Restricted glucose feed for animal cell culture

[0001] The present application claims priority over the application filed on May 15, 2003, in the United States with serial number 60 / 470,937, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention [0002] The invention relates to a method for improving the protein production efficiency of cultured animal cells. More specifically, the invention relates to a method for controlling the production of lactic acid by cultured animal cells (preferably mammalian cells) to lower its level in periodic cell culture with feeding. In some embodiments, the invention provides methods for maintaining low levels of lactate production by cultured cells by using glucose delivery systems that are not based on taking samples from the culture at regular intervals. In particular, the invention relates to the culturing of animal cells under conditions in which glucose is supplied to the farm in a restricted manner, e.g. at a rate, which is a function of the expected or modeled rate of glucose consumption by animal cells when they are exposed to a medium containing a high level of glucose. Because of the limited power supply mentioned above, production by the cultured lactate cells is kept in a controlled manner during the cultivation period at a low level. The effect of this is increased production of recombinant proteins by cultivated cells, e.g. to facilitate production on an industrial scale.

Background of the Invention Related to the Invention [0003] A large proportion of biotechnology products, commercially available or newly developed, are protein-based drugs. In addition, for the production of many forms of protein drugs (such as glycosylated proteins or monoclonal antibodies produced in hybridoma cells (Mabs)), in general, cell machinery of an animal cell is needed (as opposed to a bacterial cell). As a consequence, the need for production of these proteins in animal cell cultures is increasing.

[0004] However, when compared to bacterial cell cultures, animal cell cultures are characterized by lower rates and usually lower yields of production. It has been found that maintaining the concentration of glucose in the media for low cell culture (e.g. between 0.02 and 1.0 g / L (e.g. between 0.11 and 5.5 mM)) and culturing the cells in the production phase with osmolality about 400 to 600 mOsm increases the production of recombinant proteins in cell cultures, especially after initial culture at osmolality from about 280 to 330 mOsm (US Patent No. 5,856,179, each US patent cited in this document is incorporated herein by reference); and when the cultures in all phases are run at a selected glutamine concentration (preferably between about 0.2 and about 2 mM, US Patent No. 6,180,401).

[0005] Increasing the production of recombinant proteins may in some cases be the result of a reduction in lactate production that occurs when glucose concentrations in the medial media are maintained at low levels. Lactate is commonly known as a potent inhibitor of cell growth and protein production. Keeping glucose low in cell culture at low levels may result in low levels of lactate production. (Glacken et al. (1986) Biotechnol Bioeng. 28: 1376-89; Kurokawa et al. (1994) Biotechnol Bioeng. 44: 95-103, U.S. Patent No. 6,155,570). As a result, depending on other culture conditions, maintaining low levels of glucose in relation to cell concentration is a factor that contributes to low levels of lactate production,

[0006] The metabolism of cells exposed to low glucose concentration in the culture medium is so altered that both the glucose uptake rate and lactate generation is lower compared to cells cultured in the feed processes where the medium contained a high glucose concentration at the start of the process ( U.S. Patent No. 6 156 570). In addition, the duration of the batch process can be extended. As a result, both cell growth rate and protein production can be maintained for a longer period of time compared to a fed-batch control culture in which cells are cultured in media favoring high levels of lactate production (e.g., media containing high glucose levels in the initial phase of the culture) . [0007] One way to control lactate production in cell cultures and maintain this production at a low level is the constant continuous feeding of glucose in a fed-batch process. (Ljunggren and Haggström (1994) Biotechnol. Bioeng. 44: 808-18; Haggström et al. (1996) Annals NY Acad. Sci. 782: 40-52). Although said constant involuntary supply of glucose in a fed-batch process can help control lactate formation and keep production at low levels, maximum cell concentration, maximum growth rate and viability, and maximum protein production rate are not achieved under such conditions. This is because, usually, when the concentration of cells increases, this way of feeding glucose causes glucose hunger. is constant, continuous supply of glucose in a batch process with power supply. (Ljunggren and Haggström (1994) Biotechnol. Bioeng. 44: 808-18; Haggström et al. (1996) Annals NY Acad. Sci. 782: 40-52). Although said constant involuntary supply of glucose in a fed-batch process can help control lactate formation and keep production at low levels, maximum cell concentration, maximum growth rate and viability, and maximum protein production rate are not achieved under such conditions. This is because, usually, when the concentration of cells increases, this way of feeding glucose causes glucose hunger. is constant, continuous supply of glucose in a batch process with power supply. (Ljunggren and Haggström (1994) Biotechnol. Bioeng. 44: 808-18; Haggström et al. (1996) Annals NY Acad. Sci. 782: 40-52). Although said constant involuntary supply of glucose in a fed-batch process can help control lactate formation and keep production at low levels, maximum cell concentration, maximum growth rate and viability, and maximum protein production rate are not achieved under such conditions. This is because, usually, when the concentration of cells increases, this way of feeding glucose causes glucose hunger. Although said constant involuntary supply of glucose in a fed-batch process can help control lactate formation and keep production at low levels, maximum cell concentration, maximum growth rate and viability, and maximum protein production rate are not achieved under such conditions. This is because, usually, when the concentration of cells increases, this way of feeding glucose causes glucose hunger. Although said constant involuntary supply of glucose in a fed-batch process can help control lactate formation and keep production at low levels, maximum cell concentration, maximum growth rate and viability, and maximum protein production rate are not achieved under such conditions. This is because, usually, when the concentration of cells increases, this way of feeding glucose causes glucose hunger.

