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    • 专利摘要:
    improved cell culture process. the present invention relates to a cell culture process for the production of polypeptides in mammalian cho cells characterized by one or more changes in temperature and pH that are adjusted in relation to their moment and the step size to reduce the cell death, increase product yield and improve product quality.
    公开号:BR112012027430B1
    申请号:R112012027430
    申请日:2011-04-25
    公开日:2020-04-07
    发明作者:Leist Christian；E Joosten Christoph；Schmidt Jörg
    申请人:Novartis Ag；
    IPC主号:
    专利说明:

    Descriptive Report of the Invention Patent for “PROCESS FOR THE PRODUCTION OF A RECOMBINANT POLYPEPTIDE UNDERSTANDING CHO CELL CULTIVATION”.
    Technical Field of the Invention
    The present invention relates to the general field of biotechnology, particularly the cultivation of cells and their use for the production of polypeptides on an industrial scale.
    The present invention provides cell culture processes characterized by at least one change in temperature and at least one change in pH. These processes are suitable for the cultivation of cells with high cell viability, preferably mammalian cells such as CHO cells. The cell culture processes according to the present invention also allow to obtain high polypeptide productivity when used for the production of a polypeptide, in particular through the recombinant expression of polypeptides in mammalian cell culture systems, in particular in industrial scale.
    Technical Background of the Invention
    The preparation of polypeptides using recombinant technology has been developed in a standardized procedure over the past two decades. Access to the recombinant polypeptides by cloning the genes encoding the respective polypeptide followed by the subsequent transformation of suitable expression hosts with the gene to be expressed and the production and final purification of the obtained recombinant polypeptide product was provided for a new class of biologically planned and produced therapeutic agents.
    Pharmaceutically active compounds have been prepared in increasing numbers in the pharmaceutical industry using recombinant DNA technology followed by production processes developed in the field of bioengineering.
    Such biological products include monoclonal antibodies that have been developed into important treatment options in several fields of medicine including autoimmune diseases, inflammatory disorders, immunosuppression, oncology and the like.
    Petition 870190102985, of 10/14/2019, p. 11/19
    2/31
    The development of such therapeutic agents of biological origin requires production on an industrial scale thereby providing access to large amounts of recombinant polypeptide. Preferred expression systems are mammalian cell cultures that are superior to most other eukaryotic systems based on insect, yeast or similar cells or even traditional prokaryotic expression systems.
    However, culturing mammalian cells includes enormous challenges especially on an industrial scale. Mammalian cell culture production units require complete optimization of many process conditions.
    In particular, cell culture processes for the production of polypeptides in mammalian cells require continuous optimization of culture conditions and their adaptation to specific cell lines or products in order to achieve a high volumetric yield of the product in combination with quality optimal product.
    Much previous effort has focused on the basic parameters of cell culture media including their composition in relation to, for example, the types and concentrations of ions, amino acids, vitamins or trace elements or the osmolarity of the medium. Additional important parameters that were in focus in the research are, for example, food composition or feeding programs to achieve optimal cell growth.
    Also temperature and pH as basic physiological parameters are known to have a significant influence on the culture of mammalian cells. The temperature in general considerably affects the growth state and the viability of the cells. In addition to this, it can, however, even more specifically influence the polypeptide product and its characteristics by altering, for example, glycosylation (US 2003/0190710 A1; EP 1 373 547 A1; US 2004/0214289 A1).
    The pH at which the growth medium and cells are maintained can also influence and alter cell growth and the production of po3 / 31 lipeptides in a specific way that depends on the particular cell line and product (Sauer et al., Biotechnology and Bioengineering 2000, Vol 67, pp. 586-597; Yoon et al., Biotechnology and Bioengineering 2004, Vol 89, pp. 346-356; Kuwae et al., Journal of Bioscience and Bioengineering 2005, Vol 100, pp. 502 -510).
    Over the course of the culture time, the requirements of the cells may change. Although in the beginning it was advantageous to optimize conditions towards improved cell growth, in later stages greater cell survival and maintenance of viable cell density in association with obtaining high product titles will become important. In this regard, the introduction of one or more temperature steps during cell culture has been suggested (Chen et al., J Biosci Bioeng. 2004; 97 (4): 239-43). To do this, mammalian cells are grown at at least two different temperatures, where the first highest temperature is optimized for cell growth while the second or third lowest temperature is selected to improve cell productivity (for example , Weidemann et al., Cytotechnology. 1994; 15 (1-3); 111-6; WO 00/36092; EP 0 764 719 A2, US 2005/019859, EP 1 575 998, US 2008/081356). Other documents describe the use of temperature steps in combination with additional specific media characteristics. EP 1 757 700 A2, for example, discloses a temperature step in combination with the presence of butyrate salts as a component of the medium, whereas EP 1 789 571 A1 describes a temperature step combined with a defined amino acid content.
    Still other cell culture conditions have been changed. US 5 856 179 introduced a method for the production of polypeptides in a fed batch cell culture, in which during cultivation the osmolarity of the medium is considerably changed from approximately 280-330 mOsm in the main growth phase to approximately 400600 mOsm during the production phase.
    WO 02/101019 focused on specific components of the medium such as concentrations of glutamine and glucose, including changes
    4/31 in temperature and pH. However, it was observed that a change in pH in a medium with a high glucose content had a negative impact on the culture and that it is not recommended to reduce the pH during the growth or production phase.
    WO 2006/026445 discloses a method for producing polypeptides in which cell culture conditions are changed from one set of culture conditions to a second set in which this change is combined with specific characteristics of the medium in relation to the content of specific amino acids. The change in conditions refers specifically to changes in temperature. Other changes in conditions such as pH or osmolarity are generally mentioned as additional options, however, settings for particular parameters are not specified.
    Considering the previous challenges and the existing disadvantages, there is a continuing need in the field of industrial biotechnology for improved culture processes that allow the production of recombinant polypeptides on industrial scales with even higher yields, that is, improved specific and general productivity and higher quality of the product. product.
    A specific technical objective of polypeptide production processes is to maintain high cell viability and to maximize the final yield of the polypeptide by optimizing the parameters of the general cultivation process.
    Summary of the Invention
    The present invention relates to the combination of changes in temperature and pH in a process for producing recombinant polypeptides. Adapted to the needs of recombinant cells, in particular CHO cells, specific combinations of these two parameters lead to higher cell productivity as well as higher product quality of the recombinantly produced polypeptides. In particular, the present invention found positive effects based on the particular moment and the schedule of the change (s) in temperature and pH in
    5/31 absolute and relative terms as well as in relation to the particular magnitude of the changes.
