EA045739B1 - CULTURING MAMMALAL CELLS - Google Patents

Description

Field of invention

The present invention provides a method for culturing mammalian cells. The method gives greater control over cell growth to achieve greater production of cellular titers.

State of the art

With the increasing number of therapeutic recombinant proteins, increases in cell growth, viability and protein production can be achieved through the use of new methods to improve cell development, optimization of the working environment and process control parameters. Nowadays, great efforts are being made to optimize the process, in particular to the methods and principles of culturing, feeding and care during cell production.

Nowadays, cell culture methods that provide relative improvements in the production of recombinant proteins are very valuable when considering the large-scale costs of cell culture and the growing need for more of it and lower cost of biological products.

There is a need for improvements in cell culture processes, recombinant polypeptide isolation, titers, and cell viability that can lead to higher production levels, thereby reducing the costs associated with producing a protein therapeutic. The present invention addresses these needs by providing a simple, easy and inexpensive method for regulating cell growth while increasing protein production.

The essence of the invention

The present invention provides a method for limiting cell growth in the cultivation of mammalian cells secreting a recombinant protein that contains the formation of mammalian cells in a serum-free culture medium in a bioreactor; a method of inducing cell growth restriction by perfusion in a serum-free culture medium containing 5 millimoles or less of L-asparagine; a method of caring for mammalian cells under growth-limiting conditions by perfusion in a serum-free medium containing 5 millimoles or less of L-asparagine.

The present invention also provides a method for increasing the production of recombinant proteins by culturing mammalian cells secreting a recombinant protein, which comprises producing mammalian cells in a serum-free culture medium in a bioreactor; a method of inducing cell growth restriction by perfusion in a serum-free culture medium containing 5 millimoles or less of L-asparagine; a method of caring for mammalian cells under growth-limiting conditions by perfusion in a serum-free medium containing 5 millimoles or less of L-asparagine. In related embodiments, the production of recombinant proteins when culturing mammalian cells is increased compared to culturing in which the cells are not treated with growth-limiting L-asparagine.

The present invention also provides a method for limiting the cultivation of mammalian cells, which contains a recombinant protein with a desired volume of compacted cells, culturing mammalian cells in a serum-free culture medium in a bioreactor; a method of inducing cell growth restriction by perfusion in a serum-free culture medium containing 5 millimoles or less of L-asparagine; a method of caring for mammalian cells under growth-limiting conditions by perfusion in a serum-free medium containing 5 millimoles or less of La asparagine.

In one embodiment of the present invention, in any of the above methods, perfusion with serum-free medium containing 5 millimoles or less of L-asparagine begins on or after the third day of culture. In another embodiment, in any of the above methods, the introduction of cell growth restriction occurs before the start of the production step. In another embodiment, in any of the above methods, the introduction of cell growth restriction occurs during the manufacturing step. In another embodiment, in any of the above methods, the introduction of cell growth restriction is caused by a lack of L-asparagine. In another embodiment, in any of these methods there is a temperature change from 36 to 31 °C. In another embodiment, in any of these methods there is a temperature change from 36 to 33°C. In a related embodiment, a temperature change occurs during the transition from the growth stage to the production stage. In another embodiment, the temperature change occurs during the production stage. In another embodiment, the method contains a volume of compacted cells less than or equal to 35% during the production step. In a related embodiment, the volume of compacted cells during the production step is less than or equal to 35%.

The present invention also provides a method for culturing mammalian cells containing a recombinant protein; cultivation in a serum-free medium in a bioreactor; growth of mammalian cells during the growth stage and provision of culture medium by bead feeding of serum-free medium and care of mammalian cells during the production stage by perfusion of serum-free medium in which the volume of compacted cells during the production stage is 35% or less . In one embodiment of the present invention, perfusion begins between the fifth and ninth day of cell culture. In a related embodiment, perfusion begins on the fifth to seventh day of cell culture. In one embodiment, perfusion begins when the cells reach the production stage. In another embodiment, the method comprises limiting cell growth caused by L-asparagine deficiency following perfusion of serum-free medium containing 5 millimoles or less of L-asparagine. In another embodiment, the method comprises limiting cell growth caused by perfusion of a serum-free treatment medium containing 5 millimoles or less of L-asparagine.

In one embodiment of the present invention, the L-asparagine content of the serum-free treatment medium is 5 millimoles or less. In another embodiment, the L-asparagine content of the serum-free treatment medium is 4.0 millimoles or less. In another embodiment, the L-asparagine content of the serum-free treatment medium is 3.0 millimoles or less. In another embodiment, the L-asparagine content of the serum-free treatment medium is 2.0 millimoles or less. In another embodiment, the L-asparagine content of the serum-free treatment medium is 1.0 millimoles or less. In another embodiment, the L-asparagine content of the serum-free treatment medium is 0 millimoles. In another embodiment, perfusion occurs at a rate that increases during the production stage from 0.25 workspace to 1.0 workspace per day during cell culture. In a related embodiment, perfusion occurs at a rate of up to 1.0 workspace per day from the ninth to eleventh day of cell culture. In another related embodiment, perfusion occurs at a rate reaching 1.0 workspace per day on the tenth day of cell culture. In another embodiment, blotting of serum-free medium begins on the third or fourth day of cell culture. In another embodiment of the present invention, the method comprises changing the temperature from 36 to 31 °C. In another embodiment, the method comprises changing the temperature from 36 to 33°C. In a related embodiment, a temperature change occurs during the transition from the growth stage to the production stage. In a related embodiment, the temperature change occurs during the manufacturing stage.

In one embodiment of the present invention, the L-asparagine content of the cell culture medium is adjusted before and during L-asparagine deficiency.

In one embodiment of the present invention, the volume of compacted cells is 35% or less. In a related embodiment, the volume of the compacted cells is 30% or less.

In one embodiment of the present invention, the viable cell density in a mammalian cell culture with a compacted cell volume equal to or less than 35% is between 10 x 106 viable cells/mL and 80 x 106 viable cells/mL. In a related embodiment, the viable cell density in a mammalian cell culture is from 20x106 viable cells/ml to 30x106 viable cells/ml.

In one embodiment of the present invention, the perfusion is continuous perfusion.

In one embodiment of the present invention, the perfusion rate is constant.

In one embodiment of the present invention, perfusion occurs at a rate less than or equal to 1.0 workspace per day.

In another embodiment of the present invention, a mammalian cell culture is established by introducing into the bioreactor at least 0.5x106 to 3.0x106 cells/milliliter of serum-free medium. In a related embodiment, a mammalian cell culture is established by introducing at least 0.5x106 to 1.5x106 cells/milliliter of serum-free medium into the bioreactor.

In another embodiment of the present invention, perfusion is accompanied by a varying tangential flow.

In another embodiment of the present invention, the bioreactor capacity is at least 500 L. In a related embodiment, the bioreactor capacity is at least from 500 to 2000 L. In another related embodiment, the bioreactor capacity is at least 1000 to 2000 L.

In another embodiment of the present invention, the mammalian cells are Chinese hamster ovary (CHO) cells.

In another embodiment of the present 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.

In another embodiment of the present invention, any of the above methods comprise the step of collecting recombinant protein produced by the cell culture.

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In another embodiment of the present invention, the recombinant protein produced by the cell culture is purified and formed into a pharmaceutically acceptable formulation.

Brief description of the figures

Fig. 1 - the beginning of the process with recharge: solid square () and solid circle (·). The beginning of a periodic process: an empty square (□) and an empty circle ( ⁰ ).

Fig. 1A - density of viable cells, Fig. 1 - viability, Fig. 1C - titer.

Fig. 2 - beginning of a periodic process: open circle (°), beginning of a process with recharge with strong excitation: open square (□).

Fig. 2A - viable cell density, FIG. 2B - viability, FIG. 2C - titer, fig. 2D asparagine content.

Fig. 3 - 1.0 initial perfusion volume, without temperature change: solid circle; 1.0 initial perfusion volume, without temperature change: open circle (o); 0.75 initial perfusion volume, without temperature change: solid square (); 0.75 initial perfusion volume, no change in temperature: open square (□).

Fig. 3A - viable cell density, FIG. 3B - viability, FIG. 3C - titer.

