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    • 专利摘要:
    METHOD FOR INTERRUPTING CELL GROWTH, INCREASE PRODUCTION OF RECOMBINANT PROTEIN AND/OR LIMITING A CULTURE OF MAMMALIAN CELLS EXPRESSING A RECOMBINANT PROTEIN. The invention provides a method for culturing mammalian cells. The method provides greater control over cell growth to obtain high product titer cell cultures.
    公开号:BR112013033730B1
    申请号:R112013033730-3
    申请日:2012-06-29
    公开日:2022-01-25
    发明作者:Brian D. Follstad；Rebecca E. Mccoy；Arvia E. Morris
    申请人:Amgen Inc；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    [001] The invention provides a method for culturing mammalian cells. The method provides greater control over cell growth to obtain cell cultures with high titers of the product. FUNDAMENTALS OF THE INVENTION
    [002] As the demand for ever greater amounts of therapeutic recombinant proteins increases, positive increases in cell growth, viability and protein production are pursued through the implementation of new methods to improve cell development, optimization of the medium and parameters of process control. Much effort is now being put into process optimization, particularly methods and strategies for growing, feeding and maintaining production cell cultures.
    [003] New methods for cell culture that provide incremental improvements in the production of recombinant proteins are valuable, given the expense of large-scale cell culture processes and the growing demand for higher amounts of and lower costs for biologics. .
    [004] Improvements in cell culture processes, recombinant polypeptide expression, titer and cell viability can lead to higher levels of production, thus reducing costs associated with the production of therapeutic proteins are needed. The invention meets these needs by providing a simple, easy and inexpensive method of controlling cell growth while increasing protein production. SUMMARY OF THE INVENTION
    [005] The present invention provides a method for arresting the growth of cells in a mammalian cell culture that expresses a recombinant protein comprising culturing mammalian cells in a serum-free culture medium in a bioreactor; inducing cell growth arrest by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5mM or less; maintaining mammalian cells in a growth-stopped state by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5mM or less.
    [006] The present invention also provides a method for increasing the production of recombinant protein in a culture of mammalian cells expressing a recombinant protein, comprising establishing a culture of mammalian cells in a serum-free culture medium in a bioreactor; inducing cell growth arrest by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5mM or less; maintaining mammalian cells in a growth arrest state by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5mM or less. In a related embodiment, the production of recombinant proteins in the mammalian cell culture is increased compared to a culture where the cells are not subjected to L-asparagine-induced cell growth arrest.
    [007] The present invention also provides a method for limiting a culture of mammalian cells expressing a recombinant protein to a desired packaged cell volume, comprising establishing a culture of mammalian cells in a serum-free culture medium in a bioreactor; inducing cell growth arrest by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5mM or less; maintaining mammalian cells in a growth-stopped state by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5mM or less.
    [008] In one embodiment of the present invention, in any of the above methods the perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5mM or less begins on day 3 of culture or earlier. In another embodiment, in any of the above methods of inducing cell growth arrest occurs prior to a production phase. In another embodiment, in any of the above methods the induction of cell growth arrest occurs during a production phase. In another embodiment, in any of the above methods arrest of cell growth is induced by L-asparagine deprivation. In yet another embodiment, any of the above methods further comprises a temperature change from 36°C to 31°C. In another embodiment, any of the above methods further comprises a temperature change from 36°C to 33°C. In a related embodiment, the temperature change occurs at the transition between a growth phase and a production phase. In yet another embodiment, the temperature change occurs during the production phase. In another embodiment, the above methods further comprise a packed cell volume during a production phase of less than or equal to 35%. In a related embodiment, the packed cell volume during a production phase is less than or equal to 35%.
    [009] The present invention also provides a method for culturing mammalian cells expressing a recombinant protein comprising: establishing a culture of mammalian cells in a serum-free culture medium in a bioreactor; culturing the mammalian cells during a growth phase and supplementing the culture medium with bolus feeds of a serum-free feeding medium and maintaining the mammalian cells during a production phase by perfusion with a serum-free perfusion medium, wherein the packed cell volume during the production phase is less than or equal to 35%. In one embodiment of the present invention, the perfusion begins on or around day 5 through on or around day 9 of cell culture. In a related embodiment, the perfusion begins on or around day 5 to on or around day 7 of cell culture. In one embodiment, perfusion begins when cells have reached a production stage. In another embodiment, the method further comprises inducing cell growth arrest by L-asparagine deprivation, followed by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less. In yet another embodiment, the method further comprises inducing cell growth arrest by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less.
    [010] In one embodiment of the invention, the concentration of L-asparagine in the serum-free perfusion medium is less than or equal to 5mM. In another embodiment of the invention, the concentration of L-asparagine in the serum-free perfusion medium is less than or equal to 4.0 mM. In another embodiment of the invention, the concentration of L-asparagine in the serum-free perfusion medium is less than or equal to 3.0 mM. In yet another embodiment of the invention, the concentration of L-asparagine in the serum-free perfusion medium is less than or equal to 2.0 mM. In yet another embodiment of the invention, the concentration of L-asparagine in the serum-free perfusion medium is less than or equal to 1.0 mM. In yet another embodiment of the invention, the concentration of L-asparagine in the serum-free perfusion medium is 0 mM. In another embodiment the perfusion is performed at a rate that increases during the production phase from 0.25 working volume per day to 1.0 working volume per day during cell culture. In a related modality the perfusion is performed at a rate that reaches 1.0 working volume per day on day 9 to day 11 of cell culture. In another related embodiment, perfusion is performed at a rate that reaches 1.0 working volume per day on day 10 of cell culture. In yet another embodiment, bolus feeds of serum-free feeding medium begin on day 3 or day 4 of cell culture. In another embodiment of the invention, the method further comprises a temperature change from 36°C to 31°C. In another embodiment of the invention, the method further comprises a temperature change from 36°C to 33°C. In a related embodiment, the temperature change occurs at the transition between a growth phase and a production phase. In a related embodiment, the temperature change occurs during the production phase.
    [011] In one embodiment of the invention, the L-asparagine concentration of the cell culture medium is monitored before and during L-asparagine deprivation.
    [012] In one embodiment of the invention, the packed cell volume is less than or equal to 35%. In a related embodiment, the packed cell volume is less than or equal to 30%.
    [013] In one embodiment of the invention, the viable cell density of mammalian cell culture in a packed cell volume of less than or equal to 35% is 10 x 10 6 viable cells/ml up to 80 x 10 6 viable cells/ml. In a related embodiment, the density of viable cells from mammalian cell culture is 20 x 10 6 viable cells/ml to 30 x 10 6 viable cells/ml.
    [014] In an embodiment of the invention the perfusion comprises continuous perfusion.
    [015] In one embodiment of the invention, the perfusion rate is constant.
    [016] In one embodiment of the invention, perfusion is performed at a rate less than or equal to 1.0 work volume per day.
    [017] In another embodiment of the invention, the mammalian cell culture is established by inoculating the bioreactor with at least 0.5 x 10 6 to 3.0 x 10 6 cells/mL in serum-free culture medium. In a related embodiment, the mammalian cell culture is established by inoculating the bioreactor with at least 0.5 x 10 6 to 1.5 x 10 6 cells/mL in serum-free culture medium.
    [018] In another embodiment of the invention, the perfusion is performed by alternating tangential flow.
    [019] In another embodiment of the invention, the bioreactor has a capacity of at least 500L. In another embodiment of the invention, the bioreactor has a capacity of at least 500L to 2000L. In yet another embodiment of the invention, the bioreactor has a capacity of at least 1000L to 2000L.
    [020] In another embodiment of the invention, the mammalian cells are Chinese Hamster Ovary (CHO) cells.
    [021] In another embodiment of the invention, the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein or a cytokine.
    [022] In another embodiment of the invention, any of the above methods further comprises a step of harvesting the recombinant protein produced by the cell culture.
    [023] In another embodiment of the invention, the recombinant protein produced by cell culture is purified and formulated into a pharmaceutically acceptable formulation. BRIEF DESCRIPTION OF THE FIGURES
    [024] Figure 1 start of the fed batch: solid square (■) and solid circle (•) Batch start: open square (□) and open circle (o).
    [025] Figure 1A. Viable cell density, Figure 1B: Viability, Figure 1C: Titre.