    According to one aspect of the invention, a process for producing a recombinant polypeptide is disclosed which comprises the cultivation of CHO cells in a medium and the expression of the recombinant polypeptide in which the temperature and pH are changed during the process.
    In particular, the process according to the present invention involves at least one change in temperature and at least one change in pH. In one embodiment of the present invention, a change from a first higher temperature to a second lower temperature is performed after the cells are first grown and maintained for at least 3 days, alternatively at least 4 days or at least 5 days at a first temperature. The second lowest temperature is approximately 1 to approximately 8 ° C lower than the first temperature. In an alternative embodiment of the present invention, the change in temperature is, for example, between approximately 2 and approximately 5 ° C, in particular approximately 4 ° C or approximately 3.5 ° C. The second temperature is then maintained for at least two days. The second temperature can be maintained until collection.
    According to an embodiment of the present invention, the first temperature is preferably in the range of approximately 33 ° C to approximately 38 ° C and the second temperature is preferably in the range of approximately 30 ° C to approximately 37 ° C.
    In addition to the change in temperature, the pH is also changed from a first to a second pH. Thus, the processes according to the present invention comprise at least one change in pH, in particular, cells are grown at a first pH value for at least 2 days and the pH is then changed to a second pH value which is between approximately 0.05 and approximately 1 pH unit less than the first pH and the cells are grown at said second pH for at least 1 day, alternatively for at least 2 days. In some modalities, the second pH will be maintained until collection.
    6/31
    The first pH value is preferably in the range of approximately pH 6.8 to approximately pH 7.5. The second pH value is preferably in the range of approximately pH 6.0 to approximately pH 7.1.
    Thus, one embodiment of the present invention is a process for producing a recombinant polypeptide that comprises culturing CHO cells in a medium under conditions that comprise at least one change in temperature and at least one change in pH and expression of the recombinant polypeptide on what
    - the cells are grown at a first temperature for at least 3 days and the temperature is then changed to a second temperature which is between approximately 1 and approximately 8 ° C lower than the first temperature and the cells are maintained at said second temperature for a period of at least 2 more days;
    - the cells are grown at a first pH value for at least 2 days and the pH is then changed to a second pH value which is between approximately 0.05 and approximately 1 pH unit lower than the first pH and the cells are grown at said second pH for at least 1 day.
    The process according to the present invention can optionally comprise a second change in pH, which follows the first change in pH after at least 1 day. If the first change in pH is followed by a second change in pH after at least 1 day then the third pH value is approximately 0.05 pH unit to approximately 1 pH unit greater than the second pH value. The third pH value can be maintained until collection.
    The cell culture process according to the present invention includes active and / or passive changes in pH, that is, the pH is actively changed by a change in the pH set point to a new value and / or passively allowing a change of the pH of the medium through the accumulation of metabolic products, thus following a specific pH profile of the cell culture within the predefined pH range. In a preferred embodiment of the invention, the active change is induced by adding the respective pH change and regulating the agent (s) known to the person skilled in the art, such as acids, for example, HCI or bases, for example, NaOH. In an additionally preferred mode of the process, that is, carried out by defining a pH setpoint and a termination point at which the pH is allowed to be changed. In contrast to the active change, the passive change or the change in pH is not induced by adding the respective agent (s) that change the pH.
    In a further aspect, the process according to the invention is carried out using a medium that is free of proteins and whey. Preferably, the medium is characterized by a total amino acid content of between approximately 40 mM and approximately 100 mM, alternatively between approximately 50 and approximately 100 mM.
    A preferred process which is defined above is carried out in batch fed mode comprising feeding at least two nutrient solutions which are added to the culture. In such a process, for example, one of the feed solutions added to the culture medium is a feed comprising the cystine dipeptide and the amino acid tyrosine. It is further preferred that the feed comprises the cystine dipeptide and the amino acid tyrosine in respective concentrations in the range of approximately 6.5 g / L and approximately 8.0 g / L and in the range of approximately 9 g / L and approximately 11 g / L in an aqueous solution at a basic pH above 10. In particular, concentrations can be approximately 7.25 g / L for cystine and approximately 10.06 g / L for tyrosine. In a preferred embodiment, the feed solution comprising cystine and tyrosine is added to the culture medium in the range of approximately 0.2 and approximately 0.8% by weight of the initial weight of the culture medium per day or alternatively to approximately 0, 4% by weight of the initial weight of the cell culture medium per day.
    The process according to the invention is preferably used for the production of a recombinant polypeptide that is glycosylated.
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    According to specific modalities, the polypeptide is an antibody or an antibody fragment.
    Brief Description of Drawings
    The invention will be better understood with reference to the following examples and figures. The examples, however, are not intended to limit the scope of the invention.
    Figure 1 is an illustration of the implementation of changes in stages of an active change in pH with a change from pH 7.00 to 6.80.
    Figure 2A shows a pH profile obtained by a passive change in the pH implementation. The change in pH from 7.00 to 6.80 in the production bioreactor was reached at the set point up to 6.90 and defining an end point of 0.10. After a first change in pH to 6.80, the pH is actively maintained at 6.80 until the end of the culture.
    Figure 2B shows a pH profile with a second change in pH. In this example, the upper (previous) pH limit is not reached.
    Figure 2C shows a pH profile with a second change in pH, but here the pH meets the upper pH limit again and is maintained there.
    Figure 3 shows the effect of a constant temperature versus a change in temperature on the viable cell density of a mAb1 producing CHO cell as a function of the culture time in shake flask cultures (see example 1).
    Figure 4 shows the effect of a constant temperature versus a change in temperature on the viability of a mAb1 producing CHO cell clone (see example 1).
    Figure 5 shows the product title as a function of cultivation time for shaken flask cultures of a mAb1-producing CHO cell clone with and without a change in temperature (see example 1) ·
    Figure 6 shows the lactate concentration over time in a clone that produces mAb2 (see example 2).
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    Figure 7 shows viable cell density as a function of cultivation time in a 300-L bioreactor with a CHO cell clone. The culture conditions included a temperature step (day 5) and two changes in pH due to pH regulation with an adjustment point and an end point (see also example 2).
    Figure 8 shows the product title as a function of cultivation time in a 300-L bioreactor with a CHO cell clone. The process combined a temperature with changes in pH (see also figure 7 and example 2).