Fig. 4 - beginning of a batch process with low asparagine content: empty triangle (D). Start of batch process with controlled amount of L-asparagine: solid triangle (A). Start of process with low L-asparagine feed: open diamond (^). Start of feeding process with controlled amount of L-asparagine: solid square (♦). Processing with a pipe with holes: solid line. Treatment with sintered sprinklers: dotted line.

Fig. 4A - viable cell density, FIG. 4B - viability, FIG. 4C - titer taking into account the volume of compacted cells.

Fig. 5 - cultures grown in a medium containing 17.3 millimol or 5 millimol L-asparagine and 4.6 millimol or 10 millimol L-glutamine; 17.3 millimol L-asparagine and 4.6 millimol L-glutamine, solid diamond (♦) or 5 millimol L-asparagine, 10 millimol L-glutamine, open diamond (0).

Fig. 5A - density of viable cells; fig. 5B - titer; fig. 5C - volume of compacted cells; fig. 5D - titer taking into account the volume of compacted cells; fig. 5E - vitality.

Fig. 6 - cultivation in 2 liters of bench and 500 liters of experimental mode, with 5 millimoles of L-asparagine, 10 millimoles of L-glutamine. The medium contains 5 millimols of L-asparagine, 10 millimols of L-glutamine in 2 L bench mode is represented by a solid diamond (♦), 500 L experimental mode is represented by an open diamond (6)

Fig. 6A - density of viable cells; fig. 6B - titer; fig. 6C - volume of compacted cells; fig. 6D - titer taking into account the volume of compacted cells; fig. 6E - vitality.

Detailed Description of the Invention

During the production of recombinant proteins, it is desirable to have a regulated system in which cells are grown to the desired density and then the physiological state of the cells is switched to a growth-limiting mode, a high-throughput state in which the cells use energy and substrates to produce the recombinant protein of interest instead of producing more cells . Methods to achieve this, such as changing temperature and excitation of small molecules, are not always successful and may have undesirable effects on product quality. As described herein, the volume of compacted cells can be limited to a desired level during the production step by forcing cell growth restriction in low L-asparagine cultured cells. Limitation of cell growth can be achieved and maintained by using a treatment medium containing a limited amount of L-asparagine and maintaining a low L-asparagine content in the cell culture (5 millimoles or less).

It was also found that growth-restricted cells had greater productivity if the growth restriction was caused by low or insufficient L-asparagine, and the growth-restricted cells were sequentially treated with cell culture and treatment medium containing 5 millimoles or less of L-asparagine.

A production stage with limited growth and high productivity can be achieved by changing the concentration of L-asparagine. As described here, L-asparagine depletion results in growth restriction. In fed-batch culture, if the cell density is high enough (e.g. > 20 x 106 viable cells/mL), the cells are continuously starved of L-asparagine despite re-feeding due to L-asparagine consumption and/or conversion to L-aspartate . When growing cells, extracellular L-asparagine can be converted to L-aspartate or ammonia. L-asparagine depletion leads to cell cycle restriction. During feeding periods, when L-asparagine is present in the culture, it results in increased productivity, periods of time when L-asparagine is depleted results in decreased productivity. In the treatment system, L-asparagine is supplied continuously, which helps avoid overall depletion, and can

- 3 045739 maintain higher levels of L-asparagine, allowing cells to continue to multiply without depleting or limiting L-asparagine. Controlling L-asparagine at sufficiently low levels (eg, 5 millimoles or less) can maintain high cell productivity and establish viability and growth limitation. In a bead-fed and perfusion system, the feed medium can be changed from a composition with a high level of (growth-promoting) L-asparagine in the bead-fed and a lower level (growth-limiting) L-asparagine in the perfusion-fed. Cell cultures limited in growth by limiting L-asparagine can have high productivity by adding low levels of L-asparagine.

On a commercial scale for cell culture and therapeutic production, the ability to limit cell growth and maintain cells in this state during the production stage would be highly desirable. The presence of cells that have been introduced to increase productivity while limiting growth and maintaining increased production is ideal for production purposes.

Described herein is a method for limiting cell growth in a mammalian cell culture releasing a recombinant protein. The method comprises limiting cell growth in a mammalian cell culture by exposing the cell culture to a serum-free medium containing 5 millimoles of L-asparagine or less, containing 0 millimoles of L-asparagine. Such administration may be caused by depletion of L-asparagine or by creating a low-L-asparagine environment by perfusing the culture with serum-free medium containing 5 millimoles of L-asparagine or less, or by maintaining a low-L-asparagine environment. The cell culture is maintained in a growth-limited state by perfusion with serum-free medium containing 5 millimoles of L-asparagine or less, and the culture is maintained in medium containing low L-asparagine.

Also provided is a method for increasing the production of recombinant protein in a mammalian cell culture that releases the recombinant protein by introducing a low level of asparagine to limit cell growth in the mammalian cell culture. Mammalian cells maintained in low-asparagine growth-limiting media exhibit greater productivity (g protein/cell/day and g protein/cell mass/day) than those not growth limited by low asparagine.

This method is also useful for limiting a mammalian cell culture to a desired volume of compacted cells. The volume of compacted cells during the production stage can be limited to the desired level by reducing the level of L-asparagine during production of the culture medium. An asparagine concentration of 5 millimoles or less in the treatment medium is sufficient to control growth in culture and limits the desired volume of compacted cells.

The methods described here provide greater control over cell growth with high production titers of cell cultures; and may themselves facilitate gas release compared to large biomass perfusion processes, as well as minimize product losses during harvesting and subsequent processing processes.

The method begins with the establishment of a mammalian cell culture in a production bioreactor. It is preferable to use smaller size bioreactors, in one embodiment the volume of the bioreactors is from 500 to 2000 L. In a preferred embodiment, bioreactors with a volume of 1000-2000 l are used. The seed chamber density used in the bioreactor can have a positive effect on the level of recombinant protein produced. In one embodiment, the bioreactor is fed with at least 0.5 x 10 ⁶ to 3.0 x 10 ⁶ viable cells/milliliter in serum-free medium. In a preferred embodiment, 1.0 x 10 ⁶ viable cells/milliliter is administered.

Mammalian cells then go through a stage of exponential growth. The cell culture can be maintained without additional feed until the desired cell density is achieved. In one embodiment, the cell culture is maintained without supplementation for up to three days, followed by perfusion of serum-free medium containing 5 millimoles or less of L-asparagine to introduce and maintain low L-asparagine limitation. In another embodiment, the culture may be introduced at the desired cell density to begin the production stage without a transient growth stage with cell growth limitation induced immediately after administration by perfusion of the cell culture with serum-free medium containing 5 millimoles or less of L-asparagine to introduce and maintain the restriction low L-asparagine content. In any of the embodiments described herein, the transition from the growth stage to the production stage may be caused by a lack of L-asparagine (cells exposed to media containing 0 millimoles of L-asparagine); After this, the cell medium is perfused with an L-asparagine content of 5 millimoles or less, the concentration of L-asparagine in the cell culture is maintained

- 4 045739 at this level.

Regardless of how low growth is limited by L-asparagine, greater performance is observed in growth-limited cells in cells maintained by perfusion of low-L-asparagine medium and maintenance of cell culture containing

L-asparagine 5 millimoles or less.

As used herein, growth limitation, which may also be referred to as cell growth limitation, is the point at which the number of cells stops increasing or when the cell cycle no longer continues. Growth limitation can be monitored by determining the viable cell density of a cell culture. Some cells in a growth-limited state may increase in size but not in number, so the volume of compacted cells in a growth-limited culture may increase. Growth restriction may be somewhat reversible if cell health is not compromised by the addition of L-asparagine to the cell culture.

Growth limitation is caused by L-asparagine when the cell density of the culture reaches a level where the concentration of L-asparagine in the culture becomes limited during continuous growth or when the culture is starved of L-asparagine. L-asparagine deficiency occurs when the L-asparagine content in the cell culture medium is 0 millimole. Deficiency within 24 hours may result in growth restrictions. Deficiency for more than 48 hours can be detrimental to cellular health. To maintain cells in a growth-limited state, the content of L-asparagine in the cell culture must be maintained at 5 millimoles or less. The cell culture medium containing the L-asparagine necessary to limit cell growth depends on the cells' ability to produce their own asparagine. For crops that produce their own asparagine, lower levels or even removal of L-asparagine from the environment may be required to limit growth. For cultures unable to produce their own asparagine, such as cells lacking active asparagine synthetase enzyme, 0 to 5 millimoles of L-asparagine can be used to limit growth.