[0008] Another way of controlling lactate production in cell culture and maintaining its low level is to use glucose delivery systems based on testing its level at regular intervals. Culture samples are collected at regular intervals and after determination of glucose concentration (e.g., flow-through analysis as in Male et al. (1997) Biotechnol Bioeng. 55: 497-504, or in Siegwart et al. (1999) Biotechnol. Prog.15: 608-16, or by means of high performance liquid chromatography, as in Kurokawa et al. (1994) Biotechnol Bioeng. 44: 95-103), in order to keep the concentration of glucose in the media stable, low for culture her measured quantities are added.

[0009] Patent description WO 2004/048556 A1, which in accordance with Art. 54 (3) of the European Patent Application Convention for the present application, discloses a method of maintaining lactic acid production at low levels in a fed-batch culture supplying glucose in limited way.

[0010] Furthermore, when using feedback-based control methods based on sampling there is a significant risk of contamination with microorganisms. It is not surprising, therefore, that it was impossible to use these methods for the commercial production of recombinant proteins in cell cultures. Since the publication of the first article on the subject, feedback-based control methods based on sampling have found limited use only in scientific research. Glacken et al. (Glacken et al., Supra) in their publication report that glucose concentration in culture media was determined on an ongoing basis using an automatic analyzer in which glucose containing samples were mixed with o-toluidine, and glucose concentration determined colorimetrically at length 660 nm wave.

[0011] As can be seen from the above-mentioned indications, there is still a need for alternative methods of controlling and maintaining low levels of lactate production in breed media.

SUMMARY OF THE INVENTION [0012] The present invention provides a method for limited feeding of animal cell cultures with glucose in fed-batch processes. Due to the limited feed, production of lactate by cultured cells can be kept in a controlled manner at a low level without the need for constant feeding of glucose. In some embodiments, lactate production by cultured cells may be maintained at low levels in a controlled manner without the need to take samples from the culture at regular intervals to determine glucose concentration using a feedback control method. In particular, the present invention provides a long-awaited method of flexibly controlling lactate production by cultured cells and maintaining its low level in a controlled manner,

The present invention relates to a method of growing animal cells under conditions in which the cell culture is fed with glucose in a restricted manner (so-called limited supply), whereby the level of lactate produced by the cultured cells is low. This limited or slow power supply is achieved by constant or occasional feeding of cell culture to glucose at a rate that is lower (i.e., at a rate that is a function) than the expected or simulated rate of glucose consumption by animal cells subjected to a medium containing high levels thereof. The invention relates in particular to a method of increasing the production of recombinant protein in animal cell cultures by controlling and maintaining lactate production at low levels by limiting the glucose feeding.

[0014] Thus, some embodiments of the invention may use sample-based feedback control, other embodiments of the present invention do not require feedback-based control based on sampling. For example, estimates of the extent of the expected rate of glucose consumption by cultured animal cells can be enhanced by measuring the concentration of cells that in some embodiments are performed without sampling (e.g., photometrically). On the basis of cell concentration measurements, the rate of glucose delivery to cell cultures (in real time, if needed) can be calculated, so that glucose is administered to the cell cultures in a limited manner, i.e. at a rate of less than 100% of the expected or modeled rate of glucose consumption by animal cells in an analogous culture with similar culture conditions where the concentration of glucose is not limited, but such that its increase does not change the rate of glucose consumption by the cells. As shown in embodiments of the present invention, limited glucose feeding allows for controlled maintenance of lactic acid production by cultured cells at low levels.

[0015] In some embodiments of the invention, pH monitoring is included in methods for estimating lactate consumption and preventing glucose starvation in cell cultures. The pH monitoring uses the fact that in the absence of glucose, the cultured cells consume lactate. When cells consume lactate, the pH of the culture increases. Therefore, raising the pH signals the lack of glucose in cell culture (ie signals the state of glucose starvation of cells). Thus, in some embodiments of the invention, a feed strategy that provides a single dose of glucose feeding and / or increases the rate of limited glucose feeding following a pH increase may protect cells from glucose faming and prevent glucose demand. In some embodiments, the pH measurements are carried out without sampling (i.e.

