CN102382794B - Perfusion culture method of mammal cell - Google Patents
Perfusion culture method of mammal cell Download PDFInfo
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- CN102382794B CN102382794B CN 201010268418 CN201010268418A CN102382794B CN 102382794 B CN102382794 B CN 102382794B CN 201010268418 CN201010268418 CN 201010268418 CN 201010268418 A CN201010268418 A CN 201010268418A CN 102382794 B CN102382794 B CN 102382794B
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Abstract
The invention relates to a perfusion operation method for large-scale culture of mammal cells, which is suitable for culturing the mammal cells with recombinant protein, and more suitable for culturing the mammal cells capable of producing TNFR-Fc fusing protein. The method has the following characteristics: firstly, engineering cells are amplified gradually in the generation and amplification process; secondly, the engineering cells are cultured in a of fermentation tank of 5L; and thirdly, during the later stage of the large-scale culture, the expression amount of the protein is improved byreducing the culturing temperature, lowering the pH value, reducing the rotation speed and adjusting the filling speed rate. According to the process, the production period is prolonged, the cell density is increased, the cell viability is improved, and the expression amount of the protein is improved greatly.
Description
Technical field
The present invention relates to a kind of perfusion culture method of mammalian cell.
Background technology
Animal cell culture starts from the initial stage in this century, and its scale in 1962 begins to enlarge, and development has become the technological method that biology and medical research and Application Areas extensively adopt so far.The biological products such as enzyme, somatomedin, vaccine and monoclonal antibody that utilize animal cell culture production to have important medical value have accounted for 50% of world's biological high-technology produce market share, become the important component part of biological medicine hi-tech industry.
The animal cell large-scale culture technique is important link in the biotechnological pharmaceutics, can be divided into attached cell and suspension cell according to the growth characteristics of cell; Can be summarized as suspension culture and immobilization with regard to its cultural method cultivates; With regard to operating method, can be divided into batch-type, feed supplement-in batches or stream and add formula, semi continuous, continous way or four kinds of operating method of perfusion type.It is two kinds of the most frequently used in cell cultures pattern operating method that feeding culture and perfusion are cultivated
Produce the course of industrialization of albumen at present with the mammalian cell large scale culturing, can be regarded as with the stirring type bioreactor suspension culture as the current techique platform, use 3 kinds of cells (CHO, SP2/0, NSO) commonly used to rely on this platform technology and its throughput is improved.The characteristics of platform technology are to adopt serum-free culture and ripe stream to add and perfusion technology.
Continuously to cultivate be to be used for animal cell culture in recent years to produce a kind of mode that secretor type reorganization curative drug and genetic engineering antibodies such as chimeric antibody and humanized antibody are comparatively praised highly to perfusion.There is Genzyme in the company that uses continuous perfusion technology, Genetic Institute, Bayer company etc.Units such as domestic The Fourth Military Medical University, East China University of Science, Shanghai Inst. of Cytobiology, Chinese Academy of Sciences have been engaged in the research of this respect.
With regard to the technology of stirring type bioreactor suspension culture zooblast, present international upper reaches adds or the viable cell density of perfusion culture biotechnology cell (as Chinese hamster ovary cell CHO, hybridoma hybridoma) can reach 10
7Level.Domestic people such as the Feng Qiang of The Fourth Military Medical University report in 2002 adopts 5L CelliGen reactor continous pouring to cultivate hybridoma, the relation of growth, metabolism situation and the oxygen consumption of research hybridoma in serum free medium is cultivated the 9th day cell density and is reached 8 * 10
6More than the cells/mL.
The advantage that perfusion type is cultivated comprises: the cell closure works system can make cell or enzyme be retained in the reactor, keeps higher cell density, generally can reach 10
7-10
9Cells/mL, thus bigger raising the output of product; 2 continuous irrigation streaming systems make being in preferably the nutrient environment of cytotostatic, and harmful metabolic waste concentration accumulation is lower; 3 speed of reaction are controlled easily, and culture cycle is longer, can boost productivity target product rate of recovery height; 4 products residence time in jar is short, can in time be recovered under the low temperature and preserve, and is conducive to keep product activity.