As used herein, compacted cell volume (PCV), also called percent compacted cell volume (%PCV), is the ratio of the volume occupied by cells to the total cell culture volume, expressed as a percentage (see Settler, wt al, (2006) Biotechnol Bioeng. Dec 20 :95(6): 1228-33). The volume of compacted cells is a function of cell density and cell diameter; the increase in compacted cell volume can be caused by an increase in either cell density, cell diameter, or both. The volume of compacted cells is a measure of the solid content of a cell culture. Solids are removed during collection and subsequent cleaning processes. More solids means more energy to separate the solid from the desired product during the collection and subsequent purification steps. Also, the required product may remain in the solids and be lost during the collection process, resulting in less product being collected. Because cells themselves vary in size, and cell cultures also contain dead and dying cells and other products of cell breakdown, compacted cell volume is a more accurate way to describe the solids contained in a cell culture than cell density or viable cell density. For example, 2000 L with a cell density of 50 x 106 cells/ml may have a very different volume of compacted cells depending on the size of the cells. In addition, some cells increase in size when in a state of growth restriction, so the volume of compacted cells before and after growth restriction is likely to be different due to the increase in biomass resulting from the increase in cell size.

During the transition from the growth stage to the production stage and during the production stage, the percentage volume of compacted cells (%CCV) is 35% or less. The desired volume of compacted cells maintained during the production stage is 35% or less. In a preferred embodiment, the volume of compacted cells is 30% or less. In another preferred embodiment, the volume of compacted cells is 20% or less. In another preferred embodiment, the volume of compacted cells is 15% or less. In another preferred embodiment, the volume of compacted cells is 10% or less.

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 living cells in a given volume of culture medium, as determined by standard viability tests (such as the trypan blue exclusion test).

The desired viable cell density during the transition from the growth stage to the production stage and during the production stage results in a compacted cell volume of 35% or less. In one embodiment, the viable cell density is at least about 10x106 viable cells/ml to 80x106 viable cells/ml. In one embodiment, the viable cell density is at least about 10x106 viable cells/ml to 70x106 viable cells/ml. In one embodiment, the viable cell density is at least about 10 x 106 viable cells/ml

- 5 045739 up to 60x106 viable cells/ml. In one embodiment, the viable cell density is at least about 10x106 viable cells/ml to 50x106 viable cells/ml. In one embodiment, the viable cell density is at least about 10x106 viable cells/ml to 40x106 viable cells/ml. In one embodiment, the viable cell density is at least about 10x106 viable cells/ml to 30x106 viable cells/ml. In one embodiment, the viable cell density is at least about 10x106 viable cells/ml to 20x106 viable cells/ml. In one embodiment, the viable cell density is at least about 20x106 viable cells/ml to 30x106 viable cells/ml. In one embodiment, the viable cell density is at least about 20x106 viable cells/ml to 25x106 viable cells/ml, preferably about 20x106 viable cells/ml.

The lower density of compacted cells during the production stage helps eliminate dissolved oxygen issues that can inhibit higher density perfusion cultures. The lower density of compacted cells also allows smaller volume media to use smaller media storage vessels, and such cells can be mixed at slower flow rates. The lower density of compacted cells also has less impact on harvesting and subsequent processing compared to cultures with higher cell biomass. All of these reduce the cost associated with the production of recombinant protein therapeutics.

Three methods commonly used in commercial processes for the production of recombinant proteins by mammalian cell culture are semi-continuous culture, fed-batch culture, and perfusion culture. Semi-continuous culture, an intermittent method in which cells grow in a fixed volume of culture medium for a short period of time, followed by complete harvest. Cultures grown using a semi-continuous method experience an increase in cell density until maximum cell density is reached, after which viable cell density decreases and media components are consumed and levels of metabolic intermediates (such as lactate and ammonia) accumulate. Harvesting usually occurs at the point of maximum cell density (usually 5-10 x 106 cells/ml, depending on medium composition, cell line, etc.). Semi-continuous culture is the simplest method, however the density of viable cultures is limited by the availability of nutrients, and when cells are at maximum density, culture and production decreases. There is no need to extend the production stage because accumulation of waste products and nutrient depletion quickly lead to the cessation of cultivation (usually 3 to 7 days).

Fed culture has the advantage over a semi-continuous process in that it provides a pellet or continuous supply of media to replenish those media components that have been used. Because fed-fed cultures receive additional nutrients during operation, they can achieve higher cell densities (>10 to 30x106 cells/ml, depending on media composition, cell line, etc.) and higher product titers compared to the semi-continuous method. Unlike a semi-continuous process, a biphasic culture can be established and maintained by varying feed methods and media compositions to differentiate the timing of cell proliferation to achieve the desired cell density (growth stage) from the timing of delayed or slow cell growth (production stage). Fed-fed cultures alone cannot achieve higher product titers compared to semi-continuous cultures. Typically, fed-batch crops are used during the growth stage, the fed-fed method is used during the production stage, but the fed-fed method can be used during the entire process. However, unlike a semi-continuous process, bioreactor volume is a limiting factor in relation to feed volume. Just as with a semi-continuous process, metabolic intermediates accumulate and will result in a smaller crop, limiting the duration of the production stage to approximately 1.5 to 3 weeks. Fed crops are continuous and harvest usually occurs when metabolic intermediates or culture viability reach predetermined levels.

Perfusion methods have an advantage over semi-continuous and fed-batch processes by adding new media while removing spent media. Typical large-scale commercial cell culture methods strive to achieve high cell densities, 60-90(+)x106 cells/ml, where from a third to more than half of the reactor volume is biomass. In the case of perfusion culture, limiting cell densities >1x108 cells/ml have been achieved, and even higher densities are envisaged. Typical perfusion cultures begin with a semi-continuous culture that lasts a day or two, followed by a continuous, stepwise, and/or intermittent re-introduction of media into the culture while removing spent media during the growth and production stages of the culture, preserving cells and adding up to

- 6,045,739 additional weight components such as proteins (based on molecular weight cutoff for filters). Various techniques such as settling, centrifugation or filtration can be used to remove spent media while maintaining cell density. The perfusion flow rate of a fraction of the working volume per day is reported to various working volumes per day. The advantage of the perfusion process is that the production culture can be maintained longer than with the semi-continuous or fed-batch method. However, greater media preparation, storage, handling, and disposal are required to maintain a long-term perfusion culture, especially those with high cell densities that also require more nutrients, all of which result in even higher costs compared to semi-continuous or fed-batch methods. In addition, large cell densities can cause problems during production, such as maintaining dissolved oxygen levels and problems with high outgassing, including supplying additional oxygen and removing additional carbon dioxide, which can lead to more foaming and the need for changes in anti-foaming methods; similarly, during harvesting and subsequent processing processes, when greater energy is required to remove excess cellular material, which may result in product loss, negating the benefit of a higher titer due to excess cell mass.

Also, a large-scale cell culture method that combines feeding with feeding during the growth stage, followed by continuous perfusion during the production stage. The method targets a production step in which the cell culture is maintained at a cell compacted volume of less than or equal to 35%. The method also involves initiating and maintaining cell growth restriction at low asparagine levels.

Fed culture is a widely used method for culturing large-scale protein production from mammalian cells (See, for example, Chu and Robinson (2001), Current Opin. Biotechnol 12: 180-87). A fed-batch culture of mammalian cells is such that the culture is fed either continuously or intermittently with a concentrated feed medium containing nutrients. Feeding may occur on a predetermined schedule, such as every day, once every two days, once every three days, etc. Compared to a semi-continuous culture in which no feeding occurs, a fed-batch culture can produce more recombinant proteins. See, for example, US Patent No. 5,672,502.

In one embodiment, a bead fed fed culture is used to maintain the cell culture during the growth stage. Perfusion feed can be used during the production stage. In one embodiment, perfusion begins when the cells reach the production stage. In another embodiment, perfusion begins between days 5 and 9 of the cell culture. In another embodiment, perfusion begins between days 5 and 7 of the cell culture.