[0016] In particular, the invention provides a method of culturing cells in a fed-batch process with controlled production of lactic acid at low levels, which comprises: mixing animal cells with a culture medium to initiate a cell culture; supplying glucose to a cell culture in a limited manner. A limited supply of glucose occurs when glucose is delivered at a rate that is a function of the expected rate of glucose consumption by animal cells when they are exposed to a medium containing high glucose. This function is a multiplication by a value expressed as a percentage less than 100%, including, but not limited to, percentages such as at least 33% or not more than 45% of the expected rate.

[0017] In some embodiments of the present invention, a cell concentration sensor in culture is used to monitor the concentration of cells, and a measurement based on this sensor is used to calculate the rate at which the cell culture will be restricted in a way by glucose feeding. In other embodiments, a pH sensor is used to monitor the pH of the cell culture, wherein in the event of a pH increase above the predicted value (e.g., about 7), glucose is added to the culture (e.g., a single feed rate and / or a new feed rate in a limited manner, which is greater than the previous rate of glucose replenishment). In some embodiments, the new speed may be 15% or not more than 50% greater than the speed immediately preceding it, assuming that such a new rate does not reach 100% of the expected rate of glucose consumption by cells exposed to high glucose levels). In other embodiments, for determining the rate of limited glucose feeding of cell cultures, a cell concentration sensor system may be used together with a system based on a pH sensor. Both the system based on the cell concentration sensor and the system based on the pH sensor and both systems at the same time can be used without "sampling" from the cell culture.

According to a further embodiment, the present invention provides a method of culturing cells with controlled maintenance of low levels of lactic acid production in a fed-batch process that comprises: (a) mixing animal cells with a culture medium to initiate a cell culture; (b) determining the rate of glucose consumption (i.e., modeled rates) for cells cultured in the first culture; (c) mixing the animal cells and the culture medium to initiate the second cell culture and (d) feeding the second cell culture with glucose in a manner limited by the rate which is a function of the determined glucose consumption rate of step (b) (i.e., the function of the previously modeled glucose consumption rate) . In related embodiments, this function is a multiplication by a value expressed as a percentage lower than 100%, such as less than 33% or not more than 45% of the determined rate of glucose consumption (i.e. modeled rate). In other related embodiments, the limited glucose feeding of the second cell culture is carried out without controlled sampling with the feedback of the second cell culture.

[0019] In some embodiments of the present invention, a cell concentration sensor is used to monitor the concentration of cells in a second cell culture, where measurement based on a cell concentration sensor is additionally used to calculate the rate of limited glucose feeding of the second cell culture. In other embodiments, the pH sensor is used to monitor the pH of the second cell culture, and in response to a rise in pH above a predetermined value (e.g., about 7), glucose is added to the second culture (e.g., a single feed and / or a restricted feed rate) a method that is greater than directly preceding the glucose replenishment rate In some embodiments, the new rate may be 15% or not more than 50% greater than the rate immediately preceding it. for determining the glucose feed rate of restricted cell cultures, a system based on a cell concentration sensor together with a pH sensor based system can be used. Both the system based on the cell concentration sensor and the system based on the pH sensor, and both systems simultaneously, can be used without the need to collect cell culture samples.

In the method of the present invention, the cells are adapted to grow under conditions in which glucose is experimentally added to cell cultures at a limited rate compared to the rate of glucose consumption under control culture conditions (e.g. increase in concentration will not affect the rate of glucose consumption by animal cells). In particular, cells from two exemplary restricted-fed cultures (differing in the rates at which glucose is administered in a restricted manner to culture) produced lactate at lower levels than control cultures. They also showed different rates of growth and production of recombinant protein. Low-gradient fed crops "Low-ramps") showed lower levels of lactate production than cultures fed in a "high-gradient" restricted manner. Limited gradient-fed cultivations as compared to the "high gradient" restricted cultures were characterized by higher growth rates and production of recombinant protein.

[0021] Other characteristics and advantages of the present invention will be apparent from the following description of its embodiments and claims.

BRIEF DESCRIPTION OF THE FIGURES [0022] Fig. 1. Control compared to a limited supply of glucose: a matching curve.

[0023] Fig. 2. Growing amount of glucose: control compared to the expected increase in power.

[0024] Fig. 3. Growing amount of glucose: control compared to modeled feeds.

[0025] Fig. 4. Cell concentration: control compared to the expected increase in feed.

[0026] Fig. 5 BMP-2 titer (normalized): control compared to the expected feed increase. [0027] Fig. 6 Glucose and lactate concentration: control compared to the expected increase in feed.

[0028] Fig. 7. Cell concentration: control compared to modeled feeds.