    Figure 9 shows for three independent experiments the concentration of mAb3 obtained through a batch process fed with cultures of CHO cells grown in a glass bioreactor as a function of the viable cell integral. Culture conditions included an identical change in temperature for all three experiments and an additional change in pH in one experiment only.
    Detailed Description of the Invention
    In accordance with the present invention, a process for preparing a recombinant polypeptide comprises culturing CHO cells and expressing the recombinant polypeptide in which the temperature and pH are changed during the process. The present invention seeks to improve the process of large-scale production of polypeptides in CHO cell culture through the dynamic adaptation of cell culture conditions over the course of culture including changes in temperature and pH.
    The term large-scale production of polypeptides refers to the amounts typically required for the industrial production of recombinant polypeptides used for the preparation of therapeutically active biopharmaceutical products. Cell cultures with cell culture media of at least 500 L in volume or at least 1000 L or alternatively at least 5000 L or even larger volumes typically represent large scale production applications.
    The term cell culture medium as used herein refers to an aqueous solution of nutrients that can be used for cell growth over an extended period of time. Typically, cell culture media include the following components: an energy source, which will generally be a carbohydrate compound, preferably glucose, amino acids, preferably the basic set of amino acids, including all essential amino acids, vitamins and / or other organic compounds that are required in low concentrations, free fatty acids and inorganic compounds including trace elements, inorganic salts, buffer compounds and nucleosides and bases.
    The use of cell culture media in the field of the pharmaceutical industry, for example, for the production of therapeutically active recombinant polypeptides, generally does not allow the use of any material of biological origin due to safety and contamination issues. Therefore, the cell culture medium according to the present invention is preferably a serum and / or protein-free medium. The term serum and / or protein-free medium represents a fully chemically defined medium that does not contain additives of animal origin such as tissue hydrolysates, for example, fetal bovine serum or the like. In addition, proteins, especially growth factors such as insulin, transferrin or the like, are also preferably not added to the cell culture according to the present invention. Preferably, the cell culture medium according to the present invention is also not supplemented with a hydrolyzed protein source such as soy, wheat or rice peptone or yeast hydrolyzate or the like.
    The term change in temperature as used here refers to a change in the temperature of the culture in a bioreactor / culture vessel by actively changing the temperature setpoint to a lower value. The temperature is first controlled and stabilized at a predefined temperature over a period of time and after changing the setpoint it is then stabilized at another defined temperature over a period of time. The temperature step does not refer to small spontaneous temperature fluctuations in the culture.
    11/31
    The term change in pH as used here refers to a change in the pH of the culture in a bioreactor / culture vessel by actively changing the pH set point to a lower or higher value or allowing a change in pH to occur between an upper and lower pH limit.
    Depending on the size of the culture vessel / bioreactor and the volume of culture, the change in the respective parameter that is measured in the medium can take the form of a few minutes to several hours.
    The pH can be changed in two different ways, using an active and / or passive approach as described in more detail below.
    The term active change in pH is defined by a change in the pH set point to a new value. In a preferred embodiment of the invention, the active alteration is induced by adding the respective agent (s) that alter (s) and regulate (pH) the pH known to the expert.
    The term passive change indicates that during passive change in pH the cells alone are allowed to change the pH of the medium through the accumulation of metabolic products, thus following a specific metabolic pH profile of the cell culture within a predefined pH range . In a modality of the process this is accomplished by defining a pH set point and an end point at which the pH is allowed to be changed. In contrast to the active change, the passive change or change in pH is not induced by the addition of the respective pH change agent (s).
    The pH regulating agents are added to the cultures in order to maintain the pH at a specific set point or to change the pH during the change in pH. Typical pH regulating agents used for cell culture purposes include liquid or acid based solutions such as NaOH or HCI. Such pH regulating agents are added to the media in the culture vessel / bioreactor. Alternatively, the cell culture medium can be carbonated with CO 2 to adjust the pH.
    12/31
    According to a first aspect of the invention, there is disclosed a process for the preparation of a recombinant polypeptide which comprises the cultivation of CHO cells and the expression of the recombinant polypeptide in which the temperature and pH are changed during the process. More in particular, a change from a higher first temperature to a lower second temperature occurs after the cells are first grown and maintained for at least three days, alternatively at least 4 days or at least 5 days at a first temperature. This second lower temperature is approximately 1 to approximately 8 ° C lower than the first temperature. In another embodiment of the invention, the change in temperature can be between approximately 2 and approximately 5 ° C, in some implementations it is approximately 4 ° C or approximately 3.5 ° C. This second temperature is then maintained for at least two days. In addition to the change in temperature, the pH is also changed from a first to a second pH.
    The exact parameters in relation to the change in temperature and pH are determined in advance and adapted based on the needs of the cell line that has been transfected with one or more particular gene constructs that encode the respective polypeptide that is produced. Alternatively, requirements can be made dependent on metabolic parameters that are determined during culture for large-scale production in a bioreactor.
    Changing the temperature from a higher temperature to a lower temperature is useful because the first temperature is optimal for cell growth, while the lower temperature reduces the rate of cell death. A reduced temperature will therefore allow for a longer maintenance of the high density of viable cells. The cell-specific productivity of the polypeptide of interest at this reduced temperature is generally not drastically reduced from the initial temperature, sometimes the cell-specific productivity can be the same or sometimes even higher. Longer maintenance of high density of viable cells can in addition provide the advantage of minimizing
    13/31 the formation of product of inadequate quality. The combination of these factors allows a high volumetric productivity and the achievement of high product titles of interest of adequate quality in the collection time. In one embodiment, the first temperature is in the range of approximately 33 ° C to approximately 38 ° C. In another example, the first temperature is between approximately 36 ° C to approximately 38 ° C. The second temperature reached after the temperature change can be in the range of approximately 30 to approximately 37 ° C or between approximately 32 to approximately 34 C or alternatively between approximately 30 to approximately 32 C.
    The timing of the temperature change is important to maximize productivity. If the change in temperature is made too early, a high cell density will not be achieved or will take a long time to reach. If the change in temperature is made too late, it may not efficiently prevent a decline in the density of viable cells. Preferably, the time of change in temperature is defined in days after inoculation of the bioreactor used for large-scale production of recombinant polypeptides. In another embodiment of the invention, the timing can be defined through the cell density that is achieved in the large-scale production bioreactor. For example, the change in temperature is initiated during the phase of linear or logarithmic growth of the cells or when 40 to 90% of the maximum cell density is reached. A setpoint-dependent cell density can be expressed in relative terms (% of maximum cell density that can be achieved) or absolute terms (viable cells / mL). In a specific example, cell density is chosen to be between 60 to 90%.
