CN103320388A - Cell culture method capable of improving antibody expression levels and improving glycosylation levels - Google Patents
Cell culture method capable of improving antibody expression levels and improving glycosylation levels Download PDFInfo
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Abstract
The invention discloses a cell culture method capable of improving antibody expression levels and improving glycosylation levels. The method comprises a step of cultivating expression antibody cell strains via an optimized basic medium of expression antibody cell strains and an optimized batch feed method; the optimized basic medium comprises a mixture of a basic medium and a domestication medium, wherein the basic medium comprises a CDOptiCHO basic medium, and the domestication medium comprises CD FortiCHO, Duoning S3 and Duoning S4 mediums; and the optimized batch feed method comprises steps of: firstly adding Duoning AR7 and Duoning BR7, and adding Cell Boost 2 and Cell Boost 5. The cell culture method can both improve antibody expression levels and optimize glycosylation contents of target antibodies, and furthermore improve activities and effects of antibody medicines, so that the cell culture method is applicable to exploitation and industrialization of antibody generic medicines and novel antibody medicines.
Description
Technical field
The present invention relates to a kind of cell culture processes, particularly relate to a kind of cell culture processes that improves antibody expression amount and improvement level of glycosylation.
Background technology
Mammaliancellculture is produced antibody drug and is had clear superiority, mainly is active high, glycosylation modified correct, can guarantee the correct formation of the complicated protein structures such as disulfide linkage in the product, therefore, is most important expression system.
The structure of sugar chain and wherein the content of various glycosyls to the medicine chemical drug of monoclonal antibody medicine for characteristic so that drug effect has important effect, can affect monoclonal antibody medicine accretion rate in vivo such as the content of seminose.Therefore, level of glycosylation is the important parameter of monoclonal antibody medicine, and the level of glycosylation of antibody and glycosylation modified type directly affect antibody activity.
Antibody (comprising the Fc fusion rotein) medicine performance targeting is eliminated the vital role mode (effector function) of target cell (such as tumour cell), mainly contain two kinds: ADCC (antibody-dependent cell-mediated cytotoxicity, the cell-mediated cytotoxic effect that antibody relies on) and CDC (complement dependent cytotoxicity, the cytotoxic effect of Complement Dependent).Wherein, ADCC refers to express the effect cells such as NK cell, scavenger cell and neutrophil leucocyte of IgGFc acceptor, by being combined with the Fc section that is combined in the IgG antibody on the target cell surfaces such as virus infected cell and tumour cell, and the effect that kills and wounds these target cells, wherein, the content influence antibody-dependant cell of Fucose mediation toxicity reaction.CDC is combined with the surface of cell membrane corresponding antigens by specific antibody, forms mixture and the activating complement classical pathway, and formed membrane attack complex is to target cell performance cracking effect.ADCC is by antibody and the Fc γ R mediation that interacts, and the CDC then antibody by the extracellular combination and the protein-interacting that starts the complement effect causes.Therefore, these two kinds of mechanism of action of ADCC and CDC are in vitro study, the evaluation of antibody drug research and development, the important indicator of screening, and all have direct relation with glycosylation modified level and type.
At present, mainly concentrate in the transformation to Fc for the antibody engineering of ADCC and CDC antibody mediated effect function, to strengthen the interaction of antibody and Fc γ R, finally increase effector function.Full-length antibody has two N glycosylation sites, they have mediated Fc and have been combined the ADCC effect of bringing out with Fc γ R, and then also induced the continuous cell of Fc γ R, activated the responsing reaction of cytotoxic T lymphocyte (Cytotoxic T-lymphocyte, CTL).By the method for transgenation and glycosyl isomery, optimize Fc and Fc γ Rs and interact, can strengthen the effect of antibody inhibiting tumor.The glycosylation of IgG 297Asn has consisted of oligosaccharide and has connected the center, and connected Fucose can connect multiple monose, and the Fucose excalation can significantly strengthen the binding ability of antibody and Fc γ RIII and the ADCC effect of secondary.Use the RNA perturbation technique, the expression of downward modulation IgG glycosylation key enzyme-fucosyltransferase 8 in Chinese hamster ovary cell (CHO), antibody is gone fucosylated, the ADCC effect of this antibody has strengthened 100 times as a result, and the expression characterization of this CHO can also be hereditary to daughter cell.In addition, the successful parsing of the aminoacid sequence collection of illustrative plates that complete humanized IgG 1 is combined with Fc γ R helps to develop various IgG1 mutant, to improve the ability that they are combined with Fc γ R.These mutant in addition can with the efficient combination of Fc γ RIIIA of low-affinity, and the ADCC effect is strengthened.