[0029] Fig. 8. BMP-2 titer (normalized): control compared to modeled feeds.

[0030] Fig. 9. Glucose and lactate concentration: control compared to modeled feeds. DETAILED DESCRIPTION OF THE INVENTION [0031] Definitions: The expression "animal cells" includes the meaning of an invertebrate cell, vertebrates with the exception of mammals (e.g., birds, amphibians and reptiles) and mammalian vertebrates. Examples include, but are not limited to, invertebrate cells include the following insect cells: Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly) and Bombyx mori (silkworm larva / silkworm moth). Preferably mammalian cells include newborn hamster kidney (BHK) cells, Chinese hamster ovary (CHO) cells, human renal derived line (293) cells, normal fetal diploid (FRhL-2) cells and mouse melanoma cells (e.g. SP2). / 0 and NS0).

The expression "basic inoculum medium" means a solution or substance containing nutrients, with the exception of glucose, in which a cell culture is initiated. The "basic feed medium" contains the same nutrients as the basic inoculation medium, but is a solution or substance that the cell cultures are fed after the culture is initiated.

[0033] A "batch culture" means a cell culture in which the cells are provided with a basic glucose-containing inoculation medium at the time of cultivation and in which the product, e.g. a recombinant protein, is obtained only at the time of cultivation. Similarly, in the case of "fed-batch culture", the product is only obtained at the time of cultivation. However, in a batch culture with feeding, the basic inoculation medium containing glucose is delivered to the cells at the time of initiation of the culture, and the basic feed medium containing glucose once or several times after cultivation, but before its completion.

[0034] "High glucose" means the concentration of glucose in animal cell culture, any increase of which will not affect the rate of glucose consumption by these cells.

[0035] "Glucose consumption rate" reflects glucose consumption by the cultured animal cells at a given time point. The glucose consumption rates can be represented graphically (as above as the fitting curve in Fig. 1) or by means of a mathematical function (as in the legend of Fig. 1).

[0036] The expressions "restricted glucose feeding" and / or "restricted glucose feeding" and / or "glucose is provided in a restricted manner" and / or similar expressions means providing to the culture a limited amount of glucose that supplied a limited amount, which is determined or calculated by means of the function and is less than 100% of the amount expected or determined as the amount consumed by the cell culture. A "control culture" means a culture of the same animal cells carried out under similar conditions (e.g., culturing the same cells in a similar basal inoculum medium and feed medium at the same temperature, starting from the same initial cell concentration, etc.) except that that the glucose level in this culture is high. So the function by which a limited amount of glucose can be determined or calculated, may be a function of the expected rate of glucose consumption, or a function of the determined rate of glucose consumption in the control culture. The limited glucose feeding may take place in such a way that the glucose is delivered at a predetermined concentration or concentration over a period of time, i.e. at a particular rate or at certain rates and / or in such a way that the glucose is delivered in one or more supply portions.

[0037] The expression "function of the expected rate of glucose consumption" or "function of the designated rateand consumption "(where the determined wear rate is modeled speed) may include many mathematical relationships between the expected or modeled rate of glucose consumption and the rate of limited glucose feeding (or limited glucose addition rate), including the relationships in which the glucose consumption rate is the result (e.g. the result of the multiplication) (1) of the determined or previously modeled rate of glucose consumption at any point in time during the cell culture and (2) a percentage of less than 100%. The invention also includes many other mathematical dependencies, such as square, cubic and exponential functions. However, the functions that can be used within the scope of the invention do not include those

[0038] "Lower lactic acid" (or "lower lactate level") in cell culture means the concentration of lactic acid (or lactate) that is lower than the concentration of lactic acid (or lactate) in cell cultures characterized by high glucose levels.

[0039] "Sampling" involves taking samples containing cells from an animal cell culture (e.g., in a bioreactor) to measure the parameters of the culture medium. "Sampling" does not include cell concentration measurements taken from a culture for measuring samples that do not contain cells. For example, an approximation based on photometric measurements of cell concentration may be carried out by omitting "sampling" from a culture located in a transparent or translucent container. Furthermore, "sampling" does not include the use of a pH sensor in situ to measure the pH of the medium in which animal cells are grown, and from which samples are taken that do not contain cells for pH measurement.

[0040] According to a fixed convention as used in this patent application with reservations, the terms in English "a" and "an" mean "one or more". Although the invention has been described to a certain extent in a detailed manner, it is known in the light of the disclosure to a person skilled in the art that certain alternatives, modifications and alterations will be evident. Accordingly, it is assumed that all these alternatives, modifications and changes that fall within the scope of the invention and are in accordance with its spirit are covered by the formulated claims.