    The time between inoculation of the bioreactor / growth vessel and the change in temperature can vary between approximately 3 and approximately 14 days depending on the specific bioreactor / growth vessel and the cell line used. Alternatively, the change occurs between days 3 through 8. As an alternative to a single criterion that is presented above, a dual criterion can also be
    14/31 adjusted by combining two of the variables mentioned above, so that the conditions selected in relation to time and / or cell density have to be satisfied.
    If necessary for optimal growth and production, also more than one temperature step, for example, at least 2 steps can be used, each consisting of a change in temperature of at least approximately 1 ° C, alternatively at least approximately 2 ° C , where each temperature is maintained for at least one day. Thus, temperatures can be further reduced and a more complex temperature profile can be followed.
    According to the invention, the cells are grown at a first pH value before the change in pH for at least 2 days, alternatively for at least 3 days, for example, for at least 4 days or for at least 5 days. The pH for the first few days after starting the culture is chosen as being favorable for the rapid expansion of cell density in the bioreactor. During this time the pH of the bioreactor is controlled at a certain set point which is optimal for cell growth. Once a certain cell density is reached, it is advantageous to modify the pH of the culture. The pH is changed after this first period of time to a second pH value which is between approximately 0.051 pH unit less than the first pH. The cells are grown at said second pH for at least 2 days. In some embodiments of the present invention, the second pH value can be approximately 0.15 to approximately 1 pH unit less than the first pH. This change in pH is generally achieved by changing the pH set point of the bioreactor / culture vessel. The second pH value is selected to reduce cell death (for example, apoptosis) and to allow the maintenance of specific production rates for high quality polypeptide cells. As a consequence, in a first embodiment, the time of change in pH is defined in days after inoculation of the bioreactor which is used for the large-scale production of the recombinant polypeptide. In a second modality, the moment can be defined through the cell density that is reached in the bioreactor on a large scale. According to an additional alternative, the timing may also become dependent on the specific metabolic parameters that are measured during cultivation in the cell culture medium. In a non-limiting example, this can be the lactate concentration. Still non-direct parameters that reflect the metabolic state of the culture can be used, such as, for example, the required CO2 dosage or the acid control for time to maintain the pH at the upper pH setpoint or required NaOH dosage or caustic agent control to keep the pH at a lower pH setpoint. Instead of using just one criterion for the change in pH, alternatively, a combined criterion can be adjusted by combining parameters such as, for example, days after inoculation and cell density.
    The benefits of the pH change strategy also involve the fact that dissolved carbon dioxide levels and the addition of base can be reduced during the culture course, which consequently prevents its negative effects. At the beginning of the culture, it is advantageous to have a higher pH value (for example, 7.0) in the culture vessel or bioreactor, since a lower pH value (for example, 6.8) would require higher levels carbon dioxide in order to maintain 0 pH. These high levels of carbon dioxide, however, can have negative effects on cells and slow the growth rate. In contrast, in late stages of culture, maintaining a high pH (for example, 7.0) requires more addition of base than maintaining a low pH (for example, 6.8). This is because lactic acid is formed and the resulting acidity has to be compensated by adding base. The higher the pH set point, the greater the amount of base required. The addition of base increases the osmolarity of the culture, which can be unfavorable for the growth and maintenance of a high density of viable cells.
    The potential benefits of the pH change strategy can also be described from the perspective of minimizing lactic acid formation. CHO cells generally produce less acid
    16/31 lactic acid at lower pH values (eg 6.8) than at higher values (eg 7.0). Less lactic acid produced results in less base added, which is beneficial as previously described.
    In one embodiment, the first pH is selected to be in the range between pH 6.8 and 7.5. In another embodiment, the first pH is selected to be in the range between pH 6.8 and 7.2. In an additional embodiment, the first pH is selected to be at maximum pH 7 or alternatively below pH 7. The second pH value that is reached after the pH change is in the range between pH 6.0 and pH 7.5 or between pH 6.5 and 6.8.
    The relative moment of change in temperature and pH is selected in order to achieve the most optimal result. The optimum time of change in temperature and pH is chosen on a process-specific basis and may be dependent on the growth state or the metabolic state of the culture. The change in temperature in principle can be implemented at a time point of culture regardless of the time of change in pH. In one embodiment of the invention, the change in temperature can occur before or at the same time as the change in pH. In one embodiment of the invention, the change in temperature occurs before the change in pH. For example, the change in temperature can be initiated between 1-5 days before the change in pH occurs. In another second embodiment, the change in temperature is initiated at the same time as the change in pH of the invention. The term at the same time, here refers to a change in progress of both respective parameters from a first value to a second value. Such simultaneous changes can occur when the temperature set point and the pH set point have been changed and both parameters have not yet reached their second stable value. Alternatively, such a scenario can occur when the temperature set point has been changed during the phase of a passive pH change. This occurs in the case of a pH regulation by an adjustment point and an end point that define an upper and lower pH limit (see also below). In a further embodiment of the invention, the change in temperature can occur after the change in pH. For example, changing
    17/31 in temperature can be started between 1 to 5 days after the change in pH.
    In a further aspect of the present invention the pH is actively or passively changed between said first to said second pH value. There are a number of possible ways to control the pH of crops and to implement a change in pH. In one aspect of the invention the pH is actively changed from a first to a second pH value by changing the pH set point (without the end point) of the pH controller to a new value.
    As a result, the change from the first pH value to the second pH value is almost immediate (change in stages) in the culture. Figure 1 illustrates such an implementation of the step change in pH change, with a change from pH 7.00 to 6.80 in this particular example. In this implementation of the invention, the pH is first maintained at a higher pH value by dosing pH regulating agents accordingly (for example, CO 2 or NaOH) and then changed to a lower value by actively changing the set point (without a period). The pH of the lower set point can be achieved by dosing / actively adding an acidifying agent to the culture resulting in a rapid change or by omitting an agent that maintains the pH at the first most basic pH. Based on the size of the bioreactor and the previously mentioned method of influencing pH, the change in pH can be completed within a few minutes up to 24 hours.
