CN104004814A - Method for precisely controlling monoclonal antibody product quality through animal cell fed-batch cultivation - Google Patents
Method for precisely controlling monoclonal antibody product quality through animal cell fed-batch cultivation Download PDFInfo
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Abstract
The invention discloses a control strategy of the process of cultivating and producing monoclonal antibody medicine through mammal cells. The modes of lowering cultivation temperature, shortening the cultivation cycle and the like are adopted, so that post-translational modifying features, such as charge distribution and glycoform, of monoclonal antibodies are effectively controlled. The process has the advantages of being good in stability, high in operability, little in difference between batches, and the application range of the process is greatly widened.
Description
Technical field
The present invention relates to animal cell culture field.More specifically, the present invention relates to a kind of method that adopts zooblast feeding culture mode accurately to control monoclonal antibody quality product.
Background technology
Animal cell culture starts from the beginning of this century, to scale in 1962, start to expand, be developed so far and become the technological method extensively adopting in biology, medical research and application, utilize animal cell culture to produce monoclonal antibody, vaccine and the somatomedin etc. with important medical value, become the integral part of medicine bioengineering hi-tech industry.Due to the biological characteristics of zooblast vitro culture, the complicacy of related products structure and quality and coherence request, animal cell large-scale culture technique is still difficult to meet the demand of the scale production with important medical value biological products, in the urgent need to further research and development cell cultures technique.
Animal cell culture training method mainly contains three kinds, batch cultivation, feeding culture and perfusion culture.Also have such as the mixed mode (Hybrid) being undertaken by the ATF system of U.S. Refine company and cultivate in recent years.For batch cultivation, in reactor, add supplemented medium continuously or off and on, in the Extending culture cycle, obtain higher cell density and antibody production.And perfusion culture culture cycle is longer, conventionally, at 2-3 month, therefore culture process is had relatively high expectations, be suitable for the production of less stable protein drug.
Charge distribution and glycosylation be as the important indicator of protein post-translational modification, for folding, transportation and the location of protein, plays an important role, closely related with recombinant glycoprotein biological activity, transformation period and katabolism etc. in vivo.The charge distribution of protein and glycosylation process are subject to the impact of several factors, comprise the difference of cell category, substratum, the control of the difference of culturing process, the transhipment time of golgi body, the utilization of the precursor substances such as the state of cell and intracellular nucleic acid etc.By optimal conditions, improve the glycosylation of protein, guarantee the consistence of Protein Glycosylation Overview between different batches, will greatly increase the result for the treatment of of recombinant glycoprotein medicine.
Culture temperature, as the important control parameter of animal cell culture, directly has influence on output and the quality of monoclonal antibody.By optimizing culture temperature, can improve the output of monoclonal antibody, can change the charge distribution of antibody simultaneously, affect the ratio of acidic components and basic component, and then affect antibody pharmacokinetics behavior in animal body.Culture cycle is also most important for cell cultures, and along with the prolongation of culture cycle, cell density and motility rate decline gradually, and metabolism toxic byproduct builds up, and the glycosylation of antagonist produces material impact.High mannose can be accelerated antibody removing in animal body, shortens the transformation period, and may produce immunogenicity, and then affect the effect of antibody.Research shows, along with the prolongation of culture cycle, the ratio regular meeting of high mannose increases gradually.Therefore control the suitable production cycle extremely important for obtaining high-quality antibody.
Summary of the invention
The object of the present invention is to provide a kind of method that adopts zooblast feeding culture mode accurately to control monoclonal antibody quality product.
In order to solve the problems of the technologies described above, technical scheme provided by the invention is:
Technical scheme provided by the invention comprises the steps:
(1) cell cultures initial temperature 36-37 ℃, cultivates 5-8 days, adds fed-batch medium when Chinese hamster ovary celI grows to suitable density, carries out feeding culture;
(2) when Growth of Cells approaches plateau, reduce temperature to 31-33 ℃, carry out feeding culture, reduce temperature and finish cell cultures after 2-7 days.
After experiment finishes, carry out antibody charge distribution and sugared type ratio and detect, control antibody acidic components and be less than 25%, high mannose ratio is less than 5%.
In one embodiment, the cell inoculum density of described method is 0.3-0.6*10
6cells/ml, PH scope 6.7-7.4, dissolved oxygen 30-50% air saturation, mixing speed 50-300rpm.
In another embodiment, described method dissolved oxygen 40% air saturation, mixing speed 80-250rpm.
In another embodiment, the culture cycle of described method is 7-15 days.
The present invention selects feeding culture mode to carry out monoclonal antibody expression, reduces culture temperature between when appropriate, and selects the appropriate incubation cycle, to reach charge distribution and the glycosylation feature of accurate control antibody.
Culture temperature is a key factor that affects antibody producing.Zooblast growth is conventionally 37 ℃ of left and right.With this understanding, cell proliferation rate is very fast.After temperature declines, cellular metabolism is slack-off gradually, and in maintenance phase, cell can keep higher motility rate, now take and expresses antibody as main.In addition, temperature also can affect the charge distribution of antibody.For example, reduce the acidic components that temperature can reduce antibody.