The present invention relates to a method of carrying out animal cell cultures characterized in that said cultures are maintained at a low level of lactic acid (which prolongs cell viability) and the production of the recombinant protein takes place at a high level of yield. One skilled in the art will recognize that the method disclosed herein can be used to breed many well-known animal cells routinely used and cultured in the art, i.e. the method disclosed herein is not limited to being used only with the cells mentioned in this document in the definition animal cells.

[0042] The method of the invention relates to a limited glucose feeding of cell cultures. As further detailed in Example 2, glucose feeding may be limited in a glucose delivery rate that is a function of the expected or determined rate of glucose consumption (e.g., modeled glucose consumption rate) by the animal cells in the control culture, i.e. high glucose concentration. Glucose feeding in a restricted manner may also include administering glucose in one or more portions.

Control control conditions can be determined by one skilled in the art without unnecessary experimentation. For example, one skilled in the art understands that animal cells are typically grown in a "medium" that is generally a solution containing nutrients, including glucose. As a person skilled in the art, he will know that glucose should be added to the basic inoculum medium and the primary delivery medium respectively prior to inoculation and the start of feeding animal cells. It will be understood that the amount of glucose added to the primary inoculum may differ from the amount of glucose added to the primary feed medium. Moreover, the person skilled in the art will know which medium is suitable for growing the given cells (e.g., CHO cells), 200 or 280 g / l) is usually reduced when added to the cell culture by dilution. The person skilled in the art will also know that the optimal concentration of other nutrients (e.g., glutamine, iron, trace elements D) or factors predicted to control other culture variables (e.g., foam formation and osmolality) will vary depending on from a given animal cell. Adjusting the concentration of such nutrients or factors in the basic inoculatory or feed media is a routine activity in a given field. In addition, the person skilled in the art will know at what temperature and at what concentration to cultivate a given cell type. that the optimal concentration of other nutrients (eg glutamine, iron, trace elements D) or factors predicted to control other culture variables (eg degree of foam formation and osmolality) will vary depending on the animal cell in question. Adjusting the concentration of such nutrients or factors in the basic inoculatory or feed media is a routine activity in a given field. In addition, the person skilled in the art will know at what temperature and at what concentration to cultivate a given cell type. that the optimal concentration of other nutrients (eg glutamine, iron, trace elements D) or factors predicted to control other culture variables (eg degree of foam formation and osmolality) will vary depending on the animal cell in question. Adjusting the concentration of such nutrients or factors in the basic inoculatory or feed media is a routine activity in a given field. In addition, the person skilled in the art will know at what temperature and at what concentration to cultivate a given cell type. Adjusting the concentration of such nutrients or factors in the basic inoculatory or feed media is a routine activity in a given field. In addition, the person skilled in the art will know at what temperature and at what concentration to cultivate a given cell type. Adjusting the concentration of such nutrients or factors in the basic inoculatory or feed media is a routine activity in a given field. In addition, the person skilled in the art will know at what temperature and at what concentration to cultivate a given cell type.

[0045] In some embodiments of the present invention, the cell concentration and / or pH of the culture is monitored and used in calculating the rate of restricted feeding of glucose. Methods for measuring cell concentration and / or culture pH are well known in the art. Such methods include, but are not limited to cell concentration measurements using a Cedex measuring instrument (Innovatis GmbH, Bielefeld, Germany) and / or CASY (Scharfe GmbH system, Reutlingen, Germany) and / or for measurements using a pH sensor. Particularly useful for measuring pH in the claimed invention are methods for determining cell concentration and pH that do not require sampling, i.e. sampling cells containing cells from animal cell cultures, including but not limited to the method using a capacitive probe,

[0046] In some embodiments of the present invention, data obtained by measuring cell concentration and / or pH measurement indicate that the glucose feeding should be continued in a restricted manner at a new glucose feed rate greater than the directly earlier chosen rate. The person skilled in the art will understand that the new rate at which glucose is administered in a restricted manner should still be lower than 100% of the expected or determined rate of glucose consumption. Thus, when the immediately preceding rate is e.g. 99% of the expected or determined rate of glucose consumption, the new rate should not increase by more than 1% of the speed immediately before it. In other embodiments, the new speed increases by 1-15% of the speed used immediately before it. In some embodiments of the present invention, the new rate increases by at least 15% of the speed used immediately before it. In other embodiments of the present invention, the new rate increases by no more than 50% of the speed immediately before it.

