CN105713946A - Mammalian cell culture technology for enhancing monoclonal antibody ADCC activity - Google Patents
Mammalian cell culture technology for enhancing monoclonal antibody ADCC activity Download PDFInfo
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- CN105713946A CN105713946A CN201410712306.XA CN201410712306A CN105713946A CN 105713946 A CN105713946 A CN 105713946A CN 201410712306 A CN201410712306 A CN 201410712306A CN 105713946 A CN105713946 A CN 105713946A
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Abstract
The invention discloses a technological route suitable for mammalian cell culture and quick and effective enhancement of monoclonal antibody ADCC activity. The technology is characterized in that through the method of adding nucleoside and metal ions in a culture process, the glycosylation level of a monoclonal antibody can be controlled, and the ADCC activity of the monoclonal antibody can be enhanced. The technology has the characteristics of good stability, strong operability, easy amplification, and small result differences between batches, and can be applied to large-scale mammalian cell culture for expression of monoclonal antibodies, especially the industrial production of therapeutic monoclonal antibodies.
Description
Technical field
The present invention relates to the mammaliancellculture technique that a kind of monoclonal antibody produces, more particularly, it relates to add uridnine and manganese ion in incubation, control monoclonal antibody level of glycosylation, and then the mammalian cell feeding culture technique of raising ADCC activity, belong to field of biological pharmacy.
Background technology
Biotech drug is monoclonal antibody medicine especially, because of the targeting of its treatment, toleration, improves life in patients extending life simultaneously, obtains traction high after listing, and cookle occurs frequently, such as Rituximab, Herceptin and infliximab etc..Whole world antibody drug experienced by quick development, reach certain scale at present, and domestic antibody drug import and domestic quantity are all limited, proposing to give special assistance to biotechnology industry in country " 12 " planning, this is the boosting enthusiasm of domestic-developed monoclonal antibody medicine significantly also in addition.
Antibody-dependent cell cytotoxicity effect (ADCC) is one of important function mechanism of monoclonal antibody medicine, monoclonal antibody targeting combines to tumor cell surface molecule, effector lymphocyte is combined with monoclonal antibody Fc section by the Fc receptor (Fc γ R) on its surface, activate a series of signal path, thus discharging a series of inflammation or Cytotoxic immunoregulatory factor, including cytokine, chemotactic factor, protease and active oxygen species etc., tumor cell is produced lethal effect, thus playing the anti-tumor activity of monoclonal antibody medicine.The representative medicine utilizing this mechanisms play antitumor action has Rituximab and Herceptin etc..
The glycosylation of monoclonal antibody Fc section, as the important indicator modified after protein translation, has very important effect in antibody drug effect (such as ADCC) and medicine generation (half-life).Glycosylation modified influence factor is more, such as culture medium, host cell, training method, culture parameters (pH, temperature, DO, PCO2) etc..Report at present or applied certain methods and changed level of glycosylation or type, improve the ADCC activity of antibody, the sugar-type of monoclonal antibody is made to become high mannose type (ZhouQ as added mannosidase inhibitor (kifunensine), etal., BiotechnologyandBioengineering, 2008,99 (3): 652-665), or reduce fucose by engineered means and modify (MalphettesL, etal., BiotechnologyandBioengineering, 2010,106 (5): 774-783) method such as.Changing type of glycosylation and can affect safety and the effectiveness of monoclonal antibody accordingly, as high mannose type can accelerate antibody removing in vivo, thus affecting medicine generation (shortening the half-life), and being likely to produce immunogenicity.Engineered method then needs to rebuild cell strain, is equivalent to monoclonal antibody medicine is developed again, extends construction cycle and the cost of medicine.
Under the situation in domestic and international monoclonal antibody medicine exploitation keen competition epoch at present, in the urgent need to a kind of simple technique, do not changing on the basis of type of glycosylation, by controlling level of glycosylation, improve the ADCC activity of monoclonal antibody and do not affect other biological feature, improve the therapeutic effect of product.