    [026] Figure 2 Batch start: open circle (o), Batch start fed with high agitation: open square (□).
    [027] Figure 2A Viable Cell Density, Figure 2B Viability, Figure 2C Title, Figure 2D Asparagine Concentration.
    [028] Figure 3 initial perfusion volume 1.0, without temperature change: solid circle. Initial perfusion volume 1.0, temperature change: open circle (o). Initial perfusion volume 0.75, no temperature change: solid square (■). Initial perfusion volume 0.75, temperature change: open square (□).
    [029] Figure 3A Viable Cell Density, Figure 3B Viability, Figure 3C Title.
    [030] Figure 4 Batch start with low amount of asparagine: open triangle (Δ). Batch start with amount of L-asparagine control: solid triangle (▲). Start of Batch fed with low amount of L-asparagine: open diamond (◊). Start of batch fed with amount of L-asparagine control: solid diamond (♦). Perforated tube washing: Solid line. Washing with sintered washer: dashed line.
    [031] Figure 4A Viable Cell Density, Figure 4B Viability, Figure 4C Adjusted PCV Titer.
    [032] Figure 5 Cultures grown in medium containing 17.3 mM or 5 mM L-asparagine and 4.6 mM or 10 mM L-glutamine. 17.3 mM L-asparagine and 4.6 mM L-glutamine, solid diamond (♦) or 5 mM L-asparagine, 10 mM L-glutamine, open diamond (◊).
    [033] Figure 5A Density of Viable Cells. Figure 5B Title. Figure 5C Packed Cell Volume (PCV). Figure 5D Adjusted PCV Title. Figure 5E Feasibility.
    [034] Figure 6. 2L bench scale and 500L pilot scale cultures, with 5 mM L-asparagine, 10 mM L-glutamine. Medium containing 5 mM L-asparagine, 10 mM L-glutamine in bench scale of 2L is represented by solid diamond (♦) and pilot scale of 500L is represented by open diamond (◊).
    [035] Figure 6A Density of Viable Cells. Figure 6B Title. Figure 6C Packed Cell Volume (PCV). Figure 6D Adjusted PCV Title. Figure 6E Feasibility. DETAILED DESCRIPTION OF THE INVENTION
    [036] During recombinant protein production it is desirable to have a controlled system, where cells are grown to a desired density and then the physiological state of the cells is changed to a state of high productivity with growth halted, where cells use energy and substrates to produce the recombinant protein of interest rather than producing more cells. Methods to achieve this goal, such as temperature changes and inducing small molecules, are not always successful and can have undesirable effects on product quality. As described herein, packed cell volume can be limited to a desired level during the production phase by inducing cell growth arrest in cell cultures upon exposure to low L-asparagine conditions. Cell growth-arrest arrest can be achieved and maintained by using a perfusion culture medium that contains a limiting concentration of L-asparagine and maintaining a low concentration of L-asparagine in the cell culture (5mM or less).
    [037] It was also found that growth-arrested cells showed increased productivity when growth arrest was initiated by low L-asparagine or through L-asparagine starvation, and growth-arrested cells were subsequently maintained with the growth medium. cell culture and perfusion having an L-asparagine concentration of 5mM or less.
    [038] A high-yield production phase of arrested growth can be achieved by manipulating the concentration of L-asparagine. As described here, L-asparagine depletion resulted in growth arrest. In a fed batch culture, once the cell density was high enough (e.g. > 20x10 6 viable cells/mL), the culture was repeatedly starved of L-asparagine despite repeated feedings due to consumption of L-asparagine and/or or conversion to L-aspartate. In a cell culture, extracellular L-asparagine can be converted to L-aspartate and ammonia. L-asparagine depletion resulted in cell cycle arrest. During fed batches, periods when L-asparagine is present in the crop result in increased yields and periods when L-asparagine is depleted result in decreased yields. In the perfused system, L-asparagine is constantly supplied, total depletion is therefore avoided and higher concentrations of L-asparagine can be sustained, thus allowing the cells to continue to multiply and not be exposed to an environment with L- depleted or limited asparagine. Controlling the concentration of L-asparagine at a low enough concentration (such as concentrations of 5 mM or less) can keep cells in a state of high productivity, while maintaining viability and limiting growth. In a bolus-feed and perfusion system, the feeding medium can be switched from a formulation containing a high level (growth promoter) of L-asparagine during bolus feeds to a lower level (growth arrest) of L-asparagine during infusion feeding. Cell cultures that have stopped growth by limiting L-asparagine can be stimulated into a high-throughput state, adding back low levels of L-asparagine.
    [039] For commercial scale cell culture and the manufacture of biological therapeutics, the ability to stop cell growth and be able to maintain cells in a stopped state of growth during the production phase would be very desirable. Having cells that have also been induced to increase productivity while in the arrested state of growth, and being able to maintain this increased productivity, is ideal for manufacturing purposes.
    [040] Provided herein is a method for arresting the growth of cells in a mammalian cell culture expressing a recombinant protein. The method includes inducing cell growth arrest in a mammalian cell culture by subjecting the cell culture to serum-free medium having a concentration of L-asparagine of 5 mM or less (which includes 0 mM L-asparagine). Such induction may be initiated by depriving L-asparagine or creating a low L-asparagine environment by perfusing the culture with a perfusion of serum-free medium having an L-asparagine concentration of 5 mM or less, and maintaining the culture in a Low L-asparagine. The cell culture is then maintained in the growth-stopped state by perfusing with a serum-free perfusion medium with L-asparagine at a concentration of 5mM or less, and maintaining the culture in the low L-asparagine environment.
    [041] Also provided is a method of increasing the production of recombinant proteins in a mammalian cell culture that expresses a recombinant protein by inducing a growth-arrested cell at low asparagine in a mammalian cell culture. Mammalian cells maintained in the growth-stopped state by low asparagine had higher productivity (g protein/cell/day and g protein/cell mass/day) than those not growth-stunted by low asparagine.
    [042] Said method is also useful for limiting a mammalian cell culture to a desired packed cell volume. The cell volume packed during the production phase could be limited to a desired level by reducing L-asparagine levels in the production culture medium. Asparagine concentrations of 5mM or less in the perfusion medium were sufficient to control cell growth during culture and limit to a desired packed cell volume.
    [043] The methods described here provide greater control over cell growth for high-titer product cell cultures; and as such can simplify the gasification strategy compared to high biomass perfusion processes and minimize product loss during harvesting and downstream processing.
    [044] The method begins with the establishment of a mammalian cell culture in a production bioreactor. Preferably smaller production bioreactors are used, in one embodiment the bioreactors are from 500L to 2000L. In a preferred embodiment, 1000L-2000L bioreactors are used. The density of seed cells used to inoculate the bioreactor can have a positive impact on the level of recombinant protein produced. In one embodiment, the bioreactor is inoculated with at least 0.5 x 10 6 up to and beyond 3.0 x 10 6 viable cells/mL in a serum-free culture medium. In a preferred embodiment the inoculation is 1.0x10 6 viable cells/mL.
    [045] Mammalian cells then go through a phase of exponential growth. The cell culture can be maintained without supplemental feeding until a desired cell density is reached. In one embodiment, cell culture is maintained for up to 3 days without supplemental feeding followed by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less to induce and maintain low L-asparagine growth arrest. . In another embodiment, the culture can be inoculated at a desired cell density to begin the production phase without a brief growth phase, with cell growth arrest initiated immediately after inoculation by perfusing the cell culture with serum-free perfusion medium containing 5mM or less L-asparagine to induce and maintain low L-asparagine growth arrest. In either of the embodiments here, the change from the growth phase to the production phase can also be initiated by L-asparagine deprivation (subjecting cells to an environment of 0 mM L-asparagine) followed by perfusion with a cell culture having an L-asparagine concentration equal to or less than 5 mM. and maintaining the L-asparagine concentration in the cell culture at that level.
    [046] Regardless of how much growth arrest by low L-asparagine is induced, higher productivity is observed in growth arrested cells that are maintained by perfusion with a low L-asparagine medium and keeping the cell culture in an L-asparagine level of 5 mM or less.