EXAMPLES

EXAMPLE 1

Media

Example 1.1: Inoculation medium [0047] The basic inoculum medium contains the same components as the DMEM / F12 medium, as well as additional components: 200 mg / l dextran sulfate (US Patent 5 318 898 describes the use of dextran sulfate in the medium), 10 mg / l Nucellin (an analogue of human insulin obtained on the basis of recombinant DNA, Eli Lilly (Indianapolis, IN)) and 2.4 g / l of polyvinyl alcohol (PVA). The basic inoculation medium prepared for the needs of these experiments did not contain glucose. To prepare a control inoculation medium prior to inoculation, about 10 g / L glucose was added to the basic inoculation medium. To prepare the inoculum medium used in cultures with restricted glucose supply, 0.8 g / l glucose and 1.3 g / l NaCl were added to the basic inoculum medium. NaCl was added for

Example 1.2. Feed Media [0048] The basic feed medium contains the same ingredients as the basic inoculation medium based on the DMEM / F12 medium recipe. The basic feed medium prepared for these experiments did not contain glucose. To create a control feed medium, about 44 g / L glucose was added to the basic feed medium.

EXAMPLE 2

Determining the rate of glucose addition [0049] One way to determine the rate of glucose addition to a restricted feed involves testing the rate of glucose consumption by CHO cells during a regular fed batch control culture. The glucose concentration in a typical control culture starts with high values ​​(i.e., about 10 g / l), and then, during normal growth in the exponential phase, it gradually decreases. Glucose was supplemented after 3 days. Supplementation of glucose in the above control cultures is necessary to prevent it from being completely depleted (see glucose concentration profile for the control culture Fig. 9).

[0050] Glucose concentrations in control cultures were determined using sampling-based methods in which samples for glucose determination were taken at various time points after inoculation. Daily samples were analyzed on the Bioprofile 100 Analyzer (Nova Biomedical Corp., Waltham, MA), which measures glucose, lactate, glutamine, glutamic acid and ammonium. The glucose concentration was also determined using the Glucose HK kit (Sigma-Aldrich Co., St. Louis, MO; Cat. No. GAHK-20).

[0051] In the analysis based on sampling, the rate of glucose uptake during the exponential phase of growth in the control media is plotted as a function of time (elapsed hours and days). The exponential fit curve (i.e., y = ae) was generated using the same data (for the fit curve in Fig. 1, for example, a = 2.058 and b = 0.0064). For the purpose of preparing this curve, in order to obtain the amount of glucose needed in limited feeds at high and low gradients at any time point, the rate of glucose consumption (g / L / h) was extrapolated. For low-gradient fed farms to estimate the rate at which glucose was to be added, the values ​​in the control curve fit graph were multiplied by 33% (see the filled "low gradient" triangles in Fig. 1). Similarly, to estimate the rate at which glucose was to be added to a culture run with a limited high-gradient feed, the values ​​in the control control curve graph were multiplied by 45% (see the filled squares "high gradient" in Fig. 1). Multipliers of 33% and 45% were selected arbitrarily. All multipliers with a percentage less than 100% or functions of the dependence of the limited glucose feed rate on the expected or modeled glucose consumption rate in which the calculated rate of restricted glucose feeding is less than the expected or modeled glucose consumption rate fall within the scope of the invention (except that allowable functions do not include such relationships in which the glucose addition rate obtained is unchanged and constant throughout the duration of the cell culture). Similarly, to estimate the rate at which glucose was to be added to a culture run with a limited high-gradient feed, the values ​​in the control control curve graph were multiplied by 45% (see the filled squares "high gradient" in Fig. 1). Multipliers of 33% and 45% were selected arbitrarily. All multipliers with a percentage less than 100% or functions of the dependence of the limited glucose feed rate on the expected or modeled glucose consumption rate in which the calculated rate of restricted glucose feeding is less than the expected or modeled glucose consumption rate fall within the scope of the invention (except that allowable functions do not include such relationships in which the glucose addition rate obtained is unchanged and constant throughout the duration of the cell culture). Similarly, to estimate the rate at which glucose was to be added to a culture run with a limited high-gradient feed, the values ​​in the control control curve graph were multiplied by 45% (see the filled squares "high gradient" in Fig. 1). Multipliers of 33% and 45% were selected arbitrarily. All multipliers with a percentage less than 100% or functions of the dependence of the limited glucose feed rate on the expected or modeled glucose consumption rate in which the calculated rate of restricted glucose feeding is less than the expected or modeled glucose consumption rate fall within the scope of the invention (except that allowable functions do not include such relationships in which the glucose addition rate obtained is unchanged and constant throughout the duration of the cell culture). to estimate the rate at which glucose was to be added to a culture run with a limited high-gradient feed, the values ​​in the control control curve graph were multiplied by 45% (see the filled squares "high gradient" in Fig. 1). Multipliers of 33% and 45% were selected arbitrarily. All multipliers with a percentage less than 100% or functions of the dependence of the limited glucose feed rate on the expected or modeled glucose consumption rate in which the calculated rate of restricted glucose feeding is less than the expected or modeled glucose consumption rate fall within the scope of the invention (except that allowable functions do not include such relationships in which the glucose addition rate obtained is unchanged and constant throughout the duration of the cell culture). to estimate the rate at which glucose was to be added to a culture run with a limited high-gradient feed, the values ​​in the control control curve graph were multiplied by 45% (see the filled squares "high gradient" in Fig. 1). Multipliers of 33% and 45% were selected arbitrarily. All multipliers with a percentage less than 100% or functions of the dependence of the limited glucose feed rate on the expected or modeled glucose consumption rate in which the calculated rate of restricted glucose feeding is less than the expected or modeled glucose consumption rate fall within the scope of the invention (except that allowable functions do not include such relationships in which the glucose addition rate obtained is unchanged and constant throughout the duration of the cell culture). with which glucose was to be added to the culture carried out with a limited high-gradient feed, the values ​​in the control fit curve plot were multiplied by 45% (see filled squares "high gradient" in Fig. 1). Multipliers of 33% and 45% were selected arbitrarily. All multipliers with a percentage less than 100% or functions of the dependence of the limited glucose feed rate on the expected or modeled glucose consumption rate in which the calculated rate of restricted glucose feeding is less than the expected or modeled glucose consumption rate fall within the scope of the invention (except that allowable functions do not include such relationships in which the glucose addition rate obtained is unchanged and constant throughout the duration of the cell culture). with which glucose was to be added to the culture carried out with a limited high-gradient feed, the values ​​in the control fit curve plot were multiplied by 45% (see filled squares "high gradient" in Fig. 1). Multipliers of 33% and 45% were selected arbitrarily. All multipliers with a percentage less than 100% or functions of the dependence of the limited glucose feed rate on the expected or modeled glucose consumption rate in which the calculated rate of restricted glucose feeding is less than the expected or modeled glucose consumption rate fall within the scope of the invention (except that allowable functions do not include such relationships in which the glucose addition rate obtained is unchanged and constant throughout the duration of the cell culture).