This method greatest difficulty is that pollution probability is higher, the stability problem of emiocytosis product in the long-term cultivation, engineering problem in the scale amplification process.
The raising of animal cell large-scale perfusion culture technique level at present mainly concentrates on the aspects such as raising of change, productive rate and quality product of optimization, the cell characteristics of further expansion, the cell culture environment of cultivation scale.Wherein the most basic is the culture condition of optimizing cell, eliminates as far as possible or alleviates environment to the influence of cell, keeps the high viability of cell and efficiently expresses, and also will take into full account the subsequent purification of cell expression product simultaneously.
Theoretically, the target protein that mammalian cell expression system produces is generally glycoprotein, this type of recombination fusion protein is because complex structure, it is assembled in cell, transhipment and secreting in the process to born of the same parents, might form the albumen of false folding because the influence of culture environment produces the mispairing of disulfide linkage, perhaps form two kinds of isomer, influence the activity of protein structure domain then.Therefore, can reduce the expression amount of protein misfolding or reduction non-activity isomer by the control to fermentation condition.
A lot of documents or patent have been introduced the optimization of animal cell culture condition, comprise pH, dissolved oxygen, temperature and mixing speed etc.
Chen Zhinan is controlled to be 40-80rpm with temperature 36-37, pH6.5-7.4, stirring velocity in CN1557948A, and has adjusted the flow velocity of nutritive substance on this basis, has obtained effect preferably in using Hybridoma Cell Culture manufacture order clonal antibody.
B.M. execute woods and adopted temperature transition in the training period twice at CN101044239A " mammalian cell culture processes that is used for protein production ", temperature when wherein temperature is lower than the initial cell cultivation when incubation period finishes.High cell survival has been kept in this adjustment, can produce bigger end last titre and high quality protein matter product of protein.
Helmut is at US7, and the change of the pH that studies show that nutrient solution among 157, the 557B2 can cause that components contents changes in the target protein, and the change of temperature can reduce the false folding rate of albumen.
TEIJIN LTD discloses the technical scheme of perfusion culture mammalian cell under cell concn and glucose concn at Japanese patent application JP5146290, perfusion culture mammalian cell human B cell hybridoma, keep substratum (ITES-Erdf stationary phase, comprising Transferrins,iron complexes, Regular Insulin, thanomin, Sodium Selenite) middle glucose concn is at 3.5-30mmol/l, and cell density surpasses 8 * 10
6Cells/mL.
Chen Zhinan discloses the continous pouring formula and has cultivated the processing method that hybridoma prepares antibody in patent documentation ZL01131736, inoculating cell adopts fixed bed to fill huge carrier, cellular metabolism, growth velocity, the synthetic balance regulation of product is to glucose, index such as lactose and pH is analyzed, before cell enters the paracme by logarithmic phase, add corresponding amino acid, and reduction glucose and serum-concentration, cell is by being main transferring to product to synthesize the master with the growth metabolism, realize a plurality of stable states cultivations, hybridoma is in low/serum free medium cultured continuously 20-40 days, and maximum cell density can reach (3-9) * 10
7Cells/mL, antibody production 200-800mL/L/d.
Summary of the invention
At present, the method ubiquity expression amount that the mammalian cell large scale culturing is produced target protein is low, fermentation period short, cell density and vigor defect of insufficient, and then there are complicated operating process in some perfusion cultivation aspect new technologies, be unfavorable for problem such as enlarged culturing scale.Therefore, the invention provides the novel method that a kind of mammalian cell large scale culturing is produced target protein, concrete steps are:
(1) the target cell strain is being carried out amplification culture step by step in initial serum free medium, and every 72h rolling bottle that goes down to posterity is cultivated, behind the cell amplification through 30mL, 90mL, 300mL, 900mL, 1500mL rolling bottle, when total cellular score reaches 1 * 10
8When individual, transfer in the 5.0L fermentor tank.Rotating speed was 75 rev/mins when rolling bottle was cultivated.