    In a further aspect of the invention, the pH is allowed to be passively changed (decreased) from a first to a second pH value which corresponds to an upper and lower pH limit and thus following a specific metabolic profile of the cell culture. As a result, the change from the first pH value to the second pH value is gradual. This alternative way of controlling the pH of the cell culture medium and implementing a change in pH is achieved by programming the pH controller of the bioreactor with a set point and an end point. This defines a permissible pH range for the process, where the pH controller has no action. For example, a setpoint
    18/31 pH of 6.90 with an end point of 0.10 pH unit will set pH 7.00 as the upper pH limit and pH 6.80 as the lower pH limit. In a cell culture production bioreactor, the pH will typically be at the high limit at the beginning of the culture (first few days), where the controller prevents the pH from increasing by measuring carbon dioxide within the culture. Due to the progressive accumulation of lactic acid produced by cells, the pH will eventually drop continuously from, for example, 7.00 to 6.80, typically within a few hours. Once the lower pH limit of, for example, 6.80 is reached, the controller prevents the pH from decreasing beyond this pH by dosing the base solution within the culture. Based on the size of the bioreactor, the specific cell line, the cell density or the conditions of the medium the gradual change in pH can occur within a few hours or take up to a day.
    In a further aspect of the invention, the second pH of the culture is actively maintained after changing the pH in the second pH for the remainder of the culture time until collection. This is done by changing the pH set point to said second pH value and dosing the pH regulating agents accordingly.
    In a further aspect of the invention, the first change in pH is followed by a second change. The second change in pH occurs at least 1 day after the first and the third pH value that is reached is at least 0.05 pH unit higher than the second pH.
    In a further aspect of the invention, the second change in pH can also occur actively or passively to achieve said third pH value. In the first mode, this can be done by actively changing the pH set point as already shown for the first change in pH (see above). The timing of the second change in pH can be defined in days after the first change in pH and / or again made dependent on metabolic parameters, such as, for example, lactate concentration. Such a change can typically occur between 1 and 10 days after the first change in pH. An active change is initiated by changing the pH set point of the culture,
    19/31 pH will be dosed accordingly.
    In the second modality, the pH can also be allowed to be changed passively and follow its metabolic pH profile. This can be implemented, for example, by setting a lower and upper pH limit as already presented above. The upper pH limit in this case can correspond to the same upper pH limit that is defined for the first change in pH or can be changed to a new lower or higher value. Preferably, such a lower and upper pH limit can be achieved by programming the pH controller of the bioreactor with a set point and an end point. Such passive change in pH can occur through remetabolization of lactic acid later in the culture by the cells, which causes the pH to rise again to values above the lower limit of the pH range. In some cases, it is possible for the pH to reach the upper limit of the range again, in other cases it may also be below that limit. The magnitude of the second change in pH can include values between 0.05 and 1 pH unit. The duration of such a passive change in pH is not exactly defined since the rate of change / modification depends on the metabolic activity and remetabolization of lactic acid by cells. This can typically take 0.5 to 2 days to reach the upper pH limit, but in the case of a passive change the pH can also remain below the upper limit defined until the end of the culture.
    The choice of strategy for implementation will depend on several factors, such as the sensitivity of cells to CO 2 and the optimum pH for product formation. Different pH profiles can be obtained by defining two or more specific set points over the course of the culture or simply by defining a set point and an end point (which optionally could also be changed during the culture). The different pH profiles that can be obtained by adjusting an set point and an end point are illustrated in figure 2, for a pH set point of 6.90 with an end point of 0.10 as example values.
    20/31
    In a further aspect of the invention the total amount of the polypeptide produced through a process comprising temperature and one or more changes in pH is greater than without combining a change in temperature with one or more changes in pH. The combination of at least one change in temperature with at least one change in pH that is adapted in terms of timing and number of steps towards the needs of the particular transfected cell line has led to much better product yield.
    Still in a further aspect of the invention, a process comprising temperature and one or more changes in pH has the potential to lead to a product of improved quality compared to the quality obtained without combining the change in temperature with one or more changes in pH. One possible reason for the beneficial effect of a change in pH to lower pH values in the culture during the production phase may be as follows: ammonia typically accumulates in the cell culture medium over time of the culture and is known to affect potentially the glycosylated product, with the possible result of lower product quality. It is assumed that ammonia enters cells in the form of NH 3 , where it can influence intracellular pH by capturing H 3 O + ions. The resulting increase in intracellular pH can affect glycosylation. Changing the pH of a culture to a lower value will decrease the extracellular concentration of NH 3 through an increase in protonation of NH 3 in NH 4 + to which cells are impermeable.
    The cell culture process comprising a temperature and one or more changes in pH according to the present invention can be carried out using various cell culture media. Commonly used cell culture media that can be used are, for example, D-MEM (Dulbecco's Modified EagleMedium), D-MEM / F-12, MEMa, Fischer Medium, RPMI 1640BME, BGJb, but not limited to these examples. These media can be additionally supplemented with additional components such as, for example, nutrients, vitamins or carbohydrates.
    21/31
    Suitable media that are primarily optimized for cell growth preferably contain concentrations of amino acids.
    rinc ininiaifi according to the following tracks .--------------—------ Amino Acids Cone. (mmol / L) Aminine free base 4 0-60 preferably 4.5 - 5.5 Asparagine monohydrate 3 0-60 preferably 4.0 - 5.5 AsnártlCO 5 4 0 oreferencially 3.0 - 3.6 Glir.ina 0 3-08 preferably 0.5 - 0.7 Hyphytidine HCI H O 0 6-10 preferably 0.7 - 0.9 knlpucina 2 0-50 oreferencially 3.0 - 4.0 I pnr.ina 3 0 7 0 preferably 3.5 - 6.0 Lysine HCI 2 0 40 oreferentially 2.5 - 3.5
    Mptinnina 10-15 preferably 1.2-1.4 IVlwLIvl III IC *Pnylalanine 1 0 -2 0 oreferentially 1.3-1.8 Prnlina 2 5-60 preferably 3.0 - 5.5 .Qprina 3 0-80 oreferentially 4.0-7.0 11 IQ -T rpnnina 2 0 3 5 preferably 2.5 - 3.1 T rintnfano 0 4-10 oreferentially 0.5 - 0.8 X / alina 2 5 5 0 preferably 3.0 - 4.5 V Cl 111 1 ClTirn ^ ina 10-20 oreferentially 1.2-1.8 1 llvOH 1C4(Typhine 0 5-10 preferably 0.6 - 0.8 Glutamine 5 5-95 preferably 6.2 - 8.2
    Amino acid-containing media as defined in the preceding table can be favorably used in the improved cell culture processes according to the present invention.