    [047] As used here, “growth arrest”, which may also be referred to as “interrupted cell growth”, is the point where cells stop increasing in number, or when the cell cycle no longer progresses. Growth arrest can be monitored by determining the viable cell density of a cell culture. Some cells in a growth arrest state may increase in size but not in number, so the packed cell volume of a growth arrest culture may increase. Growth arrest can be reversed to some extent, if the cells are not in declining health, by the addition of additional L-asparagine to the cell culture.
    [048] Growth arrest is initiated by L-asparagine when the cell density of the culture reaches a level where the concentration of L-asparagine in the culture becomes limiting for continued growth or when the culture is deprived of L-asparagine. L-asparagine deprivation occurs when the concentration of L-asparagine in a cell culture medium is effectively 0 mM. Deprivation can result in growth arrest within 24 hours. Deprivation for more than 48 hours could damage the health of the cells. To maintain cells in the growth arrest state, the concentration of L-asparagine in the cell culture must be maintained at 5mM or less. The concentration of L-asparagine cell culture medium required to stop cell growth is dependent on the ability of the cells to produce their own asparagine. For cultures where cells can produce their own asparagine, a lower concentration or even removal of L-asparagine from the medium may be necessary to arrest growth. For cultures that are unable to produce their own asparagine, eg cells lacking the active asparagine synthetase enzyme, concentrations above zero up to 5 mM L-asparagine could be used to arrest growth.
    [049] As used here, “packaged cell volume” (PCV), also known as “percentage packed cell volume” (%PCV), is the ratio between the volume occupied by cells and the total volume of cell culture, expressed in percentage (see Stettler, et al., (2006) Biotechnol Bioeng. Dec 20:95(6):1228-33 ). Packed cell volume is a function of cell density and cell diameter; increases in packed cell volume could be due to increases in cell density or cell diameter or both. Packed cell volume is a measure of solid content in cell culture. Solids are removed during harvesting and downstream purification. More solids means more effort to separate the solid material from the desired product during the harvest and downstream purification steps. In addition, the desired product can become trapped in solids and be lost during the harvesting process, resulting in decreased product yield. Since host cells vary in size and cell cultures also contain dead and dying cells and other cell debris, packed cell volume is a more accurate way to describe the solids content within a cell culture than cell density or viable cell density. For example, a 2000L culture, containing a cell density of 50 x 10 6 cells/ml would have vastly different packed cell volume depending on cell size. Furthermore, some cells, when in a growth arrest state, will increase in size, so the packed cell volume before growth arrest and after growth arrest is likely to be different, due to the increase in biomass as a result of the increase in size. of the cell.
    [050] During the transition between the growth phase and the production phase and during the production phase, the percentage of packed cell volume (%PCV) is equal to or less than 35%. The desired packed cell volume maintained during the production phase is 35% or less. In a preferred embodiment, the packed cell volume is equal to or less than 30%. In another preferred embodiment, the packed cell volume is equal to or less than 20%. In another preferred embodiment, the packed cell volume is equal to or less than 15%. In yet another preferred embodiment, the packed cell volume is equal to or less than 10%.
    [051] As used herein, "cell density" refers to the number of cells in a given volume of culture medium. “Viable cell density” refers to the number of live cells in a given volume of culture medium, as determined by standard viability assays (such as the trypan blue dye exclusion method).
    [052] The desired viable cell density in the transition between the growth and production phases, and maintained during the production phase, is one that provides a packed cell volume equal to or less than 35%. In one embodiment, the viable cell density is at least about 10 x 10 6 viable cells/mL to 80 x 10 6 viable cells/mL. In one embodiment, the viable cell density is at least about 10 x 10 6 viable cells/mL to 70 x 10 6 viable cells/mL. In one embodiment, the viable cell density is at least about 10 x 10 6 viable cells/mL to 60 x 10 6 viable cells/mL. In one embodiment, the density of viable cells is at least about 10 x 10 6 viable cells/mL to 50 x 10 6 viable cells/mL. In one embodiment, the density of viable cells is at least about 10 x 10 6 viable cells/mL to 40 x 10 6 viable cells/mL. In a preferred embodiment, the density of viable cells is at least about 10 x 10 6 viable cells/mL to 30 x 10 6 viable cells/mL. In another preferred embodiment, the density of viable cells is at least about 10 x 10 6 viable cells/ml to 20 x 10 6 viable cells/ml. In another preferred embodiment, the density of viable cells is at least about 20 x 10 6 viable cells/ml to 30 x 10 6 viable cells/ml. In another preferred embodiment, the density of viable cells is at least about 20 x 10 6 viable cells/ml to 25 x 10 6 viable cells/ml, more preferably at least about 20 x 10 6 viable cells/ml.
    [053] Lower packed cell volume during the production phase helps to mitigate dissolved oxygen washout problems that can prevent higher cell density perfusion cultures. The smaller packed cell volume also allows for a smaller volume of media, which allows for the use of smaller media storage vessels and can be combined with slower flow rates. Smaller packed cell volume also has less impact on harvesting and downstream processing compared to higher biomass crops. All of which reduce the costs associated with the manufacture of therapeutic recombinant protein.
    [054] Three methods are typically used in commercial processes for the production of recombinant proteins by mammalian cell culture: batch culture, fed batch and perfusion culture. Batch culture, a batch method where cells are grown in a fixed volume of culture media for a short period of time, followed by a full harvest. Cultures grown using the batch method show an increase in cell density until maximum cell density is reached, followed by a decline in viable cell density as medium components are consumed and levels of metabolic by-products (such as lactate and ammonia) accumulate. ). Harvest typically occurs at the point when maximum cell density is reached (typically 5-10 x 10 6 cells/mL, depending on medium formulation, cell line, etc). The batch process is the simplest method of culturing, however the density of viable cells is limited by the availability of nutrients and once the cells are at maximum density, the culturing and production slows down. There is no ability to extend a production phase, because waste accumulation and nutrient depletion quickly leads to crop decline (typically around 3 to 7 days).
    [055] Fed batch culture improves the batch process by providing bolus or continuous media feeds to replenish media components that have been consumed. Since fed batch cultures receive additional nutrients during run, they have the potential to achieve higher cell density (>10 to 30x106 cells/mL, depending on medium formulation, cell line, etc.) compared to the batch method. Unlike the batch process, a biphasic culture can be created and sustained by manipulating feeding strategies and medium formulations to distinguish the period of cell proliferation to achieve a desired cell density (the growth phase) from the period of suspended cell growth. or slow (the production phase). In this way, fed batch cultures have the potential to achieve higher product titers compared to batch cultures. Typically, a batch method is used during the growth phase and a fed-batch method is used during the production phase, but a fed-batch feeding strategy can be used throughout the process. However, unlike the batch process, the volume of the bioreactor is a limiting factor that limits the amount of feed. Also, as with the batch method, the accumulation of metabolic by-products will lead to crop decline, which limits the duration of the production phase to about 1.5 to 3 weeks. Fed batch crops are discontinuous and harvest typically occurs when levels of metabolic by-product or crop viability reach predetermined levels.
    [056] Perfusion methods offer potential improvement over batch and fed-batch methods by adding fresh medium while simultaneously removing spent medium. Typical large-scale commercial cell culture strategies aim to achieve high cell densities, 60 - 90(+) x 106 cells/mL, where nearly one-third to more than one-half of the reactor volume is biomass. With perfusion culture, extreme cell densities of >1 x 10 8 cells/mL have been achieved and even higher densities are predicted. Typical perfusion cultures begin with an initial batch culture lasting a day or two, followed by continuous, stepwise and/or intermittent addition of fresh medium feeds to the culture and simultaneous removal of spent medium, with retention of cells and additional compounds. molecular weight, such as proteins (based on filter molecular weight cutoff) throughout the growth and production phases of the culture. Various methods such as sedimentation, centrifugation or filtration can be used to remove spent medium while maintaining cell density. Perfusion flow rates from a fraction of a working volume per day to many several working volumes per day have been reported. An advantage of the perfusion process is that the production culture can be maintained for longer periods than batch or fed-batch culture methods. However, increased preparation, use, storage, and disposal of medium are necessary to sustain a long-term perfusion culture, particularly those with high cell densities, which also need more nutrients, and all this makes production costs even higher. compared to batch and fed-batch methods. In addition, higher cell densities can cause problems during production, such as maintaining dissolved oxygen levels and problems with increased gassing comprising supplying more oxygen and removing more carbon dioxide, which would result in more foam and the need for changes in antifoam strategies; as well as during harvesting and downstream processing, where the efforts required to remove excessive cell material can result in product loss, negating the benefit of increased titer due to increased cell mass.
