concentration, ordinate zou 2 expression lactic acid in the right generate concentration.
Fig. 3: the amplification feeding culture process that control lactic acid generates in the 30L reactor, X-coordinate is represented incubation time, left side ordinate zou is represented viable cell concentrations, the right ordinate zou 1 expression antibody concentration, ordinate zou 2 expression lactic acid in the right generate concentration.
Fig. 4: trial production process in the 250L reactor, X-coordinate is represented incubation time, left side ordinate zou is represented viable cell concentrations, the right ordinate zou 1 expression antibody concentration, ordinate zou 2 expression lactic acid in the right generate concentration.
Embodiment
Below by specific specific examples explanation embodiments of the present invention, those skilled in the art can understand other advantages of the present invention and effect easily by the disclosed content of this specification sheets.The present invention can also be implemented or be used by other different embodiment, and the every details in this specification sheets also can be based on different viewpoints and application, carries out various modifications or change under the spirit of the present invention not deviating from.
See also Fig. 1-4.Need to prove, the diagram that provides in the present embodiment only illustrates basic conception of the present invention in a schematic way, satisfy only show in graphic with the present invention in relevant assembly but not component count, shape and size drafting when implementing according to reality, kenel, quantity and the ratio of each assembly can be a kind of random change during its actual enforcement, and its assembly layout kenel also may be more complicated.
Do not control the feeding culture process that lactic acid generates in the embodiment 17.5L reactor
Basic medium is that CD CHO Medium(is called for short CD-CHO, Gibco company), fed-batch medium is CD EfficientFeed
TMC AGT
TM(being called for short EFC, Gibco company), other cell culture reagent of using in the culturing process is all purchased the company to Sigma.
The construction process of CHO-TNF-α cell is with reference to Flora Forouzesh, Samira Shakeri Tabarian, Shaghayegh Emami, et al.Construction and Stable Expression of a Truncated Human Receptor Tyrosine Kinase Ror1 (Ror1-ECD), Avicenna J Med Biotech2012; 4 (1): 41-45, utilize electric transformation technology to import the CHO-K1 cell, through screening, subclone and study on the stability, final acquisition is efficient, the recombinant cell strain of stably express antibody molecule, and set up the cell bank of homogeneous with this cell strain, carried out the research of cultural method on this cell bank basis.
After the seed cell of above-mentioned gained recovered, in shaking bottle, adopt the CD-CHO substratum to be expanded to 300ml, to be in the cell inoculation of logarithmic phase in 7.5L reactor (NBS Bioflo310), inoculation back nutrient solution cumulative volume is 3L, and cell inoculation density is 0.61 * 10
6Cells/ml.Add 10% EFC(volume ratio at the 3rd, 6,9 day each stream of cultivation), add the glucose concentrated solution by stream glucose concn is controlled at 1-3g/L.
7.5L temperature of reactor is 36.8 ° of C; Stirring velocity is 100rpm; DO is 40%; PH is 7.0, passes through CO
2Gas and sodium bicarbonate are regulated; Deep ventilation is set to Air/CO
2/ O
2Three gas patterns, flow velocity are 0.1L/min, CO
2And O
2Ratio arranges automatic adjusting according to pH and DO.
Experimental result is seen Fig. 1, and whole culturing process was carried out 11 days, reaches maximum value at the 6th day viable cell density of cultivation, is 10.9 * 10
6Cells/ml.In culturing process, lactic acid concn is accumulated in a large number, reaches 3.78g/L at the 3rd day lactic acid concn of cultivation, maintains the level above 3g/L afterwards always.Antibody concentration reached 514mg/L at the 8th day, prolong and continue along with incubation time afterwards and accumulate, and therefore, higher lactic acid concn may limit the generation of antibody.
The feeding culture process that control lactic acid generates in the embodiment 27.5L reactor
Compared to embodiment one, in basic medium CD-CHO, added the CuSO of 50 μ mol/L
45H
2O; Reactor pH passed through CO in preceding 4 days in cultivation
2Gas and sodium bicarbonate 6.8, are not controlled its control afterwards.In addition, other culture condition, it is all the same with embodiment one that substratum, the stream that comprises use adds form, reactor control condition etc.