    A further aspect of the invention includes the use of production means designed for large-scale production of recombinant polypeptides. These means of production may optionally contain larger amounts of components, for example, amino acids. In a preferred embodiment of the invention, an initial content of amino acids is used in these media in a range of approximately 40 mM and approximates
    22/31 approximately 100 mM, alternatively between approximately 50 and approximately 100 mmoles / L. In an alternative embodiment of the invention, such means of production contain initial concentrations of amino acids according to
    with the following tracks.Amino Acids Cone. (mmol / L). Aminine free base 4 0-60 oreferentially 4.5 - 5.5 ^ Naranine monohydrate 9 0 1J 0 oreferencially 9.5 - 10.5 Ánirln asnártico 9 5 40 oreferencially 3.0 - 3.6 filirina 0 3 0 8 preferably 0.5 - 0.7 JIIVIi IQl-liQtiriin HCI H O 10 15 preferably 1.1-1.3 1 IO LI Thousand IU; > 1 1 'Z IqoIai icina 5 5 7 0 oreferentially 6.0 - 6.8 1 ai ir.ina 8 0-100 preferably 9 - 9.2 1 HCI machine 3 0 6 0 preferably 4.0 - 5.0 L.IOIIIC * · i vziMptinnina 15-25 preferably 1.5 - 2.0 PAililalanine 2 0 3 5 oreferencially 2.5 - 3.0 Prnlina 7 5 9 0 oreferentially 8.0-8.5 1 | w 111 1 C <Qorina 10.5-13.0, preferably 11.0 - 11.9 Trpnnina 3 5 55 oreferencially 4.0 - 5.0 T rintnfano 0 9-20 oreferentially 1.0-1.4 1 1 1L / Lv! v * llv—Valina 5 5 7 5 oreferentially 6.0 - 6.8
    Tirnqina 10-30 oreferencially 2.0 - 2.5 r icfj na 0 5-20 oreferentially 1.0-1.3 ríh itamina 5 5 9 5 oreferentially 6.2 - 8.2 Glutamic acid 0 5-25 oreferentially 1.0-1.2
    The production means containing amino acids as defined in the previous table can be favorably used in the improved cell culture processes according to the present invention.
    Cell cultivation can be performed in adherent culture, for example, in monolayer culture or preferably in suspension culture.
    Large-scale cell cultivation can be used, for example, by the various fermentation processes established in industrial biotechnology. Continuous and batch cell culture processes can be used using the cell culture media according to the present invention. Other known reactor technologies, for example, perfusion technologies or the like can also be used.
    Batch processes are a preferred embodiment.
    Batch cell culture includes fed batch culture or simple batch culture. Batch fed cell culture refers to cell culture in which mammalian cells and cell culture medium are supplied to the culture vessel initially and additional culture nutrients are fed continuously or in discrete increments in the culture during the process of cultivation with or without periodic collection of cells and / or products before the end of the culture. Simple batch culture refers to a procedure in which all 15 components for cell culture including mammalian cells and cell culture medium are supplied to the culture vessel at the beginning of the culture process.
    In a further aspect of the invention, the culture is fed in a fed batch process, with the food consisting of two nutrient solutions that are added to the culture. Both nutrient solutions are added to the culture vessel based on a predetermined schedule determined for the particular cell line or product or according to the metabolic needs that are determined by measuring the consumption of, for example, glucose or amino acids in the culture vessel. Both nutrient solutions can be added independently as a cake or continuously. Typically food nutrient solutions comprise amino acids, at least one carbohydrate as a source of energy, specific trace elements, vitamins or ions. It is particularly advantageous to use concentrated feeding solutions in order to avoid large volume increases and product dilution. In some embodiments, it may be useful to have at least two different a24 / 31 solutions. This allows the independent dosing of two or more different groups of nutrients and components to the cells and thus a better adjustment of feeding conditions in relation to the optimal supply of certain nutrients.
    In a further embodiment of the invention, one of the two feed solutions added to the cell culture medium is a concentrated feed comprising the cystine dipeptide and the amino acid tyrosine in respective concentrations in the range of approximately 6.5 g / L and approximately 8, 0 g / L and in the range of approximately 9 g / L and approximately 11 g / L in an aqueous solution at a basic pH above 10. In a particular embodiment, the concentrated feed comprises the cystine dipeptide and the amino acid tyrosine in the respective concentrations of 10.06 g / L of L-tyrosine and 7.25 g / L of cystine at a pH above 10.
    The feed medium comprising cystine and tyrosine which is described above can be added based on the measured consumption of the respective amino acids or according to a fixed schedule at, for example, approximately 0.2 to approximately 0.8% by weight of the weight of the initial cell culture medium per day or at approximately 0.4% by weight of the weight of the initial cell culture medium per day.
    In some examples, the other feed solution contains all other amino acids that are also present in the basic medium except tyrosine and cystine, in some examples this additional feed solution may consist of particular selected components such as, for example, amino acids or carbohydrates. In a further embodiment of the invention, this concentrated feed medium preferably contains amino acids selected according to the concentration ranges.
    Amino Acids Cone, from Feed Medium (mmol / L) Arqinina, free base 12 0-17, preferably 13.5 - 16.0 Histidine, HCI H 2 O 5.5 - 7.5, preferably 5.9 - 7.0 Isoleucine 21 - 28.0, preferably 22.0 - 27 Leucine 32 - 42, preferably 34.5 - 40.0
    25/31
    Amino Acids Cone, from Feed Medium (mmol / L) Lysine HCI 17.0-22.0, preferably 17.5-21.5 Methionine 5.5 - 8.0, preferably 6.0 - 7.5 Phenylalanine 8.5 - 12.0, preferably 9.0 - 10.5 Proline 18.0 - 24, preferably 18.5 - 22.0 Serina 39.0 - 49.0, preferably 39.5 - 46.5 Threonine 14.5 - 19.0 preferably 15.0 - 18.5 T riptofano 3.0 - 5.0, preferably 3.5 - 4.9 Valina 23.0 - 29.0, preferably 23.8 - 27.5 Glutamine 175.0 - 220.0 preferably 176.0 - 201
    Preferably, still carbohydrates such as glucose are added to this concentrated feed medium, the preferred concentrations being between approximately 1200 and approximately 1400 mmol / L or alternatively between approximately 1300 and approximately 1395 mmol / L.