Summary of the invention
Present invention aim at providing a kind of level of glycosylation effectively controlling monoclonal antibody in mammalian cell culture process, improve the production technology of monoclonal antibody body ADCC activity.
Production technology provided by the invention, it is characterised in that:
A kind of mammalian cell feeding culture technique improving monoclonal antibody ADCC activity, it is characterised in that continuous stream adds nucleoside and metal ion in cell cultivation process.
Above-mentioned process, it is characterised in that continuous stream adds uridnine and manganese ion in cell cultivation process.
Above-mentioned process, it is characterised in that stream adds the uridnine of 0.5mM-10mM total amount and the manganese ion of 1 μM of-20 μMs of total amount in cell cultivation process.
Above-mentioned process, it is preferable that stream adds the uridnine of 6mM-8mM total amount and the manganese ion of 12 μMs of-16 μMs of total amounts.
Above-mentioned process, it is characterised in that cell grows to 2-5 × 10 after cultivating 3-5 days6During cells/mL density, starting to add fed-batch medium, it is 8-10 days that stream adds the time.
Above-mentioned fed-batch medium, it is characterised in that individually stream adds, or adds with uridnine and manganese ion mixed flow.
Above-mentioned fed-batch mode, it is preferable that mixed flow adds.
Above-mentioned manganese ion, it is characterised in that manganese ion is provided by manganous salt.
Above-mentioned manganous salt, it is preferable that manganese sulfate.
Above-mentioned mammal cell line, it is preferable that NS0 cell line, SP2/0 cell line and Chinese hamster ovary celI system.
Above-mentioned mammal cell line, it is preferable that Chinese hamster ovary celI system.
Above-mentioned process produces the application in monoclonal antibody at mammalian cell.
Above-mentioned monoclonal antibody, it is characterised in that itself there is the monoclonal antibody drug of ADCC activity.
Embodiments below is gone forward one by one according to process optimization and is carried out:
In one embodiment, 6 250mL shaking flasks (Corning) are cultivated, working volume 60mL, shaking table (section is resistance to) controls: rotating speed is 120-130rpm, temperature is 36.5 DEG C, and CO2 saturation is 7%, and cell-seeding-density is 0.8-1.2 × 106Cells/mL, cultivates the 3rd to 11 day, and manganese sulfate and the uridnine of fed-batch medium and variable concentrations is added in segmentation, and total cultivation cycle is 13 days.
In another embodiment, being amplified to 3L reactor (Applikon) cell and cultivate, cell-seeding-density is 0.8-1.2 × 106Cells/mL, controlling parameter is: speed of agitator is 80-250rpm, and temperature is 36.5 DEG C, and pH is 7.0, and dissolved oxygen is 40% (air saturation), cultivates and starts for the 3rd day, and every day, stream added the fed-batch medium containing uridnine and manganese sulfate, and cultivation cycle is 13 days.
In another embodiment, it is amplified to 3 batches of 250L reactor (Thermo250LSUB) cells further to cultivate, speed of agitator is 80-120rpm, pH is 6.9-7.1, dissolved oxygen is 30-50% (air saturation), cultivating the 3rd day and start, add fed-batch medium (sulfur acid manganese and uridnine), cultivation cycle is 15-16 days.
Uridnine and manganese ion are to affect the key factor that antibody glycosylation is modified.Uridnine participates in the generation of UTP and UDP-galactose in cell, and UDP-galactose complex and albumen complete galactosylation process under the effect of galactosylation transferring enzyme, and in glycosyl galactose process, manganese ion is the important cofactor of galactosylation transferring enzyme.Therefore can reach to control the effect of level of glycosylation by adding uridnine and manganese ion, and then affect the biologic activity of antibody, such as ADCC activity.