(2) carry out the high-density culture stage in fermentor tank, the temperature of cultivating initial stage to cell log vegetative period in the high-density culture stage is that 36-37 ℃, pH are that 7.1-7.2, mixing speed are 150rpm; When cell density reaches 8 * 10
6During cells/mL, adjusting temperature and be 31-33 ℃, adjustment pH is that 6.9-7.0, adjustment mixing speed are 80-100rpm; At fermentation culture Whole Process Control glucose concn 0.5-3g/L, dissolved oxygen 50%.
The control of described step fermenting process temperature in early stage is at 36-37 ℃; Reach 8 * 10 at cell density
6Reduce the temperature to 31-33 ℃ during cells/mL, preferably reducing temperature is 33 ℃, and cellular metabolism is slowed down, product is synthetic in a large number, simultaneously can keep high cell density and vigor for a long time, reduce the formation of mispairing disulfide linkage in the purpose product, increase the expression amount of purpose product.
PH7.1-7.2 is the suitableeest growth potential of hydrogen of engineering cell strain, so at the fermentation culture initial stage pH is controlled at 7.1-7.2; When cell density reaches 8 * 10
6Cell enlargement trend is slowed down during cells/mL, reaches logarithmic growth latter stage, and express the stage of stable development for the cell target protein this period, at this moment reduces pH, more is conducive to the expression of albumen.So reach 8 * 10 at cell density
6Cells/mL reduces to 6.9-7.0 with pH by 7.1-7.2, and preferably reducing pH is 6.9.
Because the acellular wall of zooblast, to the shearing force sensitivity, so excessive speeds produces shearing force to cell easily, it is inhomogeneous that low excessively rotating speed can cause nutrient solution to mix again, and therefore suitable rotating speed is particularly important for cell cultures.The control of fermenting process rotating speed in early stage is at 150rpm; Reach 8 * 10 at cell density
6During cells/mL mixing speed is reduced to 80-100rpm, preferably reducing mixing speed is 82rpm, is beneficial to the expression of target protein, can also increase cell viability simultaneously, prolongs the time of cell cultures.
Glucose is energy supply material and the main source of carbon material of cell, yet can produce a large amount of meta-bolites lactic acid when its concentration is higher, thereby need carry out its concentration control, the concentration that is unlikely to produce a large amount of by products enough to keep cell growth is good, therefore at fermentation culture Whole Process Control glucose concn 0.5-3g/L.
The present invention has obtained good culture effect by the comprehensive regulation of these culture condition: compare with usual manner, the cell cycle has prolonged 8 days behind the change fermentating controling condition, is extended for 22 days by 14 days; Cell density was 6.12 * 10 in the time of 22 days behind the change fermentating controling condition
6Cells/mL, cell viability were 68% in the time of 22 days, and original fermentation condition cell density in the time of 14 days only is 6.02 * 10
6Cells/mL, cell viability has reduced to 61% in the time of 14 days; The target protein expression amount has improved 240mg/L after changing fermentating controling condition, has brought up to 450mg/L by 210mg/L; And the present invention also has better operability and practicality.Therefore, the present invention more is conducive to the enlarged culturing scale.
Description of drawings
Cell density under two kinds of fermentation condition controls of Fig. 1 compares, and ordinate zou is represented cell density, and X-coordinate represents to cultivate fate.It is 22 days that fermentation control 1 changes the fermentating controling condition secondary fermentation cycle, and cell density is 6.12 * 10 in the time of 22 days
6Cells/mL; Fermentation control 2 original fermentation condition fermentation periods are 14 days, and cell density is only 6.02 * 10 in the time of 14 days
6Cells/mL.
Cell viability under two kinds of fermentation condition controls of Fig. 2 compares, and ordinate zou is represented cell viability, and X-coordinate represents to cultivate fate.The fermentation 1 cell change fermentating controling condition secondary fermentation cycle of control is 22 days, and cell viability is 68% in the time of 22 days, and fermentation control 2 original fermentation condition fermentation periods are 14 days, and cell viability has reduced to 61% in the time of 14 days.