    [057] A large-scale cell culture strategy is also provided that combines batch fed during the growth phase followed by continuous perfusion during the production phase. The method is intended for the production phase, where the cell culture is maintained in a packed cell volume of less than or equal to 35%. The method also provides initiation and maintenance of cell growth arrest due to low asparagine.
    [058] Fed batch culture is a widely practiced culture method for large-scale production of proteins from mammalian cells. See, for example, Chu and Robinson (2001), Current Opin. Biotechnol. 12:180-87. A mammalian cell-fed batch culture is one in which the culture is fed, continuously or periodically, with a concentrated feeding medium that contains nutrients. Feeding can take place on a predetermined schedule of, for example, every day, once every other day, once every three days, etc. When compared to a non-fed batch culture, a fed batch culture can produce a greater amount of recombinant protein. See, for example, US Patent 5,672,502.
    [059] In one embodiment, a batch culture fed bolus feeds is used to maintain a cell culture during the growth phase. Perfusion feed can be used during a production phase. In one embodiment, perfusion begins when cells have reached a production stage. In another embodiment, the perfusion begins on or around day 5 to on or around day 9 of cell culture. In another embodiment, the perfusion begins on or about day 5 to on or about day 7 of cell culture.
    [060] In another embodiment, initiation of cell growth arrest in the fed-batch culture can be initiated by subjecting the fed-batch culture to a period of L-asparagine starvation, followed by perfusion with a perfusion of serum-free medium having a L-asparagine concentration of 5 mM or less. In one embodiment, the L-asparagine concentration of the cell culture medium is monitored before and during L-asparagine deprivation. In another embodiment, initiation of cell growth arrest in the fed batch culture can be achieved by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less.
    [061] Using bolus feeding during the growth phase allows cells to transition into the production phase, resulting in less reliance on a temperature change as a means of initiating and controlling the production phase, however a temperature change from 36°C to 31°C can occur between the growth phase and the production phase. In a preferred embodiment the change is from 36°C to 33°C.
    [062] As described here, the bioreactor can be inoculated with at least 0.5 x 10 6 up to and beyond 3.0 x 10 6 viable cells/mL in a serum-free culture medium, preferably 1.0 x 10 6 viable cells/ ml.
    [063] Perfusion culture is one in which the cell culture is perfused with fresh feed medium while simultaneously removing the spent medium. The infusion may be continuous, stepwise, intermittent, or a combination of any or all of any of these. Perfusion rates can range from less than one workload to many workloads per day. Preferably the cells are maintained in the culture and the spent medium which is removed is substantially free of cells or has significantly fewer cells than the culture. Recombinant proteins expressed by cell culture can also be retained in the culture. Perfusion can be performed by various means, including centrifugation, sedimentation or filtration, See, for example, Voisard et al., (2003), Biotechnology and Bioengineering 82:751-65. A preferred filtration method is alternate tangential flow filtration. Alternating tangential flow is maintained by pumping media through hollow fiber filter modules. See, for example, US Patent 6,544,424; Furey (2002) Gen. Eng. News. 22(7), 62-63.
    [064] As used here, “perfusion flow rate” is the amount of medium that is passed through (added and removed from) a bioreactor, typically expressed as a part or multiple of the working volume, at any given time. “Working volume” refers to the amount of bioreactor volume used for cell culture. In one embodiment, the perfusion flow rate is a working volume or less per day. The fed perfusion medium can be formulated to maximize the concentration of perfusion nutrients to minimize the rate of perfusion.
    [065] By “cell culture” or “culture” is meant the growth and propagation of cells outside of a multicellular organism or tissue. Appropriate culture conditions for mammalian cells are known in the art. See, for example, Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, spinner bottles, shaker flasks or stirred tank bioreactors, with or without microcarriers, can be used. In one embodiment, 500L to 2000L bioreactors are used. In a preferred embodiment, 1000L to 2000L bioreactors are used.
    [066] For the purposes of the present invention, cell culture medium is a medium suitable for the growth of animal cells, such as mammalian cells, in cell culture in vitro. Cell culture media formulations are well known in the art. Typically, cell culture media are composed of buffers, salts, carbohydrates, amino acids, vitamins and essential trace elements. “Serum free” applies to a cell culture medium that does not contain animal sera, such as fetal bovine serum. Various tissue culture media, including defined culture media, are commercially available, for example, any one or a combination of the following cell culture media can be used: RPMI-1640 Medium, RPMI-1641 Medium, Modified Eagle Medium Dulbecco's Medium (DMEM), Eagle Minimal Essential Medium, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco Medium, McCoy's 5A Medium, Leibovitz's L-15 Medium, and serum-free media such as EX-CELLTM Series 300 (JRH Biosciences, Lenexa , Kansas), among others. Serum-free versions of these culture media are also available. Cell culture media can be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements and the like, depending on the requirements of the cells to be cultured and /or desired cell culture parameters.
    [067] Cell cultures can be supplemented with concentrated feed media containing components, such as nutrients and amino acids, which are consumed during the course of the cell culture production phase. Concentrated feed media can be based on any cell culture media formulation. A concentrated feed medium can contain most components of the cell culture medium, for example about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X or even about 1000X their normal amounts. Concentrated feed media are often used in fed-batch culture processes.
    [068] The method according to the present invention can be used to improve the production of recombinant proteins in multi-stage culture processes. In a multi-stage process, cells are grown in two or more distinct stages. For example, cells can be grown first in one or more growth phases, under environmental conditions that maximize cell proliferation and viability, then transferred to a production phase, under conditions that maximize protein production. In a commercial process for the production of a protein by mammalian cells, there are commonly multiple, e.g. at least about 2, 3, 4, 5, 6, 7, 8, 9 or 10 growth phases that occur at different stages. culture vessels preceding a final production culture. The growth and production phases may be preceded or separated by one or more transition phases. In multi-phase processes, the method according to the present invention can be employed at least during the growth phase and production phase of the final production phase of a commercial cell culture, although they can also be employed in a growth phase. previous. A large-scale production phase can be carried out. A large scale process can be conducted in a volume of at least 100, 500,1000, 2000, 3000, 5000, 7000, 8000, 10,000, 15,000, 20,000 liters. In a preferred embodiment, production is carried out in 500L, 1000L and/or 2000L bioreactors. A growth phase can occur at a higher temperature than a production phase. For example, a growth phase can occur at a first temperature of about 35°C to about 38°C, and a production phase can occur at a second temperature of about 29°C to about 37°C, optionally , from about 30°C to about 36°C or from about 30°C to about 34°C. In addition, chemical inducers of protein production, such as caffeine, butyrate and hexamethylene bisacetamide (HMBA), can be added at the same time as, before, and/or after a temperature change. If inductors are added after a temperature change, they can be added from one hour to five days after the temperature change, optionally from one to two days after the temperature change. Cell cultures can be maintained for days or even weeks while the cells produce the desired protein(s).
    [069] Cell culture samples can be monitored and evaluated using any of the analytical techniques known in the art. Several parameters, including the quality and characteristics of the recombinant protein and the medium, can be monitored during the duration of the culture. Samples can be collected and monitored intermittently at a desired frequency, including continuous, real-time or near real-time monitoring. In one embodiment, the L-asparagine concentration of the cell culture medium is monitored before and during L-asparagine deprivation.
    [070] Typically, the cell cultures that precede the final production culture (N-x to N-1) are used to generate the seed cells that will be used to inoculate the production bioreactor, the N-1 culture. Seed cell density can have a positive impact on the level of recombinant protein produced. Product levels tend to increase with increasing seed lot density. The improvement in titer is linked not only to the higher density of the seed batch, but can be influenced by the metabolic state and cell cycle of the cells that are put into production.