Experimental result is seen Fig. 2, and culturing process was carried out 17 days altogether, reaches maximum at the 7th day viable cell density of cultivation, is 9.2 * 10
6Cells/ml.Cultivating the 3rd day, lactic acid concn reaches 3.59g/L, differently with embodiment one afterwards is, lactic acid is not the level that maintains above 3g/L, but beginning is consumed in a large number, namely is lower than 2g/L on the 7th day cultivating, and is lower than 1g/L on the 13rd day cultivating.Exactly because lactic acid concn has obtained effective control, the antibody continuous expression, final antibody production 1.57g/L has improved 2.08 times than embodiment one, and the expression that has promoted antibody for effective control of lactic acid is described.
The feeding culture process that embodiment 330L scale is amplified
According to embodiment two, the reactor scale of cultivating is amplified to 30L reactor (Sartorius Biostat Cplus) from 7.5L.Seed cell through recovery after, in shaking bottle, be expanded to 5 and shake each 300ml of bottle, 1.5L seed liquor altogether, the seed cell that will be in logarithmic growth period is seeded in the 30L reactor, inoculation back nutrient solution is 15L altogether, cell inoculation density is 0.41 * 10
6Cells/ml.Because the 30L reactor is structurally not exclusively the same with the 7.5L reactor, therefore, having difference with the 7.5L reactor aspect the reactor parameter control, the top layer is set in the 30L reactor ventilates to control tank pressure, the top layer is aeration air only, the ventilation flow velocity is 0.5L/min, and tank pressure is controlled to be 0.05bar, and other culture condition is consistent with embodiment two.
Experimental result is seen Fig. 3, and whole culturing process was carried out 17 days altogether, reaches maximum at the 7th day viable cell density of cultivation, is 11.2 * 10
6Cells/ml.In culturing process, lactic acid reached maximum 2.6g/L at the 4th day, just begin afterwards to be consumed in a large number, be lower than 1g/L cultivating namely to be reduced in the 7th day, maintain the level that is lower than 1g/L afterwards always, compared to embodiment two, lactic acid concn is lower than embodiment two, illustrates that lactic acid has obtained more effective control, causes the antibody expression level to be had been further upgraded, final antibody production reaches 1.76g/L, compares with embodiment two and has improved 12%.
The seed amplification procedure of producing is amplified in the pilot scale of embodiment 4250L scale
In the seed cell amplification procedure, adopt the mode of enlarged culturing container step by step, seed cell through and having shaken a bottle 30ml, 100ml, 400ml, WAVE2.4L, WAVE15.5L, shake the bottle and WAVE reactor culture condition see Table 1, when seed at last in the WAVE reactor cell density reach 3 * 10
6During cells/ml, be seeded in the 250L reactor and cultivate.
Production process is amplified in the pilot scale of embodiment 5250L scale
Pilot scale seeding each container culture condition that increases is as shown in table 1 in the 250L reactor.
Table 1
After the seed cell recovery, through amplification step by step, the cell inoculation of the growth period of taking the logarithm is in the 250L reactor, and inoculation back nutrient solution cumulative volume is 150L, and cell inoculation density is 0.6 * 10
6Cells/ml.Basic medium and fed-batch medium all use commercial serum free medium, and culture scheme is consistent with the feeding culture process of control lactic acid-producing, add the glucose concentrated solution by stream in whole culturing process glucose concn is controlled at 1-3g/L.
Temperature of reactor is 36.8 ° of C; Stirring velocity is 100rpm; DO is 40%; PH was set to 6.8 in preceding 4 days in cultivation, passed through CO
2Gas and sodium bicarbonate are regulated, and do not control pH afterwards; The top layer is bubbling air only, and flow velocity is 2L/min, and deep layer feeds CO
2And O
2To regulate pH and DO.
Experimental result is seen Fig. 4, and pilot scale is amplified the production cell cultivation process and carried out altogether 17 days, reaches maximum at 11 days viable cell densities of cultivation, is 9.32 * 10
6Cells/ml, final antibody production is 2.24g/L.Amplify in the production process in pilot scale, lactic acid concn has obtained effective control, reaches maximum value at the 3rd day lactic acid concn of cultivation, only be 1.41g/L, just begin afterwards to reduce gradually, just be lower than 1g/L from cultivating beginning in the 7th day, lower lactic acid concn is conducive to the generation of antibody.