Specific embodiment
Below further describe the present invention by embodiment, but the invention is not restricted to following examples.
The amplification step by step of embodiment 1 cell
The recovery of seed cell and amplification and high density fermentation stage are all selected serum free medium for use.
From the cell bank liquid nitrogen, get a frozen seed cell, it is the recombinaant CHO cell of high expression level TNFR:Fc fusion rotein, put into rapidly in 37 ℃ of warm water and recover, the nutrient solution that adds 37 ℃ of preheatings is blown and beaten gently, the centrifugal 5min of 800rpm, abandon supernatant, in precipitation, add the substratum 40mL of 37 ℃ of preheatings, put 5%CO
2Constant incubator is cultivated.Go down to posterity behind the 72hr, the centrifugal 5min of 800rpm abandons supernatant, adds the substratum of fresh 37 ℃ of preheatings in the precipitation, makes cell density reach 2.0 * 10
5More than the cells/mL.
In the passage amplification procedure, amplification culture container step by step is through the cell amplification of 30mL, 90mL, 300mL, 900mL, 1500mL rolling bottle, when total cellular score reaches 1 * 10
8When individual, transfer in the 5.0L fermentor tank and cultivate amplification.
Fermentation culture in the embodiment 25L jar
The high-density culture stage of fermentative production is still used serum free medium, keeps the stable of each stage parameter of fermentation.In whole process, adopt open perfusion culture fermentation, monitoring cell density and glucose consumption, according to the consumption of glucose, to control glucose concn be 0.5-3g/L by adding certain density glucose continuously; Dissolved oxygen is controlled to be 50% of oxygen meltage in the air.
At the 5L fermentor cultivation initial stage, the culture temperature of engineering cell strain maintains 36-37 ℃; PH is controlled to be pH7.1-7.2, by adding CO in the fermenting process
2And NaHCO
3It is stable to keep it; Mixing speed control is at 150rpm;
When cell density reaches 8 * 10
6During cells/mL, cell enlargement trend is slowed down, and reaches plateau, at this moment reduces temperature, pH and rotating speed.Temperature is reduced to 31-33 ℃ by 36-37 ℃, and preferably reducing temperature is 33 ℃; PH reduces to 6.9-7.0 by 7.1-7.2, and preferably reducing pH is 6.9; Mixing speed is reduced to 80-100rpm by 150rpm, and preferably reducing mixing speed is 82rpm.
In 5L fermentor cultivation whole process, every day sampling and measuring cell density, vigor and glucose concentration, the interpolation speed of regulating glucose according to the concentration of the glucose of surveying, making glucose concn is 0.5-3g/L, regulate perfusion and receive liquid speed according to the situation of cell density simultaneously, make cell keep higher vigor for a long time.Cell viability reduces to 70% when following, stops perfusion and receives liquid, finishes fermentation.
The simultaneous test of 3 two kinds of fermenting process controls of embodiment
One, fermentation control 1
Fermenting process is controlled to be earlier fermentation pH7.1-7.2, mixing speed 150rpm, and dissolved oxygen 50%, reaches 8 * 10 at cell density by temperature 36-37 ℃
6During cells/mL, begin to reduce pH to 6.9 when namely reaching stationary phase, reduce the temperature to 33 ℃, the reduction mixing speed is 82rpm, and dissolved oxygen is 50%, and the concentration of control glucose is 0.5-3g/L.
Two, fermentation control 2
Control pH7.1-7.2 mixing speed 150rpm in the whole fermentation process, dissolved oxygen 50%, temperature 36-37 ℃, the concentration of control glucose is 0.5-3g/L.