    [071] Seed cells can be produced by any culture method. A preferred method is a perfusion culture using alternating tangential flow filtration. An N-1 bioreactor can be run using alternate tangential flow filtration to deliver cells at high density to inoculate a production bioreactor. Stage N-1 can be used to grow cells to densities of >90 x 106 cells/mL. The N-1 bioreactor can be used to generate seed bolus cultures or it can be used as a seed bearing stock culture that could be kept to seed multiple production bioreactors with high seed cell density. The duration of the production growth phase can range from 7 to 14 days and can be designed to keep the cells growing exponentially, prior to inoculation into the production bioreactor. Perfusion rates, medium formulation and timing are optimized to grow cells and release them to the production bioreactor in a state that is most conducive to optimizing their production. Seed cell densities >15 x 106 cells/mL can be achieved to seed production bioreactors. Higher seed cell densities at inoculation can decrease or even eliminate the time needed to reach a desired production density.
    [072] The invention finds it particularly useful for improving cell culture, viability and/or protein production through cell culture processes. The cell lines (also referred to as "host cells") used in the invention are genetically modified to express a polypeptide of commercial or scientific interest. Cell lines are normally derived from a lineage derived from a primary culture that can be kept in culture for an unlimited time. Genetically engineering the cell line involves transfecting, transforming or transducing cells with a recombinant polynucleotide molecule, and/or otherwise altering (e.g., by homologous recombination and gene activation or fusion of a recombinant cell with a non-recombinant cell). ) in order to cause the host cells to express a desired recombinant polypeptide. Methods and vectors for genetically engineering cells and/or cell lines to express a polypeptide of interest are known to those skilled in the art; for example, several techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, R.J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69.
    [073] Animal cell lines are derived from cells whose progenitors were derived from a multicellular animal. One type of animal cell line is a mammalian cell line. A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and commercial suppliers. Examples of cell lines commonly used in the industry include VERO, BHK, HeLa, CV1 (including Cos), MDCK, 293, 3T3, multiple myeloma cell lines (e.g., NSO, NS1), PC12, WI38 cells, and ovarian cancer cells. Chinese hamster (CHO). CHO cells are widely used for the production of complex recombinant proteins, eg cytokines, clotting factors and antibodies (Brasel et al. (1996), Blood 88:2004-2012; Kaufman et al. (1988), J. Biol Chem 263:6352-6362; McKinnon et al (1991), J Mol Endocrinol 6:231-239 ; Wood et al (1990), J Immunol 145:3011-3016 ). Dihydrofolate reductase (DHFR) deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXB11 and DG-44, are desirable CHO host cell lines because the expression system of The selectable and amplifiable DHFR gene efficiently allows the expression of high-level recombinant proteins in these cells (Kaufman RJ (1990), Meth Enzymol 185:537-566). Furthermore, these cells are easy to manipulate as adherent or suspension cultures and have relatively good genetic stability. CHO cells and recombinant proteins expressed therein have been extensively characterized and have been approved by regulatory agencies for use in clinical commercial manufacturing.
    [074] The methods of the invention can be used in cell cultures that express recombinant proteins of interest. The expressed recombinant proteins can be secreted into the culture medium, from which they can be recovered or collected. Furthermore, proteins can be purified, or partially purified, from said culture or component (e.g., from the culture medium) using known processes and products available from commercial suppliers. The purified proteins can then be “formulated”, meaning buffer exchanged, sterilized, packaged in bulk and/or packaged for an end user. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton, PA.
    [075] As used herein, "peptide", "polypeptide" and "protein" are used interchangeably throughout the document and refer to a molecule composed of two or more amino acid residues, joined together by peptide bonds. . Peptides, proteins, and polypeptides also include modifications, including, but not limited to, glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. Polypeptides may be of scientific or commercial interest, including protein-based drugs. Polypeptides include, among other things, antibodies, fusion proteins and cytokines. Peptides, polypeptides and proteins are produced by recombinant animal cell lines using cell culture methods and may be referred to as "recombinant peptide", "recombinant polypeptide" and "recombinant protein". The expressed proteins can be produced intracellularly or secreted into the culture medium, from which they can be recovered and/or collected.
    [076] Examples of polypeptides that can be produced with the methods of the invention include proteins comprising identical or similar amino acid sequences to all or part of one of the following proteins: tumor necrosis factor (TNF), flt3 ligand (WO 94/28391 ), erythropoietin, thrombopoietin, calcitonin, IL-2, angiopoietin-2 (Maisonpierre et al. (1997), Science 277(5322): 55-60), ligand for activator of the NF-kappa B receptor (RANKL, WO 01 /36637), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, WO 97/01633), thymic stroma-derived lymphopoietin, granulocyte colony-stimulating factor, macrophage-granulocyte colony-stimulating factor (GM- CSF, Australian Patent 588819), mast cell growth factor, stem cell growth factor (US Patent 6,204,363), epidermal growth factor, keratinocyte growth factor, megakaryocyte growth and development factor, RANTES, protein t human fibrinogen type 2 (FGL2; Accession No. in NCBI NM_00682; Rüegg and Pytela (1995), Gene 160:257-62), growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, interferons, including interferon-α, interferon-Y, and consensus interferons (US Patents 4,695). .623 and 4.897471), nerve growth factor, brain-derived neurotrophic factor, synaptotagmin-like proteins (SLP 1-5), neurotrophin-3, glucagon, interleukins, colony stimulating factors, lymphotoxin-β, leukemia inhibitory factor and oncostatin M. Descriptions of proteins that can be produced according to the inventive methods can be found in, for example, Human Cytokines: Handbook for Basic and Clinical Research, all volumes (Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge, MA, 1998); Growth Factors: A Practical Approach (McKay and Leigh, eds., Oxford University Press Inc., New York, 1993); and The Cytokine Handbook, Vols. 1 and 2 (Thompson and Lotze eds., Academic Press, San Diego, CA, 2003).
    [077] Furthermore, the methods of the invention would be useful for producing proteins comprising all or part of the amino acid sequence of a receptor for any of the aforementioned proteins, an antagonist for said receptor or any of the aforementioned proteins, and /or proteins substantially similar to these receptors and antagonists. These receptors and antagonists include: both forms of the tumor necrosis factor receptor (TNFR, known as p55 and p75, US Patent 5,395,760 and US Patent 5,610,279), interleukin-1 (IL-1) receptors (types I and II; EP Patent 0460846, US Patent 4,968,607, and US Patent 5,767,064), IL-1 receptor antagonists (US Patent 6,337,072), IL-1 antagonists or inhibitors (US Patents 5,981,713). , 6,096,728, and 5,075,222), IL-2 receptors, IL-4 receptors (EP Patent 0367566 and US Patent 5,856,296), IL-15 receptors, IL-17 receptors, IL-1 receptors 18, Fc receptors, macrophage-granulocyte colony-stimulating factor receptor, granulocyte colony-stimulating factor receptor, receptors for oncostatin M and leukemia inhibitory factor, NF-kappa B receptor activator (RANK, WO 01/ 36637 and US Patent 6,271,349), osteoprotegerin (US Patent 6,015,938), receptors for TRAIL (including TRAIL receptors 1, 2, 3 and 4) and the receptors that make up the death domains, such as Fas Inducing Receptor or Apoptosis (AIR).
    [078] Other proteins that can be produced using the invention include proteins, comprising all or part of the amino acid sequences of differentiating antigens (referred to as CD proteins) or their ligands, or proteins substantially similar to any of these. Said antigens are disclosed in Leukocyte Typing VI (Proceedings of the VIth International Workshop and Conference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996). Similar CD proteins are disseminated in subsequent workshops. Examples of said antigens include CD22, CD27, CD30, CD39, CD40 and ligands thereof (CD27 ligand, CD30 ligand, etc.). Several of the CD antigens are members of the TNF receptor family, which also includes 41BB and OX40. The ligands are often members of the TNF family, as are the 41BB ligand and the OX40 ligand.
    [079] Enzymatically active proteins or their ligands can also be produced using the invention. Examples include proteins comprising all or part of one of the following proteins or their ligands or a protein similar to one of these: a disintegrin and members of the metalloproteinase domain family including TNF-alpha converting enzyme, various kinases, glucocerebrosidase, superoxide dismutase , tissue plasminogen activator, factor VIII, factor IX, apolipoprotein E, apolipoprotein AI, globins, an IL-2 antagonist, alpha-1 antitrypsin, ligands for any of the aforementioned enzymes, and numerous other enzymes and their ligands.