From feeding culture technology amplification process, from 7.5L, 30L, to the reactor amplification process of 150L different scales, because the feature of reactor self, though cause feeding culture technology unanimity, but concentration of lactic acid reduces gradually, and then the expression level of antibody is corresponding is constantly promoted, and this has further verified the high efficiency of the feeding culture technology that control lactic acid generates.
Compared to embodiment one, in basic medium CD-CHO, added the CuSO of 30 μ mol/L
45H
2O; Reactor pH passed through CO in preceding 8 days in cultivation
2Gas and sodium bicarbonate 6.6, are not controlled culturing process 18 days altogether with its control afterwards.
After the seed cell of above-mentioned gained recovered, in shaking bottle, adopt the CD-CHO substratum to be expanded to 300ml, to be in the cell inoculation of logarithmic phase in 7.5L reactor (NBS Bioflo310), inoculation back nutrient solution cumulative volume is 3L, and cell inoculation density is 0.58 * 10
6Cells/ml.Add 10% EFC(volume ratio at the 3rd, 6,9 day each stream of cultivation), add the glucose concentrated solution by stream glucose concn is controlled at 1-3g/L.
7.5L temperature of reactor is 36.3 ° of C; Stirring velocity is 80rpm; DO is 20%; Pass through CO
2Gas and sodium bicarbonate are regulated; Deep ventilation is set to Air/CO
2/ O
2Three gas patterns, flow velocity are 0.1L/min, CO
2And O
2Ratio arranges automatic adjusting according to pH and DO.
This embodiment lactic acid variation tendency is similar to embodiment 2, and antibody continues high expression level.
Embodiment 7
Compared to embodiment one, in basic medium CD-CHO, added the CuSO of 70 μ mol/L
45H
2O; Reactor pH passed through CO in preceding 4 days in cultivation
2Gas and sodium bicarbonate 7.0, are not controlled culturing process 12 days altogether with its control afterwards.
After the seed cell of above-mentioned gained recovered, in shaking bottle, adopt the CD-CHO substratum to be expanded to 300ml, to be in the cell inoculation of logarithmic phase in 7.5L reactor (NBS Bioflo310), inoculation back nutrient solution cumulative volume is 3L, and cell inoculation density is 0.64 * 10
6Cells/ml.Add 10% EFC(volume ratio at the 3rd, 6,9 day each stream of cultivation), add the glucose concentrated solution by stream glucose concn is controlled at 1-3g/L.
7.5L temperature of reactor is 37.3 ° of C; Stirring velocity is 120rpm; DO is 60%; Pass through CO
2Gas and sodium bicarbonate are regulated; Deep ventilation is set to Air/CO
2/ O
2Three gas patterns, flow velocity are 0.1L/min, CO
2And O
2Ratio arranges automatic adjusting according to pH and DO.
This embodiment lactic acid variation tendency is similar to embodiment 2, and antibody continues high expression level.
In sum, the present invention has effectively overcome various shortcoming of the prior art and the tool high industrial utilization.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not is used for restriction the present invention.Any person skilled in the art scholar all can be under spirit of the present invention and category, and above-described embodiment is modified or changed.Therefore, have in the technical field under such as and know that usually the knowledgeable modifies or changes not breaking away from all equivalences of finishing under disclosed spirit and the technological thought, must be contained by claim of the present invention.
Claims (13)
1. control the method that mammalian cell lactic acid generates for one kind, comprise the steps: in basic medium, to add CuSO
45H
2O, the mammalian cell that will be in logarithmic phase cultivated 12-18 days after being inoculated in reactor, wherein before 4-8 days control pH=6.6-7.0, fed-batch medium and glucose concentrated solution in the culturing process, and the control glucose concn is at 1-3g/L.
2. a kind of method that mammalian cell lactic acid generates of controlling as claimed in claim 1 is characterized in that, adds CuSO in the basic medium
45H
2The amount of O is 30-70 μ mol/L.