Beneficial effects of the present invention
The major advantage of the present invention: (1) present invention, by adding debita spissitudo uridnine and manganese ion in cell cultivation process, utilizes the synergism of the two, effectively raises monoclonal antibody galactosylation degree, improve the quality of monoclonal antibody；(2) present invention is while controlling level of glycosylation, significantly improves the ADCC activity of monoclonal antibody so that it is have better biological function；(3) process costs of the present invention is low, be prone to amplify, batch between stable, passed through 3 batches of 250LSUB amplification culture checkings, be very suitable for the industrialized production of therapeutic monoclonal antibodies.
Accompanying drawing explanation
Fig. 1: 3L reactor cell growth curve
Fig. 2: 3L reactor antibody expression curve
Fig. 3: 3L reactor antibody A DCC detection curve
Fig. 4: 250L reactor cell growth curve
Fig. 5: 250L reactor antibody expression curve
Fig. 6: 250L reactor antibody A DCC detection curve
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further elaborated, but is not intended to the present invention, all done within the spirit and principles in the present invention any amendment, equivalent replacement and improvement etc., should be included within protection scope of the present invention.
Experiment material: the Chinese hamster ovary celI strain (LifeTechnologies company of the U.S.) of stably express anti-CD-20 monoclonal antibody, commercial base culture medium and business fed-batch medium (LifeTechnologies company of the U.S.), uridnine and manganese sulfate (Sigma Co., USA).
Embodiment 1 adds the shake-flask culture of variable concentrations uridnine and manganese sulfate
Shake-flask culture cell-seeding-density is 0.8-1.2 × 106Cells/mL, working volume 50mL, shaking table (section is resistance to) controls: rotating speed is 120-130rpm, temperature is 36.5 DEG C, CO2 saturation is 7%, cultivates and within the 3rd, 5,7,9 and 11 days, adds fed-batch medium and the different manganese sulfate of total amount, uridnine), cultivation cycle is 13 days.Cultivation carries out quality analysis with ProteinA antibody purification after terminating.
Detection method:
Viable cell density detects: carrying out viable cell density detection with Counterstar, result is in Table 1.
Antibody titer detects: with HPLC-ProteinA method detection antibody titer, result is in Table 1.
ADCC activity detects: adopt the NK92-CD16a cell action effect cell of through engineering approaches, Wil2-s cell carries out ADCC activity detection as target cell, effect target ratio is for 2:1, lysis effect is detected by Lactic dehydrogenase detection kit, adopting the EC50 value of experimental group namely to represent than work (%) with the fiducial value of the EC50 value of matched group, result is in Table 1.
Data process:
Accumulative cell concentration IVCD computing formula is IVCDi=IVCDi-1+(ViXi+Vi-1Xi-1)(ti-ti-1)/2Vi, the i in formula refers to cultivate the same day；I-1 refers to cultivate the previous day；IVCD refers to accumulation amount of viable cell (106Cellsday/mL)；X phalangeal cell concentration (cells/mL)；V refers to volume of culture (mL)；T refers to incubation time (days)；
Antibody production capacity Qp computing formula is Qp=antibody production/IVCD, Qp phalangeal cell per unit area yield (pg/cell/day).
Results contrast:
Adding manganese sulfate, uridnine and compare cultivation results contrast in Table 1, along with interpolation uridnine and manganese ion concentration are incremented by, Growth of Cells presents downward trend；Being 10mM in uridnine concentration, when manganese ion concentration is 20 μMs, antibody production have dropped 19%；In uridnine concentration higher than 6mM, when manganese ion concentration is higher than 12 μMs, ADCC specific activity is obviously improved more than 2 times, and presents ascendant trend along with uridnine and manganese ion concentration are incremented by.So, to be tested by shake-flask culture, consider Growth of Cells, expression and ADCC activity, the uridnine selecting interpolation total amount to be 8mM and the technique of the manganese ion of 16 μMs react device process test.