[0052] Another approach to the problem of feeding glucose in a limited manner is the use of a pH-responsive system in programmed limited glucose feeding. When breeding mammalian cells (such as CHO cells) is devoid of glucose, cultured cells begin to consume lactate - an alternative source of carbohydrates and energy. The decrease in the lactate concentration in the cell culture increases the pH (to which the control system reacts and reacts to pH changes).

[0053] In practicing this approach, the syringe pump for glucose solution has been programmed to deliver glucose at a restricted rate (i.e., 0.032 g / L / hr, see initial rate of addition at low gradients Table 2). The exception is the situation when the pH of the cell culture grew by 0.02 pH units over the previously determined value of 7.00. It then began to provide a single portion of glucose feeding (0.05 to 0.2 g of glucose delivered from the feed medium per 1 liter of culture). In addition, the limited delivery rate using the syringe pump was then increased to a level of 15% to 50% higher than the earlier rate of limited delivery. For example, providing a single aliquot from 0.25 to 1.0 ml of a feed medium containing 0,

[0054] Another way of assisting the calculation of the limited glucose feeding rate is to measure cell concentration in the culture without sampling. In the initial tests according to this method, glucose was delivered to the culture in such a way that its concentration remained at a level considered to be sufficient to carry out a cell culture. A Wedgewood spectrophotometer (653 Absorbency Controller 653 i_Model BT65 Series Insertion Sensor, Wedgewood Technology Inc., San Carlos, CA) was used to determine cell concentration in real time. Alternatively, a laser probe measuring turbidity can be used to determine the cell concentration (eg Model LA-300LT, ASR Co., Ltd., Tokyo). The laser probe for turbidity measurements emits a radius that travels the light path through the cell culture. The calibration curve is used to convert the optical density value to the cell concentration value. Although cell light absorbance is not a fixed value and the cell size distribution changes during culture, Zhou and Hu ((1994) Biotechnol Bioeng. 44: 170-77) determined the total concentration of MAK hybrid mouse-line cells linearly correlated with with a laser signal of the probe measuring turbidity below 3.0x10⁹ cells / liter. To facilitate calculating the rate of limited glucose feeding, spectrophotometric measurements of cell concentration can be performed without sampling.

[0055] Another method combines a pH sensor based response system and a system based on cell concentration measurements using a power gradient program (set to approximate the expected glucose demand during cell culture).

[0056] The use of a glucose sensor probe for direct glucose measurement (and not indirectly by measuring cell pH or concentration) in real time (and in a manner that does not require the collection of culture samples) is not required for carrying out the invention. Mainly because the invention does not provide a method for keeping low glucose in the culture, but a way of avoiding adaptation of the cells to low glucose by limited rates of glucose administration. However, the use of a glucose sensor using the practical methods disclosed may be within the scope of the present invention.