    The feed medium as described now, preferably including a carbohydrate, such as glucose, can be added based on the measured consumption of the respective amino acids or according to a fixed schedule at, for example, approximately 1 to approximately 4% by weight of the weight of the initial cell culture medium per day, for example, at approximately 2% by weight of the weight of the initial cell culture medium per day.
    Polypeptides that can be produced from cell cultures and cell culture media according to the present invention are not limited. Polypeptides can be recombinant or non-recombinant. The polypeptide can be homologous to the host cell or preferably, it can be of exogenous origin. The term polypeptide as used herein encompasses molecules composed of a chain of more than two amino acids linked by peptide bonds; molecules containing two or more such chains, molecules comprising one or more such chains being further modified, for example, by glycosylation. The term polypeptide is intended to encompass proteins. The polypeptide
    26/31 Deo of interest can be from any source. Preferably the polypeptides of interest are of human origin and more preferably, the proteins of interest are therapeutic proteins.
    The preferred class of polypeptides produced by cell cultures and the cell culture media according to the present invention are recombinant antibodies.
    The term antibody is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), antibodies modified by nanobodies, antibody subunits, antibody derivatives, antibodies combinations of antibodies with proteins and antibody fragments long enough to exhibit the desired biological activity. The monoclonal antibodies as used herein can be human antibodies.
    However, non-antibody polypeptides can also be produced using cell cultures and the cell culture media according to the present invention, for example, polypeptides such as transmembrane proteins, receptors, hormones, growth factors, proteases, coagulants and anticoagulants, inhibitor proteins, interleukins, transport factors, fusion proteins and the like.
    The products obtained from such cell culture processes can be used to obtain pharmaceutical preparations. In addition, the protein (s) according to the invention can be administered together with other components of biologically active agents such as pharmaceutically acceptable surfactants, containers, carriers, diluents and vehicles.
    The invention is further illustrated by the following examples. Examples
    In the examples described below, chemically defined cell culture media 1 and 2 having the composition that is detailed in table 1 below are used. The individual components of these cell culture media are available from standard commercial sources27 / 31
    Components Cone. Middle End 1 (mg / L) Cone. Middle End 2 (mg / L) CaCI 2 , anhydrous 131 133.2 KCI, anhydrous 800 800 MgCl 2 , anhydrous 155 250.4 NaCI 850.6 500 Disodium hydrogen phosphate, anhydrous 710 1065 Sodium hydrogen carbonate, anhydrous 2500 2000 L-Arginine, free base 871 871 L-Asparagine, H 2 O 616 1501 L-Aspartic acid 461 461 L-Cystine 200.1 304.5 Hydrated sodium salt of L-glutamic acid - 182 L-Glutamic acid - __________________________________. L-Histidine, HCI-H 2 O 168 268 L-lsoleucine 394 894 L-Leucine 499 1199 L-Lysine, HCI 621 821 L-Methionine 179 279 L-Phenylalanine 264 464 L-Proline 368 968 L-Serina 432 1232 L-T reynine 333 533 L-T riptofano 102 252 L-Valina 375 775 L-Tyrosine 277.7 422.5 Glycine 38 38 L-Glutamine 1169.2 1169.2 Biotin 0.4 0.4 D-Ca-Pantothenate 4 4 Folic acid 5 5 myo-lnositol 40 140
    28/31
    Components Cone. Middle End 1 (mg / L) Cone. Middle End 2 (mg / L) Nicotinamide 4 4 Pyridoxine, HCI 2 2 Riboflavin 0.4 0.4 B12 vitamin 2 2 Thiamine, HCI 4 4 Putrescina, 2HCI 10 110 Choline chloride 40 240 Sodium selenite (Na 2 SeO 3 ) 0.03 0.03 Manganese chloride tetrahydrate 0.3 0.3 Ammonium molybdate tetrahydrate 0.3 0.3 Zinc chloride, anhydrous 3 3 Cupric chloride dihydrate 0.3 0.3 Cobalt chloride hexah id ratado 0.3 0.3 Ethanolamine 10 100 Monothioglycerol 2 - HEPES, acid form 17870 4766 Tri-sodium citrate dihydrate 911.7 1235.2 FeCI 3 x6H 2 O 54.1 54.1 Pluronic F68 1000 1000 D-Glucose, anhydrous 10000 10000 HCI - 327.6 NaOH 799.2 I 339.9
    Table 2 below shows the composition of a concentrated feed medium containing L-tyrosine and cystine. The feed medium can be added based on the measured consumption of the respective amino acids or according to a fixed schedule of, for example, 0.4% by weight per day.
    29/31
    Table 2
    Components Feed Medium (g / L) 32% NaOH 18.7 mL L-Tyrosine 10.06 Cystine 7.25
    Table 3 below shows the composition of an example of a concentrated feed medium. The feed medium can be added based on the measured consumption of amino acids or according to a fixed schedule of, for example, 2% by weight per day.
    Table 3
    Components Feed Medium (g / L) L-Arqinina, free base 2.72 L-Histidine, HCI-H 2 O 1.44 L-lsoleucine 3.44 L-Leucine 5.20 L-Lysine, HCI 3.72 L-Methionine 1.08 L-Phenylalanine 1.72 L-Proline 2.44 L-Serina 4.76 L-Threonine 2.08 L-T riptofano 0.88 L-Valina 3.16 L-Glutamine 29.23 D-glucose-monohydrate 275.00 25% HCI 8.25 mL 32% NaOH 5.6 mL
    For the experiments in the examples a parental CHO cell line CHO is used which is derived from the cell line dhfr (+) CHO-K1 ATCC CCL-61 (Kao et al., Genetics, 1967, 55, 513-524; Kao and 10 others, PNAS, 1968, 60, 1275-1281; Puck et al., J. Exp. Med., 1958, 108,
    945-959) through adaptation to conditions of serum-free media, free of
    30/31 of proteins. Three aliquots of this parental cell line are transfected to express three different monoclonal antibodies mAB1, mAB2, mAB3, respectively.
    Example 1
    In example 1, two shake bottle cultures containing medium 1 are inoculated in parallel with a CHO clone that produces mAb1. The cultures in the shake flask are incubated in a carbon dioxide incubator at 37 ° C. On day 3, a shaking flask is transferred to a carbon dioxide incubator set at 33 ° C. Both shake flasks are similarly fed with two feed solutions. Feeding was supplemented according to a fixed schedule, with the addition of 0.4% of the first feeding solution (table 2) and 2% of a second feeding (table 3) per day starting on the 5th and lasting until the end of the feeding. culture.