3. a kind of method that mammalian cell lactic acid generates of controlling as claimed in claim 1 is characterized in that described mammalian cell is Chinese hamster ovary celI.
4. a kind of method that mammalian cell lactic acid generates of controlling as claimed in claim 1 is characterized in that the mode of control pH is for passing through CO
2Gas and sodium bicarbonate control.
5. a feeding culture process of producing the Chinese hamster ovary celI of anti-TNF-Alpha antibodies comprises the steps: the CHO basic medium is added in the reactor, and add CuSO in described basic medium
45H
2O, the CHO-TNF-α cell inoculation that will be in logarithmic phase was cultivated behind reactor 12-18 days, wherein controlled pH=6.6-7.0 in preceding 4-8 days, fed-batch medium and glucose concentrated solution in the culturing process, and the control glucose concn is at 1-3g/L.
6. a kind of feeding culture process of producing the Chinese hamster ovary celI of anti-TNF-Alpha antibodies as claimed in claim 5 is characterized in that, adds CuSO in the basic medium
45H
2The amount of O is 30-70 μ mol/L.
7. a kind of feeding culture process of producing the Chinese hamster ovary celI of anti-TNF-Alpha antibodies as claimed in claim 5 is characterized in that, cell inoculation density is 0.58-0.64 * 10
6Cells/ml.
8. a kind of feeding culture process of producing the Chinese hamster ovary celI of anti-TNF-Alpha antibodies as claimed in claim 5 is characterized in that, the mode of control pH is for passing through CO
2Gas and sodium bicarbonate control.
9. a kind of feeding culture process of producing the Chinese hamster ovary celI of anti-TNF-Alpha antibodies as claimed in claim 5, it is characterized in that the control condition of reactor is as follows: temperature is 36.3-37.3 ° of C; Stirring velocity is 80-120rpm; DO is 20-60%.
10. a kind of feeding culture process of producing the Chinese hamster ovary celI of anti-TNF-Alpha antibodies as claimed in claim 9 is characterized in that the deep ventilation of reactor is set to Air/CO
2/ O
2Three gas patterns, flow velocity are 0.1L/min, CO
2And O
2Ratio arranges automatic adjusting according to pH and DO.
11. a kind of feeding culture process that Chinese hamster ovary celI lactic acid generates of controlling as claimed in claim 5 is characterized in that, when the add-on of fed-batch medium is cell inoculation 30% of the cumulative volume of nutrient solution.
12. the method that generates as the described control mammalian cell of the arbitrary claim of claim 1-4 lactic acid is in the application that improves cell cultures antibody expression field.
13. express the application in field as the feeding culture process of the Chinese hamster ovary celI of the anti-TNF-Alpha antibodies of the described production of the arbitrary claim of claim 5-11 improving anti-TNF-Alpha antibodies.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108051550A (en) * | 2017-12-22 | 2018-05-18 | 吉林冠界生物技术有限公司 | A kind of method of main acidic materials in definite cell culture medium |
| CN109517738A (en) * | 2018-12-14 | 2019-03-26 | 杭州奕安济世生物药业有限公司 | A kind of method of carbon dioxide content in regulation bioreactor |
| CN115976145A (en) * | 2022-11-28 | 2023-04-18 | 广州誉衡生物科技有限公司 | CHO cell high-efficiency perfusion feeding culture method for producing PD-1 antibody |
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| CN103282509A (en) * | 2010-12-28 | 2013-09-04 | 中外制药株式会社 | Animal cell culturing method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108051550A (en) * | 2017-12-22 | 2018-05-18 | 吉林冠界生物技术有限公司 | A kind of method of main acidic materials in definite cell culture medium |
| CN108051550B (en) * | 2017-12-22 | 2020-07-24 | 吉林冠界生物技术有限公司 | Method for determining main acidic substances in cell culture medium |
| CN109517738A (en) * | 2018-12-14 | 2019-03-26 | 杭州奕安济世生物药业有限公司 | A kind of method of carbon dioxide content in regulation bioreactor |
| CN115976145A (en) * | 2022-11-28 | 2023-04-18 | 广州誉衡生物科技有限公司 | CHO cell high-efficiency perfusion feeding culture method for producing PD-1 antibody |
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Application publication date: 20130821 |