Culture cycle is another important factor that affects antibody producing.Along with the prolongation of culture cycle, declining all appears in cell density and motility rate, and metabolism toxic byproduct builds up, and the deterioration of culture environment can affect the glycosylation feature of antibody.Research discovery, the ratio of high mannose increases along with the prolongation of culture cycle.Therefore shorten the ratio that culture cycle is conducive to reduce high mannose.
Present method adopts control by stages strategy.
First stage, i.e. step (1), cell cultures initial temperature is controlled at 36-37 ℃, cultivates 4-8 days, and cell Fast Growth is to plateau, to obtaining higher cell density;
Subordinate phase, step (2), reduces temperature to 31-33 ℃, reduces cellular metabolism on the one hand, maintains higher Cell viability, and enhancing antibody is synthetic on the other hand, reduces the acidic components of antibody simultaneously, and is conducive to the stable of albumen.
The present invention adopts feeding culture mode to carry out monoclonal antibody expression, has process stabilizing good stability, workable, batch between little etc. the feature of difference, be very suitable for animal cell culture manufacture order clonal antibody.
Accompanying drawing explanation
Fig. 1 display segment is cultivated and contrast culture cell density comparison diagram.
Fig. 2 display segment is cultivated and contrast culture antibody expression amount comparison diagram.
Fig. 3 display segment is cultivated and contrast culture charge distribution comparison diagram.
Fig. 4 display segment is cultivated and contrast culture high mannose ratio comparison diagram.
Fig. 5 display segment is cultivated and contrast culture cell density comparison diagram.
Fig. 6 display segment is cultivated and contrast culture cell density comparison diagram.
Fig. 7 display segment is cultivated and contrast culture antibody production comparison diagram.
Embodiment
The invention will be further described in description below in conjunction with accompanying drawing by embodiment, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modifications and improvement, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
Embodiment 1CHO cell expressing monoclonal antibody A
1. material: the Chinese hamster ovary celI strain (ATCC) of stably express monoclonal antibody A, commercial base substratum and supplemented medium (U.S. Thermo Fisher company, U.S. Life Technologies company).
2. bio-reactor:
3L glass biological reaction device (Dutch Applikon company), 500L disposable bioreactor (U.S. match Mo Feishier company)
3.3L reactor cell cultures sectionalized control method:
The Chinese hamster ovary celI strain of stably express monoclonal antibody A, inoculum density 0.3-0.6*10
6cells/ml, 36.5 ± 0.5 ℃ of initial culture temperature, pH is controlled at 6.7-7.4, mixing speed 150rpm, dissolved oxygen 40% air saturation, is cultured to the 5th day and reduces temperature to 32.5 ± 0.5 ℃, culture cycle 10-11 days.
4.3L reactor cell cultures sectionalized control method and the comparison of contrast culture method:
(1) contrast culture method: the Chinese hamster ovary celI strain of stably express monoclonal antibody A, inoculum density 0.3-0.6*10
6cells/ml, 36.5 ± 0.5 ℃ of initial culture temperature, pH is controlled at 6.7-7.4, mixing speed 150rpm, dissolved oxygen 40% air saturation, culturing process maintains steady temperature.
(2) segmentation culture method and contrast culture method cell density are measured: the conventional counting method of blood cell that adopts of cell cultures counting detects cell density, the results are shown in accompanying drawing 1.
(3) segmentation culture method and the flow measurement of contrast culture method protein expression: cultivate the 9th, 10,11 days, adopt ProteinA affinity chromatography method to measure, the results are shown in accompanying drawing 2.
(4) segmentation culture method and contrast culture method charge distribution detect: adopt high performance liquid phase ion exchange chromatography (WCX stratographic analysis post), the results are shown in accompanying drawing 3.
(5) segmentation culture method and contrast culture method sugar type analysis: with reference to commercially available 2-AB marker detection test kit (U.S. Glyko company), the results are shown in accompanying drawing 4.
(6) result comparison
The comparison of cell density: in two kinds of cell culture processes, from the 1st day to the 5th day, cell was all in vigorous vegetative period; The 5th day Growth of Cells approaches high-density; After this, Growth of Cells is in maintenance phase, and cell density curve presents a plateau, the high-density no significant difference of two kinds of method cells, and segmentation culture method is cultivated density while finishing higher than contrast culture method, sees accompanying drawing 1.
Antibody expression amount comparison: in two kinds of cell culture processes, the 9th, 10,11 days, the expressing quantity of segmentation culture method all, higher than contrast culture method approximately 25%, was shown in accompanying drawing 2.
Charge distribution comparison: in two kinds of cell culture processes, the 9th, 10,11 days, the acidic components proportion of segmentation culture method antibody was starkly lower than contrast culture method, sees accompanying drawing 3.