The present antibody present situation of China compares with world level that to also have certain gap, main manifestations be that the antibody expression amount is low, and product level of glycosylation and standard substance are misfitted, and then causes the active undesirable or toxic side effect of antibody product large etc.; Although some antibody expression amount is greatly improved, the glycosylation modified of antibody never well solves, and caused antibody activity lower, thereby limited the development of China's antibody medicine industrialization.Such as the study hotspot of present biological imitation medicine, it mostly is the monoclonal antibody medicine that goes on the market the America and Europe about the nineties in last century.Whether the quality level of biological imitation medicine is consistent with former medicine, it is the prerequisite of toxicological study and clinical experiment, also be the key of examining, namely require every mass parameter of this biology imitation medicine can reach consistent with original product or similar, the drug effect of guarantee same level.
Yet present stage, the domestic method that does not also have effectively to adjust level of glycosylation as adjust the method for Fucose content by changing cell cultures technique, improves the expression amount of antibody drug.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of cell culture processes that improves antibody expression amount and improvement level of glycosylation.For the shortcoming of existing mammaliancellculture technique, the cell culture processes that the present invention sets up can improve glycosylation modified type, and then improves antibody activity, reduction antibody toxic side effect; Simultaneously, can change that antibody activity is high and shortcoming that the antibody expression amount is low is a kind of antibody high expression level, highly active cell culture processes.
For solving the problems of the technologies described above, the cell culture processes of raising antibody expression amount of the present invention and improvement level of glycosylation, comprise: basic medium and the feed supplement batch method of the cell strain by the expression antibody optimized, express the cultivation of the cell strain of antibody, thereby improve antibody expression amount and the level of glycosylation of improving antibody;
Wherein, the basic medium of optimization comprises: the mixture of basic medium and domestication substratum; Described basic medium comprises: CD OptiCHO basic medium (Invitrogen company); The domestication substratum comprises: CD FortiCHO (Invitrogen company), many peaceful S3 (Duoning company), many peaceful S4 substratum (Duoning company); And preferred many peaceful S4 substratum.
The feed supplement batch method of optimizing comprises: after adding first many peaceful AR7 (Duoning company) and many peaceful BR7 (Duoning company), add Cell Boost 2 (Hyclone company) and Cell Boost 5 (Hyclone company) again.
In the mixture of described basic medium and domestication substratum, basic medium is 1: 1 with the volume ratio of domestication substratum.
Among the present invention, the nutritive substances of above three kinds of domestication substratum, for example aminoacids content is abundant than the original base substratum, and the level of glycosylation that is conducive to improve the expression amount adjustment purpose antibody of purpose antibody makes it to be similar to former medicine.
In the feed supplement of the described optimization batch method, preferably: in the cell strain process of culture expression antibody, added more than 1.5% peaceful AR7 and more than 0.5% peaceful BR7 at the 3rd day, added more than 3% peaceful AR7 and more than 1% peaceful BR7 on the 6th day, added more than 3% peaceful AR7 and more than 1% peaceful BR7 on the 8th day, add 10% Cell Boost 2 and the mixture (volume ratio of Cell Boost 2 and Cell Boost 5 is 1: 1) of Cell Boost 5 on the 10th day, all added 5% Cell Boost 2 and the mixture (volume ratio of Cell Boost 2 and Cell Boost 5 is 1: 1) of Cell Boost5 in the 13rd day and the 15th day.
Wherein, the present invention also finds in to the screening study of feed supplement batch:
1) in original basic medium CD OptiCHO, added 5% CHO CD Efficient Feed B (Invitrogen company on the 3rd day, Cat#A10241), the 6th day and the 8th day add this kind feed supplement of 10% when cultivating, it is on the low side that the result demonstrates output, and glycosylation content and former medicine standard substance gap are large;
When 2) cell strain being tamed 50%CD OptiCHO+50%S3 substratum, the feed supplement method is that output and quality still do not reach needs the expection level at the Cell Boost 2 of adding 5% in the 3rd, six, eight day and the mixture (volume ratio of Cell Boost 2 and Cell Boost 5 is 1: 1) of Cell Boost 5.
The method of existing change glycosylation content is mainly by transgenation, the method that RNA disturbs knocks out or the glycosylation relevant enzyme of engineered host cell, make its activity decreased or forfeiture, with change glycosyl content, but this kind method cycle is long, and process is complicated, have a big risk, the stability of improved host cell, security need for a long time monitoring, are unfavorable for examining of antibody medicine.Yet, the present invention is the improvement to the cell strain culture technique, by the optimization to basic medium and the feed supplement batch experimental technique of cell strain, can when improving the antibody expression amount, optimize the glycosylation content of purpose antibody, thereby improve activity and the drug effect of antibody (such as monoclonal antibody) medicine.