Table 1 shake-flask culture result collects
Embodiment 23L reactor process is tested
Cell being seeded to 3L glass reactor (Applikon company of Holland) after shaking flask expands and carries out process test, cell-seeding-density is 0.8-1.2 × 106Cells/mL, controlling parameter is: speed of agitator is 80-250rpm, temperature is 36.5 DEG C, pH is 7.0, dissolved oxygen is 40% (air saturation), cultivates and added the fed-batch medium fed-batch medium containing uridnine, manganese sulfate to the 12nd day on the 3rd day, and cultivation cycle is 13 days, it is 8mM that uridnine stream adds total amount, and it is 16 μMs that manganese ion stream adds total amount.Matched group only adds fed-batch medium.In incubation sampling every day carry out growing, metabolism and titre detection.Cultivation carries out quality analysis with affinity chromatograph, cation-exchange chromatography and anion-exchange chromatography antibody purification after terminating.
Detection method:
Viable cell density detects: carrying out viable cell density detection with Counterstar, result is shown in Fig. 1.
Antibody titer detects: with HPLC-ProteinA method detection antibody titer, result is shown in Fig. 2.
ADCC activity detects: adopt the NK92-CD16a cell action effect cell of through engineering approaches, Wil2-s cell carries out ADCC activity detection as target cell, effect target ratio is for 2:1, lysis effect is detected by Lactic dehydrogenase detection kit, adopting the EC50 value of experimental group namely to represent than work (%) with the fiducial value of the EC50 value of matched group, result is shown in Fig. 3.
Sugar-type detects: with reference to commercially available 2-AB marker detection test kit (Glyko company of the U.S.), is separated by N-glucosides by endoglycosidase (N-Glycanase), be enriched with through atresia graphite pillar from monoclonal antibody.N-glucosides is through vacuum concentrate drying, by dried N-glucosides 2-Amino-benzamide (2-AB) labelling, is applied to HPLC-FLD and separates analysis.By compare the N-glucosides of 2-AB labelling with saccharide retention time in liquid chromatograph, the N-glucosides type of monoclonal antibody is belonged to；Different types of N-glucosides is normalized at the peak area of HPLC-FLD spectrogram, each N-glucosides type of monoclonal antibody is carried out quantitative analysis.Result is in Table 2.
Comparative result:
Growth fraction is relatively: if Fig. 1, UM experimental group (adding the experimental group of uridnine and manganese ion) is to cultivate early growth very fast, late stage of culture, cell density and motility rate are lower by about 10% than matched group, overall no significant difference；
Titre compares: such as Fig. 2, UM experimental group with matched group Titer over time, cultivating under 13 days conditions, experimental group titre is 1493.40mg/L, and matched group is 1550.55mg/L simultaneously, and cell is expressed and had no significant effect by this technique；
ADCC activity compares: if Fig. 3, UM experimental group EC50 is 1.282ng/mL, matched group is 3.024ng/mL, and experimental group ADCC activity is 2.36 times of matched group.
Sugar-type compares: if the G0F ratio of table 2, UM experimental group is lower than matched group, and G1F and G2F ratio is above matched group, illustrates that uridnine and manganese ion have promoted the galactosylation of antibody.Galactosylation can affect the other biological activity of antibody, such as the cytotoxicity (CDC) of Complement Dependent, thus improves galactosylation degree and is conducive to improving the biologic activity of antibody.UM experimental group G0, G1 and the G2 ratio without core fucose is slightly above matched group, and core fucose can reduce the ADCC activity of antibody, one of this reason being probably ADCC activity of the present invention raising.
Table 2 sugar-type analysis
Embodiment 3250LSUB bioreactor culture process certification
In 250L level, repeat 3 batches of Chinese style scale amplification culture.
Thermo250LSUB bioreactor culture: cell-seeding-density is 0.8-1.2 × 106Cells/mL, controlling parameter is: speed of agitator is 80-120rpm, temperature is 36.5 DEG C, and pH is 6.9-7.1, and dissolved oxygen is 30-50% (air saturation), initialization volume 150L, when cultivating the 3-4 days, add fed-batch medium (containing uridnine and manganese ion), maintain concentration of glucose to 2g/L, cultivation cycle is 14-16 days, containing 8mM uridnine and 16 μMs in final culture fluid.