In another embodiment, the onset of cell growth restriction in a fed culture may be caused by exposing the fed culture to a deficiency of L-asparagine, followed by perfusion of serum-free medium containing 5 millimoles or less of L-asparagine. In one embodiment, the L-asparagine content of the cell culture is observed before and during L-asparagine deficiency. In another embodiment, the onset of limiting cell growth in a fed culture can be achieved by perfusion of serum-free medium containing 5 millimoles or less of L-asparagine.

The use of bead feed during the growth stage allows cells to enter the production stage, resulting in less dependence on temperature changes to induce and control the production stage. However, a temperature change of 36 to 31°C can occur between the growth and production stages. In a preferred embodiment, the change is from 36 to 33°C.

The bioreactor described herein may contain at least 0.5 x 106 to 3.0 x 106 viable cells/ml in serum-free medium, preferably 1.0 x 106 viable cells/ml.

Perfusion culture is a culture in which the cell culture receives a new supply of treatment medium while removing spent medium. Perfusion can be continuous, stepwise, intermittent, or a combination of any of these options. The perfusion rate may be less than the working volume for many working volumes per day. Preferably, the cells are maintained in culture and the spent medium removed is substantially free of cells or contains significantly fewer cells than the culture. Recombinant proteins isolated by cell culture can also be maintained in culture. Perfusion can be accomplished by a certain number of means, which include centrifugation, sedimentation or filtration. See, for example, Voisard et al., (2003), Biotechnology and Bioengineering 82:751-65. The preferred filtration method is variable tangential flow filtration. Variable tangential flow is maintained by forcing media through half-fiber filter modules (See, for example, US Pat. No. 6,544,424; Furey (2002) Gen. Eng. News. 22 (7), 62-63).

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As used herein, perfusion flow rate refers to the volume of media (added and removed) passed through the bioreactor and typically released as a fraction or unit of working volume in a given time. Working volume refers to the volume of the bioreactor used to accommodate the cell culture. In one embodiment, the perfusion flow rate is one working volume per day or less. The perfusion media can be formulated to maximize nutrients to minimize the perfusion rate.

Cell culture and culture refers to the growth and propagation of cells outside a multicellular organism or tissue. Suitable conditions for culturing 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, roller bottles, shake flasks, or mechanically stirred bioreactors, with or without microcarriers, may be used. In one embodiment, bioreactors ranging from 500 to 2000 L are used. In a preferred embodiment, bioreactors from 1000 to 2000 L are used.

For purposes of the present invention, a cell culture medium is a medium suitable for the growth of animal cells, such as mammalian cells, in cell culture outside the body. Cell culture medium compositions are well known in the art. Typically, the cell culture medium contains buffer mixtures, salts, carbohydrates, amino acids, vitamins and essential impurities. Serum-free medium means a cell culture medium that does not contain animal serum, such as fetal bovine serum. Various tissue culture media, including specific culture media, are commercially available, such as any one or combination of the following cell culture media: RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Essential Minimum Essential Medium (DMEM), Needle Minimum Essential Medium , F-12K medium, Ham's F12 medium, Iscove's modified Dulbecco's medium, McCoy's 5A medium, Leibowitz's L-15 medium, and serum-free medium such as EX-CELL™ Series 300 (JRH Biosciences, Lenexa, Kansas), among others . Serum-free versions of these culture media are also available. The cell culture medium may be accompanied by additional or increased levels of components such as amino acids, salts, buffers, sugars, vitamins, hormones, growth factors, antibiotics, lipids, impurities, etc., depending on the requirements of the cultured cells and/or the desired parameters cell culture.

Cell cultures may be supplied with a concentrated medium containing components, such as nutrients and amino acids, consumed during the cell culture production step. The supply of concentrated medium can be based on virtually any cell culture medium composition. This concentrated medium supply can contain most of the components of the cell culture medium, with 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X or even 1000X nominal quantities. Concentrated media feeding is often used in fed-culture processes.

The method according to the present invention can be used to increase the production of recombinant proteins at various stages of cultivation processes. In a multi-step process, cells are cultured in two or more separate steps. For example, cells may be cultured first in one or more growth stages under environmental conditions that maximize cell proliferation and viability, and then may be transferred to a production stage where protein production is maximized. In the commercial mammalian cell protein production process, there are varying numbers of growth steps, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, which occur in different culture vessels prior to final culture production. The growth and production stages may be followed by, or separated by, one or more transition stages. In multi-phase processes, the method of the present invention can be used at a minimum during the growth and production stages of the final production stage of a commercial cell culture, although it can also be used in a previous growth stage. The production stage can be carried out on a large scale. A large scale process may involve at least 100, 500, 1000, 2000, 3000, 5000, 7000, 8000, 10,000, 15,000, 20,000 liters. In a preferred embodiment, production takes place in bioreactors with a volume of 500 L, 1000 L and/or 2000 L. The growth stage may occur at a higher temperature than the production stage. For example, the growth step may occur at a first temperature of 35 to 38°C, and the production step may occur at a second temperature of 29 to 37°C, in some embodiments, 30 to 36°C, or 30 to 34°C. In addition, chemical stimulants for protein production, such as butyrate, caffeine and hexamethylene biacetamide (HMBA), can be added simultaneously, before and/or after the temperature change. If stimulants are added after a temperature change, they can be added from one hour to five days after the temperature change, with variations ranging 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).

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Cell culture samples can be monitored and evaluated using any of the analytical methods known in the art. Various parameters including recombinant protein quality, media quality and characteristics can be monitored during cultivation. Samples can be taken and monitored transiently at the desired frequency, including continuous monitoring in real time or near real time. In one embodiment, the L-asparagine content of the cell culture medium is monitored before and during L-asparagine deficiency.

Typically, cell cultures that precede final culture production (N-x to N1) are used to form seed chambers that will be used to introduce the N-1 culture for production in the bioreactor. Seed chamber density may have a positive effect on the level of recombinant protein production. Production levels increase with seed density. The increase in titer is not only associated with higher seed density, but may well be influenced by the cell cycle and metabolism of cells in production.

Seed chambers can be produced by any cultivation method. The preferred method is perfusion culture using variable tangential flow filtration. The N-1 bioreactor can operate with variable tangential flow filtration presenting cells at high density for introduction into the bioreactor for production. Stage N-1 can be used to grow cells to densities >90 x 10 ⁶ cells/ml. The N-1 bioreactor can be used to produce ball seed crops or can be used as a rolling seed culture used to produce seeds in bioreactors with high density seed chambers. The duration of the growth stage of production can vary from 7 to 14 days, and can be arranged to maintain exponential cell growth before introduction into the bioreactor for production. Perfusion rate, media composition and timing are optimized to grow and deliver cells to the bioreactor in the best condition to optimize production. Seed chamber densities > 15 x 10 ⁶ cells/ml can be achieved for seed production in bioreactors. Higher seed chamber densities when introduced can reduce or even eliminate the time required to achieve the desired production density.

The present invention finds particular application in improving cell growth, viability and/or protein production through cell culture processes.

The cell lines (also called host cells) used in this invention are genetically processed to isolate polypeptides of commercial or scientific interest. Cell lines are usually derived from a primary culture and can be maintained in culture indefinitely. A genetically engineered cell line comprises transfecting, transforming and converting cells with recombinant polynucleotide molecules and/or otherwise altering (eg, homologous recombination and gene activation or fusing a recombinant cell with a non-recombinant cell) to cause the host cell to secrete the desired recombinant polypeptide. Methods and directions for genetically altered cells and/or cell lines that secrete a polypeptide of interest are well known to those skilled in the art; for example, various methods are illustrated in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly editions); 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.

Animal cell lines are derived from cells whose precursors were derived from multicellular animals. 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 from commercial manufacturers. Examples of cell lines commonly used in industry include VERO, BHK, HeLa, CV1 (including Cos), MDCK, 293, 3T3, myeloma cell lines (eg, NSO, NS1), PC12, WI38 cells and Chinese hamster ovary cells ( YAKH). HAC cells are widely used for the production of complex recombinant proteins, such as 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 host cells of ICC because the effective DHFR of the system is selected and enhanced gene expression allows these cells to release high levels of recombinant proteins (Kaufman R.J. (1990), Meth Enzymol 185:537-566). In addition, these cells are easy to control in adherent or suspension cultures and exhibit relatively high genetic stability. JCC cells and their isolated recombinant proteins have been characterized and approved for use in clinical commercial production by regulatory authorities.