Therefore, the present invention is by autotelic adjustment basic medium and feed supplement method, set up a kind of mammalian cell supplying technics of easy handling, the cell cultures technique of comparing traditional, expression amount and level of glycosylation all are improved, glycosylation modified type improves, thus, the present invention has changed the level of glycosylation of biological imitated antibody drug, make it consistent with former medicine standard substance, and improved protein yield, and method of the present invention can be applicable to exploitation and the industrialization of antibody imitation medicine and antibody new drug widely.
Description of drawings
The present invention is further detailed explanation below in conjunction with accompanying drawing and embodiment:
Fig. 1 is the viable cell density figure of the cell strain A feed supplement batch experiment among the embodiment 1; Wherein, the cell strain A-CDOptiCHO among the figure refers to that substratum is 100%CD OptiCHO substratum; Cell strain A-50%S3 refers to that substratum is the 50%CDOptiCHO+50%S3 substratum; Cell strain A-50%CD FortiCHO refers to 50%CD OptiCHO+50%CD FortiCHO substratum; Cell strain A-50%S4 refers to that substratum is 50%CD OptiCHO+50%S4 substratum;
Fig. 2 is the Cell viability figure of the cell strain A feed supplement batch experiment among the embodiment 1;
Fig. 3 is the antibody expression spirogram of the cell strain A feed supplement batch experiment among the embodiment 1;
Fig. 4 is that the glycosylation of cell strain A in 50%CD OptiCHO+50%S3 substratum among the embodiment 1 contains spirogram;
Fig. 5 is that the glycosylation of cell strain A in 50%CD OptiCHO+50%S4 substratum among the embodiment 1 contains spirogram;
Fig. 6 is the glycosyl structure iron among Fig. 4-5, the 9-10, and wherein, Man-5 is the seminose of high-content;
Fig. 7 is the viable cell density figure of the cell strain B feed supplement batch experiment among the embodiment 2; Wherein, the cell strain B-CDOptiCHO among the figure refers to that substratum is 100%CD OptiCHO substratum; Cell strain B-50%S3 refers to that substratum is the 50%CDOptiCHO+50%S3 substratum; Cell strain B-50%CD FortiCHO refers to 50%CD OptiCHO+50%CD FortiCHO substratum; Cell strain B-50%S4 refers to that substratum is 50%CD OptiCHO+50%S4 substratum;
Fig. 8 is the Cell viability figure of the cell strain B feed supplement batch experiment among the embodiment 2;
Fig. 9 is the antibody expression spirogram of the cell strain B feed supplement batch experiment among the embodiment 2;
Figure 10 is that the glycosylation of cell strain B in CD OptiCHO substratum among the embodiment 2 contains spirogram;
Figure 11 is that the glycosylation of cell strain B in 50%CD OptiCH0+50%S4 substratum among the embodiment 2 contains spirogram.
Embodiment
1, the optimization of serum free medium and screening
For expression amount and the optimization glycosylation content that improves Trastuzumab, with cell strain A[Chinese hamster ovary cell (CHO), available from Eureka company] from original basic medium (CD OptiCHO, Invitrogen company, Cat#A12681), being optimized to 3 kinds contains in the original basic medium of 50% (volume percent) and the 50% new substratum.3 kinds of new substratum be respectively CD FortiCHO (Invitrogen company, Cat#A11483), many peaceful S3 (Duoning company, Cat#M012-001) and many peaceful S4 (Duoning company, Cat#M012-002) substratum.
2, optimization and the screening of feed supplement batch experimental technique
By the optimization to feed supplement kind and add-on, improved the output of purpose monoclonal antibody, also optimize glycosylation content simultaneously.Wherein, in the feed supplement batch experiment, inoculated with dilution in 1: 5 with 4 days cultured cells in the 0th day, cell density is in 6-8 * 10
5About cells/ml, initial volume is controlled at 30ml.At the 3rd, 6,8,10,13 day, detect cell density, survival rate and nutrition.If glucose concn is lower than 2g/L, can add to 8-10g/L.When motility rate is lower than 60%, or reaches 17-18 days and collect supernatant.After collecting supernatant, with the method for high performance liquid chromatography (HPLC), by the content of Protein A post test purpose monoclonal antibody albumen Trastuzumab.