Sugar type analysis comparison: in two kinds of cell culture processes, along with the prolongation of incubation time, high mannose ratio increases gradually, and segmentation culture method, compared with contrast culture Fa Lvegao, reaches 7% left and right, sees accompanying drawing 4.Because antibody production under segmentation culture condition is higher, and acidic components ratio is lower, therefore by shortening the method for culture cycle, effectively controls the seminose ratio of antibody.
The 500L one-time reaction device cell culture processes of culture cycle is cultivated and is shortened in 5 segmentations:
The Chinese hamster ovary celI strain of stably express monoclonal antibody A, inoculum density 0.3-0.6*10
6cells/ml, 36.5 ± 0.5 ℃ of initial culture temperature, pH is controlled at 6.7-7.4, mixing speed 80rpm, dissolved oxygen 40% air saturation, cultivates and within the 5th day, reduces temperature to 33 ℃, culture cycle 7 days.
Cultivation results:
Cell density: cultivate cooling in the 5th day, cell arrives high-density, and after this Growth of Cells is in maintenance phase, and Growth of Cells presents plateau, sees accompanying drawing 5.
Expressing quantity: cultivate the 7th day, antibody production is 262mg/L.
Antibody charge distribution: cultivate the 7th day, acidic components ratio is 19.0%.
Sugar type analysis: cultivate the 7th day, high mannose ratio is 4.2%.
Embodiment 2CHO cell expressing monoclonal antibody B
1. material:
The Chinese hamster ovary celI strain (ATCC) of stably express monoclonal antibody B, commercial base substratum and supplemented medium (U.S. Thermo Fisher company, U.S. Life Technologies company).
2. bio-reactor
3L glass biological reaction device (Dutch Applikon company).
3.CHO cell segmentation culture method:
By the Chinese hamster ovary celI strain of stably express monoclonal antibody B, inoculum density 0.3-0.6*10
6cells/ml, 36.5 ± 0.5 ℃ of initial culture temperature, pH is controlled at 6.7-7.4, mixing speed 250rpm, dissolved oxygen 40% air saturation, is cultured to the 3rd day and starts flow feeding substratum, be cultured to the 8th day and reduce temperature to 31.5 ± 0.5 ℃, culture cycle 14-15 days.
4. the present invention reaches the object that improves product production and improve quality product by segmentation culture method.
(1) contrast culture method, cell cultures temperature maintains 36.5 ± 0.5 ℃, and other culture condition are identical with cooling culture method.
(2) segmentation culture method and contrast culture method cell density are measured: the conventional counting method of blood cell that adopts of cell cultures counting detects cell density, the results are shown in accompanying drawing 6.
(3) segmentation culture method and the flow measurement of contrast culture method protein expression: adopt ProteinA affinity chromatography method to measure, the results are shown in accompanying drawing 7.
(4) segmentation culture method and contrast culture method charge distribution are measured: adopt high performance liquid phase ion exchange chromatography (WCX stratographic analysis post), the results are shown in accompanying drawing 3.
(5) result comparison
Compare with contrast culture technique, segmentation is cultivated and obviously to be extended cell plateau, cultivates 15 days Cell viabilities still more than 75%, and expression amount can reach 1.63g/L, and output improves approximately 25%.After cooling, antibody acidic components also have obvious reduction.In addition, under two kinds of culture condition, high mannose ratio is all less than 6%, the results are shown in Table 1.
Table 1
| Culture process | Acidic components ratio (%) | High mannose ratio (%) |
| Segmentation is cultivated | 11.0 | 4.1 |
| Contrast culture | 15.3 | 4.9 |
Claims (4)
1. one kind adopts stream of cells to add the method that training method is prepared monoclonal antibody, it is characterized in that described method comprises the steps: 1) cell cultures initial temperature is 36-37 ℃, cultivate 5-8 days, when Chinese hamster ovary celI grows to suitable density, add fed-batch medium, carry out feeding culture; 2) reduce temperature to 31-33 ℃, carry out feeding culture, reduce temperature and finish cell cultures after 2-7 days.
2. method according to claim 1, is characterized in that, the cell inoculum density of described method is 0.3-0.6*10
6cells/ml, PH scope 6.7-7.4, dissolved oxygen 30-50% air saturation, mixing speed 50-300rpm.
3. method according to claim 1, is characterized in that, described method dissolved oxygen 40% air saturation, mixing speed 80-250rpm.
4. method according to claim 1, is characterized in that, the culture cycle of described method is 7-15 days.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107904200A (en) * | 2017-10-20 | 2018-04-13 | 通化东宝生物科技有限公司 | A kind of Combined culture base for expressing adalimumab and its application |
| CN108463730A (en) * | 2015-11-05 | 2018-08-28 | 格纳西尼有限公司 | Including the composition of recombinant human thyroid-stimulating hormone and the method for producing recombinant human thyroid-stimulating hormone |
| CN112011514A (en) * | 2019-05-31 | 2020-12-01 | 百济神州(苏州)生物科技有限公司 | Cell culture process for improving ADCC activity of antibody |
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