The methods of the present invention can be used for cell cultures that secrete

- 9 045739 recombinant proteins of interest. The isolated recombinant proteins can be secreted into the culture medium from which they can be recovered and/or collected. In addition, proteins can be purified, or partially purified, from such cultures or components (eg, culture media) using known processes and products available from commercial manufacturers. The purified proteins can then be formed, which means that the buffer solution is changed, sterilized, placed in a multi-dose package and/or packaged for the end user. Suitable formulations for pharmaceutical formulation include those described in Remington's Pharmaceutical Sciences, 18 ed., 1995, Mack Publishing Company, Easton, PA.

As used herein, the terms peptides, polypeptides and proteins are used interchangeably and refer to a molecule containing two or more amino acid residues attached to each by peptide bonds. Peptides, polypeptides and proteins also contain, but are not limited to, modifications including protein glycosylation, lipid attachment, sulfation, gammacarboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. Polypeptides may be of scientific or commercial interest, including protein-based drugs. Polypeptides contain antibodies, fusion proteins, and cytokines, among other things. Peptides, polypeptides and proteins are produced by recombinant animal cell lines using cell culture techniques and may be referred to as recombinant peptides, recombinant polypeptides and recombinant proteins. The isolated protein can be produced intracellularly or secreted into the culture medium from which it can be recovered and/or harvested.

Examples of polypeptides that can be produced by the methods of the present invention are proteins containing amino acid sequencing identical or very similar 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), Science277(5322): 5560), ligand for receptor activator of transcription factor kappa B (NRAF (nuclear factor receptor activator ligand), WO 01/36637), apoptosis-inducing ligand associated with tumor necrosis factor (TNF) (TNF-apoptosis-inducing ligand, WO 97/01633), thymic stroma-derived lymphopoietin, granulocyte colony-stimulating factor (G-CSF, Australian patent No. 588819 ), mast cell growth factor, stem cell growth factor (US patent No. 6204363), epidermal growth factor, keratinocyte growth factor, megakaryocyte growth and development factor, cytokine, human fibrinogen-like protein-2 (HFP-2; NCBI No. accession number NM_00682; Ruegg and Pytela (1995), Gene 160:257-62) growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, interferons, including α-interferon, γ-interferon and general interferons (US Pat. No. 4,695,623 and 4897471), neurogrowth factor, synaptotagmin-like proteins (SPB 1-5), nephrotrophin-3, glucagon, interleukins, colony-stimulating factor, fff lymphotoxin, leukemia inhibitory factor and oncostatin-M. Descriptions of proteins that can be produced according to the methods of the invention can be found, for example, in the books 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).

In addition, the methods of the present invention may be useful for the production of proteins containing all or part of the receptor amino acid sequencing for any of the above proteins, an antagonist of such a receptor or any of the above proteins and/or proteins substantially similar to such receptors or antagonists. These receptors and antagonists contain: both forms of the tumor necrosis factor receptor (TNF receptor, defined on p. 55 and p. 75 of US patent No. 5395760 and US patent No. 5610279), interleukin-1 (IL-1) receptors (types I and II; European Patent No. 0460846, US Patent 4968607 and US Patent 5767064) IL-1 receptor antagonist (US Patent No. 6337072), IL-1 antagonists or inhibitors (US Patent Nos. 5981713, 6096728 and 5075222), IL-2 receptors, IL-1 receptors 4 (European Patent No. 0367566 and US Patent No. 5856296), IL-15 receptors, IL-17 receptors, IL-18 receptors, Fc receptors, granulocyte-monocyte stimulating factor receptor, receptors for Oncostatin-M and leukemia inhibitory factors, receptor activator transcription factor B (RAFT, WO 01/36637 and US Pat. No. 6,271,349), osteoprotegerin (US Pat. No. 6,015,938), TNF-apoptosis-inducing ligand receptors (including TNF-apoptosis-inducing ligand receptors 1, 2, 3 and 4), and receptors containing death domains, such as the apoptosis-inducing receptor (AIR).

Other proteins that can be produced using this invention contain all or part of the amino acid sequencing of various antigens (called CD proteins) or their ligands or proteins similar to essentially any of them. These antigens are described in Leukocyte Typing VI (Proceedings of the VIth International Workshop and Conference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996). Similar CD proteins have been described in relevant seminars. Examples of such antigens include CD22, CD27, CD30, CD39, CD40 and their ligands (CD27 ligand, CD30 ligand, etc.). Some of the CD antigens belong to the TNF receptor family, which also contains 41BB and OX40. Ligan

- 10 045739 ys often belong to the TNF family, like ligands 41BB and OX40.

Proteins with active enzymes and their ligands can also be obtained using the present invention. Examples contain proteins containing part or all of the protein or its ligand, or a protein similar to one of the following: members of the disintegrin and metalloprotease family region, including alpha-TNF converting enzyme, various kinases, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, factor VIII , factor IX, apilopoprotein E, apilopoprotein A-I, globins, IL-2 antagonist, alpha-1 antitrypsin, ligands of any of the above enzymes and various other enzymes and their ligands.

The term antibody contains a reference to both glycated and non-glycated immunoglobulins of any isotype or subclass, or a reference to an antigen-binding region complementing a healthy antibody for a specific connection, unless otherwise noted, with a human, humanized, chimeric, polyspecific, monoclonal, polyclonal protein and oligomers or antigens that bind their parts. Also contains proteins with an antigen-binding part, or such as Fab, Fab', F(ab') ₂ , Fv, diabodies, Fd, dAb, maxibodies, individual chains of antibody molecules, fragments of hypervariable regions (HVR), scFv, deates, tribodies, tetrabodies and polypeptides containing at least a portion of the immunoglobulin sufficient to bind the specific antigen to the target polypeptide. The term antibody includes, but is not limited to, antibodies prepared, isolated, generated or isolated by recombinant means, such as antibodies isolated from a host cell transfected to produce the antibody.

Examples of antibodies include, but are not limited to, those that recognize any one or more proteins containing, but not limited to, the above proteins and/or the following antigens: CP2, CP3, CP4, CP8, CP11a, CP14, CP18, CP20, CP22, CP23, SR25, SR33, SR40, SR44, SR52, SR80 (V7.1), SR86 (V7.2), SR147, IL-1a, IL-1b, IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-2 receptors, IL-4 receptor, IL-6 receptor, IL-13 receptor, IL-18 receptor subelements, FGL2, PDGF-β and their analogs (see patents US No. 5272064 and 5149792), VEGF receptors, TGF, TGF-β2, TGFβ 1, EGF (see US Patent No. 6235883), VEGF receptor, hepatocyte growth factor, osteoprotegerin ligands, interferon-gamma, B-lymphocyte stimulator (SV-L , also known as BAFF, THANK, TALL-1 and ζΤΦΡ4; see Do and Chen-Kiang (2002), Cytokine Growth Factor Rev. 13(1): 19-25), C5 complement, IgE, tumor antigen CA125, tumor antigen MUC1, PEMantigen, LCG (which is a gene product isolated along with lung cancer), HER-2, HER-3, tumor associated glycoprotein, TAG-72, SK-1 antigen, tumor associated antigenic determinants present at elevated levels in the serum of patients with colon and/or pancreatic cancer, cancer-associated antigenic determinants or proteins isolated from breast, colon, prostate, pancreatic, lung, squamous and/or kidney cancer cells and/or cells melanomas, gliomas, neuroblastomas, necrotic tumor center, integrin alpha 4 beta 7, integrin VLA-4, integrins B2, TNF apoptosis-inducing ligand receptors 1, 2, 3 and 4, RAFT, RAFT ligand, TGF-a, adhesion molecule VAP -1, epithelial cell adhesion molecule (AMEC), intermolecular adhesion molecule-3 (MAM-3), leucointegrin adhesin, platelet glycoprotein gr. IIb/IIIa, cardiac myosin heavy chain, parathyroid hormone, rNAPc2 (which is an inhibitor of tumor factor VIIa), MHC I, oncoembryonic antigen (OEA), alpha-fetoprotein (AFP), tumor necrosis factor (TNF), CTLA-4 ( which is a cytotoxic T-lymphocyte-associated antigen), Fc-yyy-1 receptor, HLA-DR 10 beta, HLA-DR antigen, sclerotin, L-celestine, respiratory syncytial virus, human immunodeficiency virus (HIV), hepatitis B virus (HBV) -B), Streptococcus mutans and Staphlycoccus aureus. Specific examples of known antibodies can be produced using the methods of the present invention, including, but not limited to, adalimumab, bevacizumab, infliximab, absiximab, alemtuzumab, bapineizumab, basiliximab, belimumab, briakiumab, canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, labetuzumab, matuzumab, matumumab, mepolizumab, motavizumab, muromonab-SRZ, natalizumab, nimotuzumab, ofatumumab, omalizumab, oregovomab, palivizumab, panitimumab, pemtumomab, pertuzumab, ranibizumab, roveliz umab, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizomab , zalutumumab and zanolimumab.