Wherein, the glycosyl analytical procedure is: (PNGase F) separates oligosaccharides from monoclonal antibody albumen Trastuzumab with endoglycosidase, obtains containing the supernatant liquor of oligosaccharides behind ice ethanol protein precipitation.The ethanol in the supernatant liquor is removed in vacuum-drying, and dried polysaccharide jelly with fluorophor on 2-amino-benzamide (2-AB) indicia band, is applicable to the HPLC-FLD isolation identification.By the color atlas of comparative sample and standard substance, identify the oligosaccharides kind of sample protein, and absorb its relative percentage value of calculated by peak area according to dissimilar oligosaccharides.
HPLC instrument model in the present embodiment is: Agilent Technologies 1260 Infinity with FLD (G1321);
Feed supplement method: added 1.5%AR7 (Duoning company at the 3rd day, Cat#F007-001) and 0.5%BR7 (Duoning company, Cat#F008-001), added 3%AR7 and 1%BR7 on the 6th day, added 3%AR7 and 1%BR7 on the 8th day, added 10% Cell Boost 2 (Hyclone company on the tenth day, Cat#SH30596) and Cell Boost 5 (Hyclone company, Cat#SH30865) mixture (volume ratio of Cell Boost 2 and Cell Boost 5 is 1: 1) all added 5% Cell Boost 2 and the mixture (volume ratio of Cell Boost 2 and Cell Boost5 is 1: 1) of Cell Boost 5 in the 13 day and the 15 day.Wherein, the per-cent that relates in the feed supplement method is batch per-cent of experiment initial volume.
Wherein, cell strain A at the viable cell density of feed supplement batch experiment and Cell viability respectively shown in Fig. 1-2, can find out and work as cell strain A in 50%CD OptiCHO+50%S4 and 50%CD OptiCHO+50%FortiCHO, after the 13rd day, other two kinds of substratum still keep higher viable cell density and motility rate relatively in feed supplement batch test.
The expression amount of the Herceptin antibody of cell strain A in original basic medium and feed supplement batch experimental technique only has 0.7g/L (as shown in Figure 3), through the optimization to 3 kinds of different culture medias, find that the upgrowth situation of this cell strain in 50%CD OptiCHO and 50%S4 is best, expression amount is up to 1.4g/L (as shown in Figure 3).
In addition, glycosyl type and the content of this cell strain A in 50%CD OptiCHO+50%S3 substratum and 50%CDOptiCHO+50%S4 substratum has been compared in the glycosylation analysis, the results are shown in Figure shown in the 4-5.Wherein, in utilizing the 50%CDOptiCHO+50%S4 substratum, the motility rate of cell maintains higher level for a long time, the batch cultivation time lengthening, output increased, and the glycosylation of cultivating the purpose monoclonal antibody obtain in 50%CD OptiCHO+50%S4 conforms to the standard substance Trastuzumab, the content of GOF and Fucose G1F (as shown in Figure 6) particularly, and this content has very important directive function to activity and the drug effect of monoclonal antibody medicine.
Embodiment 2 cell strain B carry out the expression of Trastuzumab (Herceptin) antibody and cultivate
According to the method for embodiment 1, utilizing cell strain B[Chinese hamster ovary cell (CHO), available from Eureka company] expression of carrying out Trastuzumab (Herceptin) antibody cultivates.
Wherein, cell strain B, can find out when cultivating in 50%CD OptiCHO+50%S4 respectively shown in Fig. 7-8 at the viable cell density of feed supplement batch experiment and Cell viability, and the highest viable cell concentrations of cell strain B reaches approximately 8 * 10
5Cells/ml is higher than other substratum, and motility rate also can remain in the long period and be higher than 90% level.
The expression amount of cell strain B in original CD OptiCHO basic medium and feed supplement batch experimental technique has 1.2g/L (as shown in Figure 9), but level of glycosylation and standard substance differ a lot of (as shown in figure 10).Through the optimization at 3 kinds of different culture medias, the expression amount of cell strain B in 50%CD OptiCHO+50%S4 substratum rises to 1.4g/L (as shown in Figure 9), and conforms to (as shown in figure 11) with the glycosylation standard substance.
Claims (5)
1. a cell culture processes that improves antibody expression amount and improvement level of glycosylation is characterized in that, comprising: basic medium and the feed supplement batch method of the cell strain by the expression antibody optimized, express the cultivation of the cell strain of antibody;
Wherein, the basic medium of optimization comprises: the mixture of basic medium and domestication substratum; Described basic medium comprises: CD OptiCHO basic medium; The domestication substratum comprises: CD FortiCHO, many peaceful S3, many peaceful S4 substratum;
The feed supplement batch method of optimizing comprises: after adding first many peaceful AR7 and Duo Ning BR7, add Cell Boost 2 and Cell Boost 5 again.