    Changing the temperature to 33 ° C allows for longer maintenance of viable cell density and culture viability over time (figures 3 and 4) and the achievement of a higher product titer (figure 5) compared to culture which is maintained at 37 ° C for the duration of the experiment. This example illustrates the benefit of implementing a change in temperature to 33 ° C during the production process of the cell culture based on a CHO host cell line.
    Example 2
    In this example, a 300-L bioreactor containing medium 2 is inoculated with a CHO clone that produces mAb2. On day 5, the temperature of the bioreactor is changed from 36.5 ° C to 33 ° C. The pH set point is 6.90 and the end point is 0.10. As a result, the culture starts at pH 7.00, the pH decreases to 6.80 between day 2 and day 4 and then progressively returns to 7.00 due to the consumption of lactic acid by the cells (figure 6). The change to pH 6.80 makes it possible to reduce the addition of base compared to a scenario with a constant pH 7.00. The return to pH 7.00 makes it possible to reduce the concentration of CO2 in the medium compared to a scenario in which the pH is maintained at 6.80 after the first change. In this process that combines
    31/31 changes in temperature and pH, a high density of viable cells is achieved and the decrease in density of viable cells over time is minimized (figure 7), allowing a high titre (figure 8) of product of adequate quality. The feed is applied similarly as in example 1.
    Example 3
    In this example, three independent batched batch culture processes are performed in a glass bioreactor using a CHO cell clone that produces mAb3 and medium 2 (see table 1). The feeding scheme of example 1 is applied again. Two independent cultures include a change in temperature without an additional change in pH, that is, the pH in both cell cultures is maintained at a pH value of 7.0 throughout the culture period. The third cultivation experiment differs from the first two experiments by an additional change in pH from pH 7.0 to pH 6.8 applied on day 3 of cultivation. The change in temperature performed in all experiments occurred at a cell density of 4-6 x 10 6 viable cells / mL, respectively. In figure 9, the concentration of mAb3 obtained as the expression product from the corresponding CHO clone is represented as a function of the viable cell integral (IVC) which is an integral of all living cells calculated from the cell concentration / mL of VCD in a milliliter of cell culture. The y / x slope is the specific productivity of the qp cell [g / VC / h] which indicates the amount of the recombinant mAb3 product that a single living cell can produce in an hour. As a result, figure 9 illustrates an increase in cell-specific productivity in a cell culture when subjected to an additional change in pH. Due to the additional change in pH the cells grow more slowly, but exhibit greater productivity.
    权利要求:
    Claims (5)
    [1]
    1. Process for the production of a recombinant polypeptide, characterized by the fact that it comprises the cultivation of CHO cells in a medium under conditions that comprises at least one change in temperature and at least one change in pH and the expression of the recombinant polypeptide, in what
    - the cells are grown at a first temperature for at least 3 days and the temperature is then changed to a second temperature which is between 1 and 8 ° C lower than the first temperature and the cells are kept at said second temperature for a period of at least 2 more days;
    - the cells are grown at a first pH value for at least 2 days and the pH is then changed to a second pH value that is between 0.05 and 1 pH unit lower than the first pH and the cells are grown at said second pH for at least 1 day.
    [2]
    2/3 between pH 6.0 and pH 7.1.
    Process according to any one of claims 1 to 7, characterized by the fact that said second pH is actively maintained until the end of the culture.
    Process according to any one of claims 1 to 7, characterized in that the first change in pH is followed by a second change in pH after at least 1 day with the third pH value being 0.05 unit of pH up to 1 pH unit higher than the second pH value.
    10. Process according to claim 9, characterized by the fact that the pH is actively changed from said second to said third pH values.
    Process according to claim 9, characterized in that the pH is passively changed from said second to said third pH values.
    Process according to any one of claims 1 to 11, characterized in that the medium is free of protein and serum and characterized by a total amino acid content of between 40 and 100 mM.
    Process according to any one of claims 1 to 12, characterized in that the culture is carried out in a fed batch mode which comprises feeding at least two nutrient solutions which are added to the culture.
    Process according to claim 13, characterized in that one of the feed solutions added to the culture medium is a feed comprising the cystine dipeptide and the amino acid tyrosine.
    Process according to claim 14, characterized in that the feed comprises 7.25 g / l of the cystine dipeptide and 10.06 g / l of the amino acid tyrosine in an aqueous solution at a basic pH above 10.
    16. Process according to claim 14 or 15, characterized by the fact that the amount of the feed solution you buy
    Petition 870190102985, of 10/14/2019, p. 13/19
    2. Process according to claim 1, characterized by the fact that the pH is actively changed between said first and said second pH values.
    [3]
    3/3 of cystine and thiprosine is added to the culture medium at 0.4% by weight of the weight of the initial culture medium per day.
    Process according to any one of claims 1 to 16, characterized in that the polypeptide produced is glycosylated.
    3, characterized by the fact that the first temperature is in the range between 33 ° C and 38 ° C.
    Process according to any one of claims 1 to
    Process according to claim 1, characterized by the fact that the pH is passively changed between said first and said second pH values.
    [4]
    4, characterized by the fact that the second temperature is in the range of between 30 ° C and 37 ° C.
    Process according to any one of claims 1 to
    5, characterized by the fact that the first pH value is in the range between pH 6.8 and pH 7.5.
    Process according to any one of claims 1 to
    6, characterized by the fact that the second pH value is in the range of
    Petition 870190102985, of 10/14/2019, p. 12/19
    Process according to any one of claims 1 to
    [5]
    18. Process according to any one of claims 1 to 17, characterized in that the polypeptide is an antibody or an antibody fragment.
    类似技术:
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    BR112012027430B1|2020-04-07|process for the production of a recombinant polypeptide comprising the cultivation of cho cells
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    同族专利:
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    DK3330370T3|2021-05-10|
    PT3330370T|2021-05-11|
    JP2016214254A|2016-12-22|
    PL2563906T3|2018-04-30|
    LT3330370T|2021-06-10|
    EP2563906B1|2017-11-08|
    IL222454D0|2012-12-31|
    EP3862423A1|2021-08-11|
    US8765413B2|2014-07-01|
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    法律状态:
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-07-16| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2020-02-11| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-04-07| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 25/04/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US32784610P| true| 2010-04-26|2010-04-26|
    PCT/EP2011/056507|WO2011134919A2|2010-04-26|2011-04-25|Improved cell cultivation process|
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