    [080] The term “antibody” includes reference to glycosylated and non-glycosylated immunoglobulins of any isotype or subclass or to an antigen-binding region thereof that competes with the intact antibody for specific binding, unless otherwise specified, including human, humanized, chimeric, multispecific, monoclonal, polyclonal, and oligomers or antigen-binding fragments thereof. Also included are proteins containing an antigen-binding fragment or region such as Fab, Fab', F(ab')2, Fv, diabodies, Fd, dAb, maxibodies, single chain antibody molecules, region determinant fragments (CDR), scFv, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to a target polypeptide. The term "antibody" includes, but is not limited to, those that are prepared, expressed, raised, or isolated by recombination, such as antibodies isolated from a host cell transfected to express the antibody.
    [081] Examples of antibodies include, but are not limited to, those that recognize any one or a combination of proteins, including but not limited to the aforementioned proteins and/or the following antigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1α, IL-1β, IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-13 receptor, IL-1 receptor subunits 18, FGL2, PDGF-β and analogues thereof (see US Patent 5,272,064 and 5,149,792), VEGF, TGF, TGF-β2, TGF-β1, EGF receptor (see US Patent 6,235,883) VEGF receptor , hepatocyte growth factor, osteoprotegerin ligand, interferon gamma, B lymphocyte stimulator (BlyS, also known as BAFF, THANK, TALL-1 and zTNF4; see Do and Chen-Kiang (2002), Cytokine Growth Factor Rev. 13 (1): 19-25), complement C5,IgE, tumor antigen CA125, tumor antigen MUC1, PEM antigen, LCG (which is a gene product which is expressed in association with lung cancer), HER-2, HER-3, a tumor-associated glycoprotein TAG-72, the SK-1 antigen, tumor-associated epitopes that are present at high levels in sera from cancer patients of colon and/or pancreatic cancer, epitopes or cancer-associated proteins expressed in breast, colon, squamous cell, prostate, pancreas, lung and/or kidney cancer cells and/or melanoma, glioma or neuroblastoma, the necrotic nucleus of a tumor, integrin alpha 4 beta 7, the integrins VLA-4, integrins B2, TRAIL receptors 1, 2, 3 and 4, RANK, RANK ligand, TNF-α, the adhesion molecule to VAP-1, epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesin, the platelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain, parathyroid hormone, rNAPc2 (which is an inhibitor of tissue factor VIIa), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), necrosis factor t (TNF), CTLA-4 (which is a cytotoxic T lymphocyte-associated antigen), Fc-Y-1 receptor, HLA-DR 10 beta, HLA-DR antigen, sclerostin, L-selectin, Respiratory Syncytial Virus, Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Streptococcus mutans and Staphlycoccus aureus. Specific examples of known antibodies that can be produced using the methods of the invention include, but are not limited to, adalimumab, bevacizumab, infliximab, abciximab, alemtuzumab, bapineuzumab, basiliximab, belimumab, briakinumab, canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab, eculizumab, gemtuzumabe ozogamicin, golimumab, ibritumomabe tiuxetan, labetuzumabe, mapatumumabe, matuzumab, mepolizumabe, motavizumabe, muromonabe-CD3, natalizumab, nimotuzumab, ofatumumab, omalizumab, oregovomabe, palivizumab, panitumumab, pemtumomabe, Pertuzumab, ranibizumab, rituximab, rovelizumabe, tocilizumab, Tositumomab, trastuzumab, ustekinumab, vedolizomab, zalutumumab, and zanolimumab.
    [082] The invention can also be used for the production of recombinant fusion proteins comprising, for example, any of the aforementioned proteins. For example, recombinant fusion proteins comprising one of the aforementioned proteins plus a multimerization domain, such as a leucine zipper, a coiled coil, an Fc portion of an immunoglobulin, or a substantially similar protein, can be produced using the methods of the invention. See, for example, WO94/10308; Lovejoy et al. (1993), Science 259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury et al. (1994), Nature 371:80-83; Hâkansson et al. (1999), Structure 7:255-64. Specifically included among these recombinant fusion proteins are proteins in which a portion of a receptor is fused to an Fc portion of an antibody such as etanercept (a p75 TNFR:Fc) and belatacept (CTLA4:Fc).
    [083] While the terminology used in this application is standard within the art, definitions of certain terms are provided here to ensure clarity and definition for the meaning of the claims. Units, symbols and prefixes may be indicated in their accepted SI form. Numeric ranges mentioned include the numbers defining the range and include and support each integer within the defined range. The methods and techniques described herein are generally performed according to conventional methods known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification, unless otherwise indicated. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (nineteen ninety). All documents, or parts of documents, cited in this application, including but not limited to patents, patent applications, articles, books and treaties, are expressly incorporated by reference. What is described in one embodiment of the invention may be combined with other embodiments of the invention.
    [084] The present invention is not to be limited in scope by the specific embodiments described herein which serve as simple illustrations of individual aspects of the invention, and functionally equivalent components and methods are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the description that precedes and accompanies the drawings. These modifications are intended to be within the scope of the appended claims. EXAMPLES Example 1
    [085] This experiment compares different initial conditions using batch feeding or batch fed methods followed by continuous perfusion using alternating tangential flow filtration. Perfusion was started early during the early exponential growth phase (“initial batch”) with no additional feeds prior to perfusion, or at the end of the exponential phase and entering the stationary or production phase (“initial fed batch”) receiving multiple feeds. bolus of a serum-free feeding medium prior to infusion.
    [086] On day 0, CHO cells expressing a recombinant antibody were inoculated into 2L production bioreactors at 1 x 10 6 viable cells/mL in a working volume of 1500 mL of serum-free defined medium for the start of the fed batch and 1800mL to the batch start. Cultures were maintained at 36°C, in 30% dissolved oxygen (DO), shaking at 215 RPM. Glucose was maintained above 0 g/L and below 8 g/L.
    [087] Perfusion was started on day 4 (0.25 Vol/day) for batch cultures and on day 7 (0.75 Vol/day) for fed batch cultures. Perfusion was performed using an alternate tangential flow perfusion and filtration system ((Refine Technologies, Hanover, NJ, 50 kDa hollow fiber filter). Concentrated serum-free defined feeding medium on day 4 (7.5% of initial working volume) and day 6 (10% of initial working volume) Perfusion rates are given in Table 1. Table 1. Perfusion rate
    Values are based on working volumes disclosed above * Day 0-7 for fed batch start
    [088] During the cultivation process, daily samples were collected to evaluate the culture. Viable cell density (VCD) and viability were determined using Vi-Cell (Beckman Coulter, Brea, CA). Titer was measured by HPLC analysis. Packed cell volume was determined using VoluPAC (Sartorius, Goettingen, Germany).
    [089] A temperature shift (36.0°C to 33.0°C) was applied when the density of viable cells exceeded by 20 x 106 viable cells/mL, which was at day 7 and day 11 for the initial conditions. batch and fed batch, respectively.
    [090] For initial batch conditions, viable cell density continued to increase after perfusion was initiated; for the initial fed-batch conditions, perfusion was started after the cell culture had reached a plateau or stationary phase with little growth. On day 15, the density of viable cells for the fed batch was between 27.7 and 30.7 x 10 6 viable cells/mL while the VCD of the batch culture was between 22.5 and 27.4 x 10 6 viable cells/mL (Figure 1A). Fed batch culture viability was between 73.9% and 77.5%, while batch culture viability was between 72.5 and 83.1% (Figure 1B). The fed batch culture titer was between 15.3 and 16.1 g/L, while the batch culture titer was between 10.6 and 12.3 g/L (Figure 1C). Since the integrated variable cell density (IVCD) values were similar for all four cultures at day 15 (approximately 230 x 10 6 cell days/mL), the specific productivity was higher between the initial fed-batch conditions. Fed batch cultures were continued until day 24. A concentration of 20 g/L was reached within 20 days.