2. the method for claim 1 is characterized in that: described domestication substratum is many peaceful S4 substratum.
3. the method for claim 1 is characterized in that: in the mixture of described basic medium and domestication substratum, basic medium is 1: 1 with the volume ratio of domestication substratum.
4. the method for claim 1, it is characterized in that: in the feed supplement of the described optimization batch method, added more than 1.5% peaceful AR7 and more than 0.5% peaceful BR7 at the 3rd day, added more than 3% peaceful AR7 and more than 1% peaceful BR7 on the 6th day, added more than 3% peaceful AR7 and more than 1% peaceful BR7 on the 8th day, add 10% Cell Boost 2 and the mixture of Cell Boost 5 on the 10th day, all added 5% Cell Boost 2 and the mixture of Cell Boost 5 in the 13rd day and the 15th day.
5. method as claimed in claim 4, it is characterized in that: in the mixture of described Cell Boost 2 and Cell Boost 5, its volume ratio is 1: 1.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106190948A (en) * | 2015-05-07 | 2016-12-07 | 上海津曼特生物科技有限公司 | A kind of CHO-S cell semisolid culturemedium and compound method thereof and application |
| CN106459185A (en) * | 2014-04-02 | 2017-02-22 | 普雷斯蒂奇生物制药私人有限公司 | Method for preparing antibody through regulation of sugar content of antibody |
| CN106755096A (en) * | 2016-12-20 | 2017-05-31 | 上海药明生物技术有限公司 | The method for obtaining the stable cell mass of expression target protein in Chinese hamster ovary celI using piggyBac transposon |
| CN111954719A (en) * | 2018-03-26 | 2020-11-17 | 美国安进公司 | Total defucosylated glycoforms of antibodies produced in cell culture |
| CN114195904A (en) * | 2021-12-27 | 2022-03-18 | 成都蓉生药业有限责任公司 | Fed-batch culture method for producing long-acting recombinant human coagulation factor VIII recombinant cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1761746A (en) * | 2003-01-22 | 2006-04-19 | 格黎卡特生物技术股份公司 | Fusion constructs and use of same to produce antibodies with increased Fc receptor binding affinity and effector function |
| CN101624614A (en) * | 2009-08-14 | 2010-01-13 | 上海抗体药物国家工程研究中心有限公司 | Culture method for anti-CD25 antibody |
| CN102021217A (en) * | 2006-12-20 | 2011-04-20 | 上海国健生物技术研究院 | Method for high efficient expression of monoclonal antibody by means of animal cell fed-batch culture |
-
2012
- 2012-03-20 CN CN201210074420.5A patent/CN103320388B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1761746A (en) * | 2003-01-22 | 2006-04-19 | 格黎卡特生物技术股份公司 | Fusion constructs and use of same to produce antibodies with increased Fc receptor binding affinity and effector function |
| CN102021217A (en) * | 2006-12-20 | 2011-04-20 | 上海国健生物技术研究院 | Method for high efficient expression of monoclonal antibody by means of animal cell fed-batch culture |
| CN101624614A (en) * | 2009-08-14 | 2010-01-13 | 上海抗体药物国家工程研究中心有限公司 | Culture method for anti-CD25 antibody |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106459185A (en) * | 2014-04-02 | 2017-02-22 | 普雷斯蒂奇生物制药私人有限公司 | Method for preparing antibody through regulation of sugar content of antibody |
| CN106190948A (en) * | 2015-05-07 | 2016-12-07 | 上海津曼特生物科技有限公司 | A kind of CHO-S cell semisolid culturemedium and compound method thereof and application |
| CN106190948B (en) * | 2015-05-07 | 2020-09-25 | 上海津曼特生物科技有限公司 | CHO-S cell semisolid culture medium and preparation method and application thereof |
| CN106755096A (en) * | 2016-12-20 | 2017-05-31 | 上海药明生物技术有限公司 | The method for obtaining the stable cell mass of expression target protein in Chinese hamster ovary celI using piggyBac transposon |
| CN111954719A (en) * | 2018-03-26 | 2020-11-17 | 美国安进公司 | Total defucosylated glycoforms of antibodies produced in cell culture |
| CN114195904A (en) * | 2021-12-27 | 2022-03-18 | 成都蓉生药业有限责任公司 | Fed-batch culture method for producing long-acting recombinant human coagulation factor VIII recombinant cells |
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