The present invention can also be used to produce recombinant fusion proteins containing, for example, any of the above proteins. For example, recombinant fusion proteins containing one of the above proteins plus a multimerization domain such as a leucine zipper, double helix, Fc portion of an immunoglobulin or similar protein can be produced using the methods of the present invention. For example, see International Application Publication 94/10308; Lovejoy et al. (1993), Science 259:1288-1293; Harbury et al. (1993), Science 262:140105; Harbury et al. (1994), Nature 371:80-83; Hakansson et al. (1999), Structure 7:255-64. Among these recombinant fusion proteins are proteins in which a portion of the receptor is coupled to the Fc portion of an antibody, such as etanercept (page 75 TNFR:Fc) and belatacept (CTLA4:Fc).

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Because the terminology used in this application is standard in the art, definitions of certain terms are provided herein to provide clarity and certainty to the meaning of the application. Units, prefixes and symbols can be designated in the SI system. The number ranges specified here contain the numbers that define the range and also contain and support each integer in the specified range. The methods and techniques described herein are generally performed in accordance with conventional methods well known in the art and as described in the various general and more specific references that are cited and discussed herein unless otherwise indicated. See, for example, Sambrook et al. Sabrook, 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. (1990). All documents, or portions of documents, set forth in this application are incorporated herein by reference and include, but are not limited to, patents, patent applications, books, textbooks. What is described in the embodiment of the invention can be combined with other embodiments of the present invention.

The present invention should not be limited in scope by the specific embodiments described herein, which are provided to illustrate particular cases of the invention; functionally equivalent methods and components are applicable within the scope of the present invention. Indeed, various modifications of the present invention other than those described herein will become apparent to those skilled in the art from the preceding description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Examples

Example 1.

This experiment compares different initial conditions of semi-continuous feed and fed feed methods before perfusion using variable tangential flow filtration. Perfusion began either early during the initial exponential growth stage (semi-continuous start) without additional perfusion feeds, or at the end of the exponential stage, and enters the stationary stage, or into the production stage (fed start) with a bead feed of serum-free medium of a certain feed before perfusion.

On day 0, recombinant antibody-secreting HCC cells were introduced into 2 L production bioreactors at 1 x 10 ⁶ viable cells/ml in a working volume of 1500 ml serum-free defined medium for the fed start and 1800 ml for the semi-continuous start. Cultures are kept at 36°C, dissolved oxygen (DO) at 30%, agitation at 215 rpm. Glucose is maintained above 0 g/l and below 8 g/l.

Perfusion began on the fourth day (0.25 volume/day) for semi-continuous cultures and on the seventh day (0.75 volume/day) for fed cultures. Perfusion was performed using a variable tangential flow and filtration system (Refine Technologies, Hanover, NJ, 50 kilodalton hollow fiber filter). Before perfusion begins, the fed cultures receive a pellet feed of concentrated serum-free medium on day 4 (7.5% of the initial working volume) and day 6 (10% of the initial working volume). Perfusion rates are given in table. 1.

Table 1. Perfusion rate

Day Perfusion rate (volume/day) 0-4* 0.00 4-6 0.25 6-7 0.50 7-10 0.75 10- 1.00

Values are based on the working volumes described here *Day 0-7 for fed start

During culture growth, samples were taken daily to evaluate the culture.

Viable cell density (VCD) and viability were determined using Vi-Cell (Beckman Coulter, Brea, CA). The titer was measured using high performance liquid chromatography analysis. The volume of compacted cells was determined using VoluPAC (Sartorius, Goettingen, Germany).

A temperature change (36.0 to 33.0°C) was applied when the viable cell density exceeded 20 x 10 ⁶ viable cells/ml, which occurred on days 7 and 11 for the semi-continuous and fed start, respectively.

For semi-continuous onset, viable cell density continued to increase after onset

- 12 045739 perfusion; for fed start, perfusion began after the cell culture had reached a steady-state or stationary stage with little growth. At day 15, the viable cell density was between 27.7 and 30.7 x 106 viable cells/mL, while the semi-continuous culture VFA was between 22.5 and 27.4 x 106 viable cells/mL (Fig. 1A). The viability of the fed culture was between 73.9 and 77.5%, while the viability of the semi-continuous culture was between 72.5 and 83.1% (Fig. 1B). The fed-batch culture titer was between 15.3 and 16.1 g/L, while the semi-continuous culture titer was between 10.6 and 12.3 g/L (Fig. 1C). Since integrated variable cell density (IVC) values were similar for all four cultures at day 15 (approximately 230x106 cells/ml), specific productivity was greater under fed-start conditions. Fed culture was continued until 24 days. Within 20 days, a titer of 20 g/l was obtained.

Variable tangential perfusion flow resulted in increased production with fed start, maintaining cells in a more productive state compared to semi-continuous start.

Example 2.

On day 0, HCC cells secreting recombinant antibody were introduced into 2 L production bioreactors at 1 x 10 ⁶ viable cells/ml in a working volume of 1500 ml serum-free defined medium for the fed start and 1300 ml for the semi-continuous start. Cultures are at a temperature of 36°C, DO at 30%, stirring at 215 rpm. The fed culture was rotated at 430 rpm. The fed-batch culture was supplied with 7 g/L glucose daily before perfusion; All cultures maintained glucose levels at 4 g/L during perfusion. Perfusion (variable tangential flow) began on the fourth day (0.25 volume/day) for semi-continuous cultures and on the eighth day (0.75 volume/day) for fed culture. Before perfusion begins, the fed culture receives a pellet feed of concentrated serum-free defined medium on the fourth day (7.5% of the initial working volume) and the sixth day (10% 30 of the initial working volume). The perfusion rate is given in table. 2. Cultures were maintained for 21 days.

Table 2. Perfusion rate

Day Perfusion rate (volume/day) 4-6 0.25 6-8 0.50 8-10 0.75 10 - 1.00

Values are based on the displacements described here

During culture growth, samples were taken daily to evaluate the culture. Temperature variation (36.0 to 33.0°C) was used with semi-continuous cultures on the sixth day when the viable cell density exceeded 20x10 ⁶ viable cells/ml as in Example 1. The fed culture was maintained at 36.0°C during cultivation time.

For the semi-continuous start, the viable cell density was similar to the case described above, reaching approximately 20 to ^25x106 viable cells/ml, with no growth after day 10. The fed culture reached almost ^30x106 viable cells/ml on day 20 after most of the cultures containing less than 20x10 ⁶ viable cells/ml, see FIG. 2A. Viability remained above 80% until day 10 and then dropped to approximately 40% by day 20 for semi-continuous culture and 60% for fed culture; see fig. 2B. The highest titer value was almost 15 g/l for semi-continuous cultures, see Fig. 2C. It was observed that the start of a semi-continuous culture with about 3-4 millimoles of L-asparagine occurred on the third day and was not affected by the asparagine-limiting environment. However, the fed start perfusion culture was starved of L-asparagine on day 6 before perfusion started on day 7. The culture was then perfused with medium containing 2.0 g/L (or 13.3 millimol) L-asparagine, which did not result in further L-asparagine limitation after day 8 (Fig. 2D). Glucose concentrations were generally maintained between 4 and 10 g/L.