Test-results sees Table 1, Fig. 1 and Fig. 2.The result shows that the present invention has obtained good culture effect by the comprehensive regulation of these culture condition: compare with usual manner, the cell cycle has prolonged 8 days behind the change fermentating controling condition, is extended for 22 days by 14 days; Cell density was 6.12 * 10 in the time of 22 days behind the change fermentating controling condition
6Cells/mL, cell viability were 68% in the time of 22 days, and original fermentation condition cell density in the time of 14 days only is 6.02 * 10
6Cells/mL, cell viability has reduced to 61% in the time of 14 days; The target protein expression amount has improved 240mg/L after changing fermentating controling condition, brought up to 450mg/L by 210mg/L, and the present invention has better operability and practicality.Therefore, the present invention more is conducive to the enlarged culturing scale.
Fermentation period under two kinds of fermentation condition controls of table 1 and expression amount are relatively
| Fermentation control | Fermentation period (my god) | Expression amount (mg/L) |
| 1 | 22 | 450 |
| 2 | 14 | 210 |
Claims (6)
1. the perfusion working method that mammalian cell is cultivated comprises the steps:
(1) the target cell strain is carried out in initial medium step by step the amplification culture stage and
(2) in fermentor tank, carry out the cell cultures stage, it is characterized in that temperature that the cell cultures stage cultivates initial stage to cell log vegetative period is that 36-37 ℃, pH value are that 7.1-7.2, mixing speed are 150rpm; When cell density reaches 8 * 10
6During cells/mL, temperature is adjusted to 33 ℃, and the pH value adjusts to 6.9, and mixing speed is adjusted to 82rpm; Described target cell strain is Chinese hamster ovary celI.
2. perfusion working method as claimed in claim 1 is characterized in that described initial medium is serum free medium.
3. perfusion working method as claimed in claim 1 is characterized in that in the described stage of amplification culture step by step that every 72h rolling bottle that goes down to posterity cultivates, behind the cell amplification through 30ml, 90ml, 300ml, 900 ml, 1500 ml rolling bottles, when total cellular score reaches 1 * 10
8When individual, transfer in the 5.0L fermentor tank.
4. perfusion working method as claimed in claim 3, shaking speed is 75 rev/mins when it is characterized in that described rolling bottle is cultivated.
5. perfusion working method as claimed in claim 1 is characterized in that cell cultures stage Whole Process Control glucose concn 0.5-3g/L.
6. perfusion working method as claimed in claim 1 is characterized in that cell cultures stage Whole Process Control dissolved oxygen 50%.
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| SG10201802023RA (en) | 2013-03-26 | 2018-05-30 | Coherus Biosciences Inc | Protein production method |
| EP3529350A1 (en) * | 2016-10-19 | 2019-08-28 | H. Hoffnabb-La Roche Ag | Method for producing an immunoconjugate |
| CN107974432B (en) * | 2017-08-11 | 2021-04-23 | 浙江海隆生物科技有限公司 | Culture method and application of CHO cell strain capable of efficiently secreting and expressing hog cholera E2 protein |
| CN110713970A (en) * | 2019-10-23 | 2020-01-21 | 金宇保灵生物药品有限公司 | Continuous production method for suspension culture, preservation and recovery of BHK21 cells by using bioreactor and application thereof |
| CN110894494B (en) * | 2019-11-22 | 2022-09-27 | 广西梧州制药（集团）股份有限公司 | Method for large-scale high-density suspension culture of 293 cell high-yield adenovirus |
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| CN1869214A (en) * | 2005-05-24 | 2006-11-29 | 中国人民解放军军事医学科学院基础医学研究所 | Method for high efficiency expressing destination protein |
| CN101205530B (en) * | 2006-12-20 | 2011-02-09 | 上海国健生物技术研究院 | Method for high-effectively expressing recombinant protein by employing animal cell fed-batch culture mode |
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Effective date of registration: 20161230 Address after: 276005 Hongqi Road, Shandong, Linyi, No. 209 Patentee after: Lunan Pharmaceutical Group Co., Ltd. Address before: 276005 Shandong province Feixian County North Ring Road No. 1 Patentee before: Shangdong New Time Pharmaceutical Co., Ltd. |