    [091] Alternating tangential flow perfusion with a fed batch start resulted in increased productivity, keeping the cells in a more productive state compared to the batch start method. Example 2
    [092] On day 0, CHO cells expressing a recombinant antibody were inoculated into 2L production bioreactors at 1 x 10 6 viable cells/mL in a working volume of 1500 mL of serum-free defined medium for the start of batch culture and 1300mL for the start of the fed batch. Cultures were maintained at 36°C, 30% OD, shaking at 215 RPM for batch cultures. The fed batch culture was shaken at 430 RPM. The fed batch culture was fed 7 g/L glucose daily prior to perfusion and all cultures were maintained at or above 4 g/L glucose during perfusion. Perfusion (alternating tangential flow) was started on day 4 (0.25 Vol/day) for the batch cultures and on day 8 (0.75 Vol/day) for the fed batch culture. Prior to initiating perfusion, fed-batch cultures received bolus feeds of a concentrated serum-free defined feeding medium on day 4 (7.5% of initial working volume) and day 6 (10% of initial working volume) . Perfusion flow rate settings are provided in Table 2. Cultures were maintained for 21 days. Table 2. Perfusion rate

    [093] Values are based on work volumes disclosed above
    [094] During the culture process, daily samples were collected, as described above, to evaluate the culture.
    [095] A temperature shift (36.0°C to 33.0°C) was applied to the batch cultures on day 6 when the density of viable cells exceeded by 20 x 10 6 viable cells/mL as in Example 1. The fed batch culture was maintained at 36.0°C for the entire duration of the culture.
    [096] Batch start method cultures had similar results as described above, with cells reaching approximately 20 to 25 x 10 6 viable cells/mL with no growth after day 10. Fed batch culture reached nearly 30 x 10 6 viable cells/ml on day 20, after spending most of the culture duration below 20 x 10 6 viable cells/ml, see Figure 2A. All viabilities remained above 80% until day 10 and then dropped to about 40% at day 20 for the batch starter cultures and 60% for the fed batch culture, see Figure 2B. The titer peaked at almost 15 g/L for the batch starter cultures, but did not reach more than 20 g/L for the high agitation fed batch culture, see Figure 2C. It was observed that the batch starter cultures had an L-asparagine concentration of about 3 to 4mM on day 3 and did not experience an asparagine limited culture environment. However, the fed-batch start perfusion culture experienced a limited L-asparagine environment on day 6, prior to the start of perfusion on day 7. The culture was then perfused with medium containing L-asparagine at a concentration of 2. 0 g/L (or 13.3 mM), which resulted in no more L-asparagine limitations after day 8 (Figure 2D). Glucose concentrations were mainly maintained between 4 and 10 g/L.
    [097] Batch starter fed high shake perfusion culture reached highest titer (more than 20 g/L) in 20 days, more than 5 g/L higher than batch starter cultures, which was similar to the results described above. No negative effects of a higher agitation rate were observed. Maintaining a constant temperature did not appear to negatively affect the fed batch culture. Example 3
    [098] This experiment characterizes the effects of perfusion volume and temperature changes in an alternating tangential flow perfusion with a fed-batch start, as described above. All cultures were started in fed batch with the start of perfusion on day 7. Perfusion flow rates moving working volumes from three-quarters of the full volume or all of the working volume to three-quarters of the working volume were tested . A temperature change from 36°C to 33°C on day 14 was also tested.
    [099] On day 0, CHO cells expressing a recombinant antibody were inoculated into 2L production bioreactors at 1 x 10 6 viable cells/mL in a working volume of 1200 mL of serum-free defined medium. Cultures were maintained at 36°C, at 30% OD. Prior to infusion, glucose was fed at 7 g/L daily, and during infusion glucose was maintained above 1 g/L. The culture was maintained for 20 days.
    [100] Cultures received bolus feeds of a concentrated serum-free defined feeding medium on day 4 (7.5% of initial working volume) and day 6 (10% of initial working volume). Perfusion started on day 8. Perfusion rates are given in Table 3. One culture from each group had a temperature change from 36°C to 33°C on day 15, the other cultures remained at 36°C for the entire duration. of the experiment. Table 3. Perfusion rate

    [101] Values are based on working volumes disclosed above
    [102] Change in temperature and perfusion rate had no impact on viable cell density, see Figure 3A. However, a change in temperature appears to help preserve viability at later time points in a culture. There seems to be an outlet between the changing temperature conditions starting on the 15th onwards. The viability of the temperature shift cultures dropped more slowly than that of the cultures that remained at 36°C, see Figure 3B. As for titer, three cultures had very similar titers on day 15 (17.1-17.9 g/L) as well as on day 20 (22-24 g/L), but one culture had a higher titer on day 20 (22-24 g/L). 15 (21.58 g/L) as well as on day 20 (28.33 g/L) (see Figure 3C). Both temperature and perfusion rates did not appear to have any impact on titer yield, suggesting that cultures can be maintained at different perfusion rates. Example 4
    [103] This experiment was designed to investigate the effects of perfusion medium asparagine concentrations and perfusion initiation conditions with limited or unlimited L-asparagine culture environments on viable cell density during the production phase.
    [104] On day 0, CHO cells expressing a recombinant antibody were inoculated into 2L production bioreactors at 1 x 10 6 viable cells/mL in a working volume of 1500 ml for the batch start and fed-batch methods. Cultures were maintained at 36°C, dissolved oxygen (DO) at 30%, shaking at 400 RPM. Washing was performed using a perforated tube or a sintered washer. Glucose was maintained above 0 g/L and below 8 g/L.
    [105] Perfusion (alternating tangential flow) was started on day 3 (0.29 Vol/day) for batch-start “unlimited asparagine cultures” and on day 7 (0.48 Vol/day) for “ cultures with limited asparagine” in fed batch. The batch culture medium contained 10mM L-asparagine. Prior to initiating the perfusion, the fed-batch cultures received bolus feeds of a concentrated serum-free defined feeding medium on days 3 and 6 (7% of the initial working volume) containing 113.4 mM L-asparagine. Asparagine concentrations of the perfusion medium were either a control concentration (17.3 mM Asn in a defined perfusion medium without serum) or a low concentration (5 mm Asn in a defined perfusion medium without serum). Perfusion was performed as described above. Perfusion rates are given in Table 4. Table 4. Perfusion rate

    [106] During the culture process, samples were taken daily to assess the culture. Viable cell density (VCD) and viability were determined using Vi-Cell (Beckman Coulter, Brea, CA). Titer was measured by HPLC analysis. All cultures were maintained at 36.0°C.
    [107] Reduction in cell growth and increased productivity were achieved during the production phase by limiting asparagine in the culture medium. At day 15, the maximum viable cell density was approximately 17.0 x 10 6 viable cells/ml for the low asparagine fed batch starter cultures (Figure 4A). Control-level asparagine cultures achieved viable cell densities exceeding 40 x 10 6 viable cells/mL (>30% packed cell volume). Low asparagine fed batch culture viability was 67.1%, while batch culture viability was 55.1% and the control was 69% (Figure 4B). The packed cell volume adjusted titer of the low asparagine fed batch culture was 17.0 g/L (adjusted for packed cell volume), while the batch culture titer was about 15.4 g/L (Figure 4C) . Controls had a concentration of 10.2 to 12.9 g/L (batch start) and 14.2 to 15.9 g/L (fed batch start).
    [108] Maintaining asparagine levels at 5mM or less during production resulted in growth arrest, stimulated productivity, and maintained viability during the production phase.
    [109] Example: 5 This experiment compares medium conditions during perfusion. In this 2L bioreactor experiment, cells were inoculated into a chemically defined batch medium in a working volume of 1.5 L, cultured for 3 days and then perfused for 12 days using a chemically defined perfusion medium containing 17, 3 mM L-asparagine and 4.6 mM L-glutamine or 5 mM L-asparagine and 10 mM L-glutamine. Perfusion was performed using an alternating tangential flow perfusion and filtration system ((Refine Technologies, Hanover, NJ), with a 30 kDa hollow fiber filter (GE Healthcare, Little Chalfont, UK). , at a rate of 0.3 culture volumes per day. The perfusion rate was increased on days 4, 9, and 11, as indicated in Table 6 below. Cultures were maintained at 36oC, at 30% OD, with pH at 7.0 and stirring at 400 rpm.