A fed start with strong rotation of the culture gave the maximum titer (more than 20 g/L) in 20 days, more than 5 g/L more than a semi-continuous start, which was similar to the results described above. No negative effects of faster rotation speed were observed. Maintaining a constant temperature did not lead to a negative impact on the fed crop.

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Example 3.

This experiment characterizes the effects of perfusion volume and temperature changes on variable tangential flow with a fed start as described above. All perfused cultures were started with a fed start on day 7. Perfusion rates from three-quarters volume to full volume or from full working volume to three-quarters working volume were tested. The temperature was changed from 36 to 33°C on day 14.

On day 0, ICC cells secreting the recombinant antibody were introduced into 2-liter production bioreactors at a rate of 1 x 10 ⁶ viable cells/ml in a working volume of 1200 ml of serum-free defined medium. The cultures were kept at a temperature of 36°C, DO at 30%. Before perfusion, 7 g/L glucose was supplied; All cultures were maintained at 1 g/L glucose during perfusion. The culture was maintained for 20 days.

Cultures received a pellet feed of concentrated serum-free medium on the fourth day (7.5% of the initial working volume) and the sixth day (10% of the initial working volume). Perfusion began on day 8. Perfusion rates are given in table. 3. One culture from each group had a temperature change from 36 to 33°C on day 15, the other cultures remained at 36°C during the experiment

Table 3. Perfusion rate

Condition Day Perfusion rate (volume/day) Condition 1 8-12 0.75 (n=2) 12- 1.00 Condition 2 8-10 1.00 (n=2) 10- 0.75

Values are based on the displacements described here

Changes in temperature and perfusion rate did not affect the density of viable cells, see Fig. 3A. However, changing the temperature helps keep cells alive longer in culture. There is a gap between the temperature change conditions starting from day 15. The viability of temperature-changed crops declines more slowly than that of 36°C crops, see FIG. 3B. In terms of titer, three cultures showed very similar titers on day 15 (17.1-17.9 g/L), and on day 20 (22-24 g/L), but one culture had a higher titer on day 15 (21 .58 g/L) and on day 20 (28.33 g/L) (see Fig. 3C). Neither temperature nor perfusion rate affected titer production, suggesting that cultures can be maintained at different perfusion rates.

Example 4.

This experiment examines the effect of perfusion medium with asparagine concentration and perfusion initiation conditions with either L-asparagine limitation or no culture medium limitation on viable cell density during the production stage.

On day 0, HCC cells secreting recombinant antibody were introduced into 2 L production bioreactors at 1 x 10 ⁶ viable cells/ml in a working volume of 1500 ml for semi-continuous and fed-batch methods. The cultures were kept at a temperature of 36°C, DO at 30%, rotation speed 400 rpm. Diffusion occurred either through a perforated tube or through sintered sprinklers. Glucose levels were maintained above 0 g/L and below 8 g/L.

Perfusion (variable tangential flow) began on day three (0.29 volume/day) for semi-continuous asparagine-limited cultures and on day seven (0.48 volume/day) for asparagine-limited fed culture. The semi-continuous culture medium contained 10 millimoles L-asparagine. Before perfusion begins, the fed culture receives a pellet feed of concentrated serum-free defined medium on days three and six (7% of the initial working volume) containing 113.4 millimoles of L-asparagine. The asparagine content of the treatment medium is either a controlled level (17.3 millimoles of asparagine in a defined serum-free treatment medium) or a low level (5 millimols of asparagine in a defined serum-free treatment medium). Perfusion was performed as described above. Perfusion rates are given in table. 4.

- 14 045739

Table 4. Perfusion rate

Condition Day Perfusion rate (volume/day) Day 3 3-4 0.29 Start 4-7 0.48 perfusion culture with 7-9 0.48 unlimited 9-11 0.67 asparagine content 11-20 0.96 Day 7 7-9 0.48 Start 9-11 0.67 fed culture with limited 11-20 0.96 asparagine content

During culture growth, samples were taken daily to evaluate the culture. Viable cell density (VCD) and viability were determined using Vi-Cell (Beckman Coulter, Brea, CA). The titer was measured using high performance liquid chromatography analysis. All cultures had a temperature of 36.0°C.

Reduced cell growth and increased productivity were achieved during the production stage by limiting asparagine in the culture medium. At day 15, the maximum viable cell density was approximately 17.0 x 106 viable cells/ml for low-asparagine fed cultures (Fig. 4A). The level of asparagine in the culture reached a viable cell density of more than 40x106 viable cells/ml (>30% of the volume of compacted cells). The viability of the fed culture with low content of the controlled parameter was 67.1%, and the semi-continuous culture was 55.1%, the content of asparagine of the controlled parameter was 69% (Fig. 4B). The compacted cell volume had a low asparagine fed culture titer of 17.0 g/L (for the compacted cell volume), while the fed culture titer was about 15.4 g/L (Figure 4C). Culture titers ranged from 10.2 to 12.9 g/L (semi-continuous start) and 14.2 to 15.9 g/L (fed start).

Maintaining asparagine levels of 5 millimoles or less during production resulted in limited growth, increased productivity, and maintained viability during the production stage.

Example 5.

This experiment compares environmental conditions during perfusion. In this 2 L bioreactor experiment, cells were introduced into a chemically defined semi-continuous medium with a working volume of 1.5 L, cultured for 3 days, and then sprayed for 12 days with a chemically defined treatment medium containing either 17.3 millimol L- asparagine and 4.6 millimols L-glutamine, or 5 millimols L-asparagine and 10 millimols L-glutamine. Perfusion was performed using a variable tangential flow perfusion and filtration system (Refine Technologies, Hanover, NJ) with a 30 kilodalton hollow fiber filter (GE Healthcare, Little Chalfont, UK). Perfusion began on the third day at a rate of 0.3 culture volumes per day. The perfusion rate increased on days 4, 9 and 11, as indicated in the table. 6 below.

Cultures were maintained at 36°C, 30% DO, pH 7.0, and 400 rpm.

During culture growth, samples were taken daily to evaluate the culture. Viable cell density (VCD) and viability were determined using Vi-Cell (Beckman Coulter, Brea, CA). The titer was measured using high performance liquid chromatography analysis. The volume of compacted cells was determined using VoluPAC (Sartorius, Goettingen, Germany).

Table 5. Perfusion rate

Day Perfusion rate (volume/day) 3 0.30 4 0.50 9 0.67 eleven 0.96

Restricting asparagine resulted in fewer cells accumulating and increased productivity. Cultures treated with medium containing 5 millimoles of asparagine achieved a maximum FFA of 8.16x107-8.54x107 cells/ml, while cultures treated with medium containing

- 15 045739

17.3 millimoles of asparagine reached 11.9x107-12.2x107 cells/ml (Fig. 5A). Although 17.3 millimols of asparagine contained more cells, cultures in 5 millimols of asparagine produced more product. Cultures treated with medium containing 17.3 millimoles of asparagine produced 6.89-7.18 g/L (4.33-4.67 g/L compacted cell volume) compared to 7.59-8.15 g/L l (5.01-5.38 g/L compacted cell volume) for cultures treated with medium containing 5 millimoles of asparagine (Figs. 5B and 5D). The final compacted cell volume (TCV) of 5 millimoles of asparagine contained slightly less than 17.3 millimoles of asparagine (Fig. 5C), with no difference in culture viability (Fig. 5E).

Interestingly, in this example, the more than doubling of glutamine concentration under low-asparagine conditions (4.6 millimolar vs. 10 millimolar glutamine) is not due to the ability of low-asparagine media to limit crop growth.

Example 6.