Such as Fig. 4 and Fig. 5,3 batches of 250LSUB reactor growth all keep consistent with expressing, and EC50 respectively 1.189ng/mL, 1.449ng/mL and 1.362ng/mL, the CV of the ADCC of expression product < 10%, there is good repeatability.
The present invention adds the feeding culture technique of aminoacid and UMG and all reaches product development target at Growth of Cells, cell expression and antibody mass, and passed through 3 batches of 250L amplification culture process certifications, this process stabilizing mature and reliable, favorable reproducibility, the present invention has good industrialization potential.
Claims (13)
1. the mammalian cell feeding culture technique improving monoclonal antibody ADCC activity, it is characterised in that continuous stream adds nucleoside and metal ion in cell cultivation process.
2. process according to claim 1, it is characterised in that continuous stream adds uridnine and manganese ion in cell cultivation process.
3. process according to claim 1, it is characterised in that stream adds the uridnine of 0.5mM-10mM total amount and the manganese ion of 1 μM of-20 μMs of total amount in cell cultivation process.
4. process according to claim 1, it is preferable that stream adds the uridnine of 6mM-8mM total amount and the manganese ion of 12 μMs of-16 μMs of total amounts.
5. the process according to claim 1-4, it is characterised in that cell grows to 2-5 ' 10 after cultivating 3-5 days6During cells/mL density, starting to add fed-batch medium, it is 8-10 days that stream adds the time.
6. fed-batch medium according to claim 5, it is characterised in that individually stream adds, or adds with uridnine and manganese ion mixed flow.
7. fed-batch mode according to claim 6, it is preferable that mixed flow adds.
8. the manganese ion according to claim 1-7, it is characterised in that manganese ion is provided by manganous salt.
9. manganous salt according to claim 8, it is preferable that manganese sulfate.
10. mammalian cell according to claim 1, it is preferable that NS0 cell line, SP2/0 cell line and Chinese hamster ovary celI system.
11. mammal cell line according to claim 1, it is preferable that Chinese hamster ovary celI system.
12. the process described in claim 1-11 any one produces the application in monoclonal antibody at mammalian cell.
13. monoclonal antibody according to claim 10, it is characterised in that itself there is the monoclonal antibody drug of ADCC activity.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321188A (en) * | 2018-12-17 | 2020-06-23 | 嘉和生物药业有限公司 | Formula for modifying antibody glycoform, cell culture method and application in industrial production |
| CN112779307A (en) * | 2021-01-11 | 2021-05-11 | 苏桥生物(苏州)有限公司 | Method for two-stage regulation of CHO expression exogenous protein glycoform |
| CN113549667A (en) * | 2021-07-21 | 2021-10-26 | 杭州奕安济世生物药业有限公司 | Method for reducing galactosylation of antibody |
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2014
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321188A (en) * | 2018-12-17 | 2020-06-23 | 嘉和生物药业有限公司 | Formula for modifying antibody glycoform, cell culture method and application in industrial production |
| CN112779307A (en) * | 2021-01-11 | 2021-05-11 | 苏桥生物(苏州)有限公司 | Method for two-stage regulation of CHO expression exogenous protein glycoform |
| CN112779307B (en) * | 2021-01-11 | 2022-04-19 | 苏州药明生物技术有限公司 | Method for two-stage regulation of CHO expression exogenous protein glycoform |
| CN113549667A (en) * | 2021-07-21 | 2021-10-26 | 杭州奕安济世生物药业有限公司 | Method for reducing galactosylation of antibody |
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Address after: The 856000 Tibet autonomous region in southern area of Zedang town Xiang Qu Road No. 8 Applicant after: Haisike Pharmaceutical Group Limited by Share Ltd Address before: The 856000 Tibet autonomous region in southern area of Zedang town Xiang Qu Road No. 8 Applicant before: Tibet Haisco Pharmaceutical Group Co., Ltd. |
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Application publication date: 20160629 |