    [110] During the culturing process, samples were taken daily to assess the culture. Viable cell density (VCD) and viability were determined using Vi-Cell (Beckman Coulter, Brea, CA). Titer was measured by HPLC analysis. Packed cell volume was determined using VoluPAC (Sartorius, Goettingen, Germany).Table 5. Perfusion rate schedule

    [111] Asparagine limitation resulted in fewer cells accumulating and increased productivity. Cultures perfused with medium containing 5 mM asparagine reached a maximum VCD of 8.16 x 10 7 - 8.54 x 10 7 cells/mL, while cultures perfused with medium containing 17.3 mM asparagine reached 11.9 x 10 7 - 12, 2 x 10 7 cells/ml (Figure 5A). Although cultures in 17.3 mM asparagine had more cells, cultures in 5 mm asparagine produced more product. Cultures perfused with medium containing 17.3 mM asparagine yielded 6.89-7.18 g/L (adjusted packed cell volume 4.33-4.67 g/L) compared to 7.59-8.15 g /L (packaged cell volume adjusted from 5.01-5.38 g/L) for cultures perfused with medium containing 5mM asparagine (Figures 5B and 5D). The final packed cell volume (PCV) of cultures with 5 mM asparagine tended slightly less than cultures with 17.3 mM asparagine (Figure 5C) and there was no difference in culture viability (Figure 5E).
    [112] Interestingly, in this example, increasing the glutamine concentration by more than twice in the low asparagine condition (4.6 mM versus 10 mM glutamine) did not interfere with the ability of the low asparagine medium to stop the culture growth. Example 6
    [113] This example compares the performance of a clonal CHO cell line expressing antibody grown in an ATF perfusion process using asparagine limitation to control growth at bench and pilot scales. The bench scale model used 2L bioreactors and the 500L pilot scale model. At bench scale, cells were inoculated into a chemically defined batch medium at a working volume of 1.5 L and at pilot scale, the working volume was about 378 L. Cells were cultured for 3 days in batch medium and then perfused for 12 days using a chemically defined perfusion medium containing 5 mM L-asparagine and 10 mM L-glutamine. Perfusion was performed using an alternating tangential flow perfusion and filtration system (Refine Technologies, Hanover, NJ) with a 30 kDa hollow fiber filter (GE Healthcare, Little Chalfont, UK). Perfusion was started on day 3 at a rate of 0.3 culture volume per day. The infusion rate was increased on days 4, 9 and 11 as shown in Table 7 below. Cultures were maintained at 36°C, 30% DO and pH 6.9.
    [114] During the culture process, daily samples were taken to assess the culture. Viable cell density (VCD) and viability were determined for bench scale using Vi-Cell (Beckman Coulter, Brea, CA) and for pilot scale using a CEDEX (Roche Applied Science, Indianapolis, IN). Titer was measured by HPLC analysis. Packed cell volume was determined using VoluPAC (Sartorius, Goettingen, Germany).Table 6. Perfusion rate schedule

    [115] Data from four bench scale cultures and two pilot scale cultures are provided. VCD curves were similar on both scales, growth control was achieved (Figure 6A), and total cell mass (packed cell volume) was kept below 30% on both scales (Figure 6C). Although VCD reached a plateau around day 10 or day 11, packed cell volume continued to increase until about day 13 or 14 (Figure 6C). Productivity was also similar between scales. Cultures perfused with medium containing 5mM asparagine produced 14.2-15.7 g/L (adjusted packed cell volume 10.7-11.4 g/L) on a 2L bench scale compared to 15.0 - 17 .3 g/L (packaged cell volume adjusted from 10.6-12.8 g/L) in 500L pilot scale (Figures 6B and 6D). Viability tended slightly downwards on the pilot scale (Figure 6E).
    权利要求:
    Claims (21)
    [0001]
    1. A method for culturing mammalian cells expressing a recombinant protein, wherein said mammalian cells are 293 cells or CHO cells, the method comprising: (a) establishing a culture of mammalian cells in a serum-free culture medium in a bioreactor, inoculating the bioreactor with at least 0.5 x 10 6 to 3.0 x 10 6 cells/mL in a serum-free culture medium; (b) culturing the mammalian cells during a growth phase and supplementing the culture medium with bolus feeds of a serum-free feeding medium, (c) initiating perfusion on day 5 through day 9 of cell culture, or when the viable cell density (VCD) is at least 10 x 10 6 cells/mL, and (d) maintaining mammalian cells during the production phase by perfusion with a serum-free perfusion medium, wherein: (i) the packed cell volume during the production phase is maintained at less than or equal to 35%, or (ii) the VCD does not exceed 1 x 10 8 cells/mL during the production phase; and wherein said method is defined to further comprise: (1) inducing cell growth arrest by L-asparagine deprivation followed by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less; (2) inducing cell growth arrest by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less; (3) change the temperature, where the growth phase takes place at a temperature of 35°C to 38°C and the production phase takes place at a temperature of 30°C to 34°C, or where the temperature is reduced from a range between 35°C and 38°C, to a range between 30°C and 34°C.
    [0002]
    Method according to claim 1, characterized in that the perfusion starts on day 5 to day 7 of cell culture.
    [0003]
    Method according to claim 1 or 2, characterized in that the perfusion starts when the cells have reached a production stage.
    [0004]
    Method according to claim 1, characterized in that the concentration of L-asparagine in the serum-free perfusion medium is less than or equal to 5 mM, less than or equal to 4.0 mM, less than or equal to 3.0 mM , less than or equal to 2.0 mM, less than or equal to 1.0 mM or be 0 mM.
    [0005]
    Method according to claim 1, characterized in that the concentration of L-asparagine in the cell culture medium is monitored before and during the deprivation of L-asparagine.
    [0006]
    Method according to any one of claims 1 to 5, characterized in that the packed cell volume is less than or equal to 30%.
    [0007]
    Method according to any one of claims 1 to 5, characterized in that the density of viable cells from the mammalian cell culture in a packed cell volume less than or equal to 35% is 10 x 10 6 viable cells/ml at 80 x 106 viable cells/ml.
    [0008]
    Method according to any one of claims 1 to 7, characterized in that the infusion comprises continuous infusion.
    [0009]
    Method according to any one of claims 1 to 8, characterized in that the perfusion rate is constant.
    [0010]
    A method according to any one of claims 1 to 9, characterized in that the perfusion is performed: (a) at a rate less than or equal to 1.0 working volume per day; or (b) at a rate that increases during the production phase from 0.25 working volume per day to 1.0 working volume per day during cell culture.
    [0011]
    A method according to any one of claims 1 to 10, characterized in that the perfusion is performed at a rate reaching 1.0 working volume per day on day 9 to day 11 of cell culture.
    [0012]
    A method according to any one of claims 1 to 11, characterized in that bolus feeds of the serum-free feeding medium begin on day 3 or day 4 of cell culture.
    [0013]
    Method according to any one of claims 1 to 12, characterized in that the mammalian cell culture is established by inoculating the bioreactor with at least 0.5 x 10 6 to 1.5 x 10 6 cells/ml in culture medium. without serum.
    [0014]
    Method according to any one of claims 1 to 13, characterized in that it further comprises a temperature change from 36°C to 31°C or from 36°C to 33°C.
    [0015]
    Method according to claim 14, characterized in that the temperature change occurs in the transition between the growth phase and the production phase or during the production phase.
    [0016]
    Method according to any one of claims 1 to 13, characterized in that it further comprises a temperature shift, wherein the growth phase takes place at a temperature of 35°C to 38°C and the production phase takes place at a temperature of 35°C to 38°C. temperature from 30°C to 34°C.
    [0017]
    Method according to any one of claims 1 to 16, characterized in that the perfusion is carried out by alternating tangential flow.
    [0018]
    Method according to any one of claims 1 to 17, characterized in that the bioreactor has a capacity of at least 500L.
    [0019]
    Method according to any one of claims 1 to 18, characterized in that the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein or a cytokine.
    [0020]
    Method according to any one of claims 1 to 19, characterized in that it additionally comprises a step of harvesting the recombinant protein produced by the cell culture.
    [0021]
    21. Method according to any one of claims 1 to 20, characterized in that the bioreactor has a capacity of at least 500L, at least 1000L or at least 2000L.
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    法律状态:
    2019-06-04| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2019-12-17| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|
    2020-03-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-10-27| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2021-05-04| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2021-11-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2022-01-25| 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 29/06/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201161503737P| true| 2011-07-01|2011-07-01|
    US61/503,737|2011-07-01|
    PCT/US2012/045070|WO2013006479A2|2011-07-01|2012-06-29|Mammalian cell culture|
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