This example compares the performance of a cell line isolated from a cloned HCC antibody in a process using asparagine restriction to control growth in laboratory and pilot scale conditions. The laboratory model used 2 L bioreactors and the pilot model used 500 L bioreactors. In the laboratory model, cells were introduced into a chemically defined semi-continuous medium with a volume of 1.5 L, and in the pilot model, the bioreactor volume was approximately 378 L. Cells were cultured for 3 days in semi-continuous medium and then treated for 12 days with a chemically defined treatment medium containing 5 millimol L-asparagine and 10 millimol L-glutamine. Perfusion was performed using a variable tangential flow perfusion and filtration system (Refine Technologies, Hanover, NJ) with a 30 kilodalton hollow fiber filter (GE Healthcare, Little Chalfont, UK). Perfusion began on day 3 at a rate of 0.3 culture volumes per day. The perfusion rate was increased on days 4, 9 and 11, as indicated in the table below. 7. Cultures were kept at 36°C, 30% DO and pH 6.9.

During culture growth, samples were taken daily to evaluate the culture. Viable cell density (VCD) and viability were determined in vitro using Vi-Cell (Beckman Coulter, Brea, CA) and in pilot conditions using CEDEX (Roche Applied Science, Indianapolis, IN). The titer was measured using high performance liquid chromatography analysis. The volume of compacted cells was determined using VoluPAC (Sartorius, Goettingen, Germany).

Table 6. Perfusion rate graph

Day Perfusion rate (volume/day) 3 0.30 4 0.50 9 0.67 eleven 0.96

Data from four laboratory cultures and two pilot cultures are presented. PLC curves are similar in both conditions, growth control was achieved (Fig. 6A) and total cell mass (volume of compacted cells) was maintained below 30% in both cultures (Fig. 6C). Although the PLC reached a steady state around days 10 or 11, the volume of compacted cells continued to increase until approximately days 13 or 14 ( Fig. 6C ). The performance of the two crops was also similar. A culture treated with medium containing 5 millimols of asparagine produced 14.2-15.7 g/L (10.7-11.4 g/L of compacted cell volume) in a laboratory setting with a 2-L reactor, compared to 15.0 -17.3 g/L (10.612.8 g/L packed cell volume) under 500 L pilot conditions (Figures 6B and 6D). Viability was slightly lower under pilot conditions (Fig. 6E).

Claims (39)

1. A method for limiting cell growth in a mammalian cell culture releasing a recombinant protein, which includes establishing a mammalian cell culture in a serum-free culture medium in a bioreactor; imposing cell growth restriction by treatment with serum-free medium containing 5 millimoles or less of L-asparagine; maintaining mammalian cells in a growth-limited state by treatment with serum-free medium containing 5 millimoles or less of L-asparagine. 2. A method for increasing the production of a recombinant protein in a mammalian cell culture that produces a recombinant protein, which comprises establishing the mammalian cell culture in a serum-free culture medium in a bioreactor; introduction of cell growth restriction by treatment with serum-free medium containing - 16 045739 L-asparagine 5 millimoles or less; maintaining mammalian cells in a growth-limited state by treatment with serum-free medium containing 5 millimoles or less of L-asparagine. 3. A method for limiting the growth of a mammalian cell culture releasing a recombinant protein to a packed cell volume (PVC) that is 35% or less, which comprises establishing the mammalian cell culture in a serum-free culture medium in a bioreactor; imposing cell growth restriction by treatment with serum-free medium containing 5 millimoles or less of L-asparagine; maintaining mammalian cells in a growth-limited state by treatment with serum-free medium containing 5 millimoles or less of L-asparagine. 4. A method for limiting the growth of a mammalian cell culture that produces a recombinant protein with a limiting cell density of >1x108 cells/ml or less, which includes establishing a mammalian cell culture in a serum-free culture medium in a bioreactor; introduction of cell growth restriction using L-asparagine deficiency; maintaining mammalian cells in a growth-limited state by treatment with serum-free medium containing 5 millimoles or less of L-asparagine. 5. The method according to any one of claims 1 to 4, characterized in that it includes treatment with serum-free medium containing 5 millimoles of L-asparagine or less on or before the third day of culture. 6. Method according to any one of claims 1 to 4, which is characterized in that it includes the introduction of cell growth restriction before the start of the production stage. 7. Method according to any one of claims 1 to 4, which is characterized in that it includes the introduction of cell growth restriction during the production stage. 8. Method according to any one of claims 1 to 4, characterized in that the restriction of cell growth is caused by a lack of L-asparagine. 9. The method according to any one of claims 1-4, further including a change in temperature, in which the temperature decreases from 35-38°C to 30-34°C. 10. The method according to any one of claims 1 to 4, further comprising a temperature change in which the temperature is reduced from 35-38°C to 30-34°C, and the temperature change occurs during the transition between the growth stage and the production stage, during production stage or before the production stage. 11. The method according to any one of claims 1 to 4, further comprising changing the temperature, in which the growth stage occurs at a temperature of from about 35 to about 38°C, and the production stage occurs at a temperature from about 30 to about 34°. 12. The method according to any one of claims 1-4, which differs in that it includes changing the temperature from 36 to 31°C. 13. The method according to any one of claims 1-4, which differs in that it includes changing the temperature from 36 to 33°C. 14. The method according to claim 9, which is characterized in that the temperature change occurs during the transition from the growth stage to the production stage. 15. The method according to claim 9, which is characterized in that the temperature change occurs during the production stage. 16. Method according to any one of claims 1 to 15, characterized in that the content of L-asparagine in the serum-free medium is 5 millimoles or less. 17. The method according to any one of claims 1 to 15, characterized in that the content of L-asparagine in the serum-free medium is 4.0 millimoles or less. 18. The method according to any one of claims 1 to 15, characterized in that the content of L-asparagine in the serum-free medium is 3 millimoles or less. 19. The method according to any one of claims 1 to 15, characterized in that the content of L-asparagine in the serum-free medium is 2.0 millimoles or less. 20. The method according to any one of claims 1 to 15, characterized in that the content of L-asparagine in the serum-free medium is 1.0 millimoles or less. 21. Method according to any one of claims 1 to 20, characterized in that the content of L-asparagine in the cell culture medium is monitored before and during L-asparagine deficiency. 22. Method according to any one of claims 1 to 21, characterized in that the volume of compacted cells during the production step is 30% or less. 23. The method according to claim 22, wherein the density of viable cells of the mammalian cell culture in the volume of compacted cells is 35% or less and ranges from 10x106 viable cells/ml to 80x106 viable cells/ml. 24. The method according to any one of claims 1-23, which is characterized in that the processing includes continuous - 17 045739 processing. 25. Method according to any one of claims 1 to 24, characterized in that the perfusion rate is constant. 26. Method according to any one of claims 1 to 25, characterized in that the treatment is carried out at a rate less than or equal to 1.0 working volumes per day. 27. Method according to any one of claims 1 to 25, characterized in that the treatment is carried out at a rate increasing during the production stage from 0.25 working volume per day to 1.0 working volume per day during cell culture. 28. The method according to any one of claims 1 to 27, characterized in that the mammalian cell culture is established by introducing into the reactor at least from 0.5 x 10 ⁶ to 3.0 x 10 ⁶ cells/ml in a serum-free culture medium. 29. The method according to any one of claims 1 to 27, characterized in that the mammalian cell culture is established by introducing into the reactor at least 0.5 x 10 ⁶ to 1.5 x 10 ⁶ cells/ml in a serum-free culture medium. 30. Method according to any one of claims 1 to 29, characterized in that the processing is carried out by a variable tangential flow. 31. Method according to any one of claims 1 to 30, characterized in that the bioreactor capacity is at least 500 liters. 32. The method according to any one of claims 1 to 30, characterized in that the bioreactor capacity is from 500 to 2000 liters. 33. The method according to any one of claims 1 to 30, characterized in that the bioreactor capacity is from 1000 to 2000 liters. 34. The method according to any one of claims 1 to 33, characterized in that the mammalian cells are Chinese hamster ovary (CHO) cells. 35. The method according to any one of claims 1 to 34, characterized in that the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody or a cytokine. 36. Method according to any one of claims 1 to 34, characterized in that the recombinant protein is a recombinant fusion protein. 37. The method according to any one of claims 1 to 36, which is characterized in that it includes the step of collecting the recombinant protein produced by the cell culture. 38. The method according to any one of claims 1 to 37, which is characterized in that the recombinant process produced by the cell culture is purified and formed into a pharmacologically acceptable composition. 39. The method according to claim 2, wherein the production of the recombinant protein in a mammalian cell culture increases compared to a culture in which the cells were not treated with growth-limiting L-asparagine.