EXAMPLE 3

Preparation of BMP-2 in CHO cells [0057] The purpose of these experiments was to implement a strategy of limited glucose feeding to control lactate production and maintain its low level in a fed-batch culture (more specifically in a one liter (1 L) culture) CHO cells (more specifically EMCG5 cells) for the production of bone morphogenetic protein (BMP-2) (U.S. Patent No. 5,318,898; U.S. Patent No. 5,618,244; U.S. Patent No. 5,631,142 provides more data on BMP-2 protein and its production). During the experiment, the effect of limited glucose feeding strategies on cell concentration and viability, lactate production, protein production and extended culture period was monitored.

[0058] The sterile glucose solution was limited to the bioreactor using a syringe pump programmed to increase the amount of glucose fed during the fed-batch culture. In one series of tests, glucose was added to the culture as a function of the relationship (e.g., as a percentage) from the previously determined rate of glucose consumption by animal cells when subjected to high glucose (e.g. as a function of the previously modeled rate).

[0059] Glucose starvation moments were monitored using a pH sensor (Bradley-James Corp.) whose use did not require sampling. Accordingly, raising the pH by 0.02 units over the pre-determined value of 7.00 was supposed to be a signal that the glucose resource in the culture medium was completely depleted and the cells started to consume lactic acid. Lowering the level of lactic acid in cultures increases the pH of the culture medium. This dependence allows us to predict the moments of glucose hunger using a system based on a pH sensor.

[0060] To evaluate the effectiveness of a pH sensor based system in preventing glucose starvation moments in glucose restricted experiments, the syringe pump was programmed to deliver a single portion of glucose feeding to the bioreactor if the pH rose above the previously determined value of 7.00. An increase in the pH of 0.02 units causes in some tests to give a portion of glucose feeding and to increase the limited continuous glucose administration by 15%, and in other tests by 50%.

[0061] In these experiments, Applikon® 2 lo bioreactors with an operating volume of 1 l (Applikon Biotechnology, Foster City, CA) were used, and aeration was aerated to maintain dissolved oxygen at 23% of the air saturation, if necessary. C for medical purposes ((Dow Coming Corporation, Midland, MI.) For the duration of the fed-batch culture, the temperature was maintained at 37 ° C. To supply glucose to control cultures or cultures fed in bioreactors in a restricted manner, Becton Dickinson® syringes (Becton, Dickinson and Company, Franklin Lakes, NJ) were filled with a control or experimental glucose solution, respectively.

[0062] In the first experiment (i.e. in the experiment with the expected feed increase), an increasing daily amount of glucose was used in a representative control culture, and an exponential fit curve was used to estimate the amount of glucose consumed each day by the control culture. Using the fitting curve as the basis, the syringe pump (Yale Apparatus, Wantagh, NY) was set up to supply glucose to the experimental culture at a limited feed rate, i.e., about 50-70% of the amount of glucose consumed by the control culture. Every day the rate of limited power was changed considering the increasing density of cells. The glucose feed concentration was 200 g / l.

[0063] The initial glucose concentration in the control culture (1 L) was 10.38 g / L. After a period of slightly longer than three days, glucose (2.2 g) was added at 24-hour intervals to the control culture (i.e., at 75.5, 99.5 and 123.5 hours of culture) (Table 1, Fig. 2 and Figures 6). The initial glucose concentration in the culture with restricted feeding (1L) was 1.1 g / L. The rate of constant limited feeding of glucose to the restricted feeding culture was increased four times (27.5, 51.5, 75.5 and 99.5 hrs of culture) starting from an initial constant rate of restricted feed of 0.046 g / l / h. (which was maintained during culturing from 20 to 27.5 hours) (Table 1).

[0064]

Table 1. Experiment of expected power increase: glucose replenishment

<td colspan="2">Supplementing glucose up to 11 control cultures (initial glucose concentration: 10.38 g / l)</td><td colspan="2">Speed ​​of addition to limited feeding cultures (initial glucose concentration: 1.1 g / l)</td>

<td>Time point (hours)</td><td>Quantity (g)</td><td>Time periods (hours)</td><td>Speed ​​(g / l / hour)</td>

<td>-</td><td>-</td><td>0-20</td><td>0</td>

<td>-</td><td>-</td><td>20-27.5</td><td>0.046</td>

<td>-</td><td>-</td><td>27.5-51.5</td><td>0.068</td>

<td>75.5</td><td>2.2</td><td>51.5-75.5</td><td>0.088</td>

<td>99.5</td><td>2.2</td><td>75.5-99.5</td><td>0.104</td>

<td>123.5</td><td>2.2</td><td>99.5-147.5</td><td>0.12</td>

[0065] The rate of glucose consumption in the batch fed-batch was similar to the limited glucose delivery rate over the entire duration of the continuous limited feed of glucose in a restricted feed. This is evidenced by the concentration of glucose in the culture with a limited supply, which remained close to zero when after 20 hours. the culture was subjected to continuous restricted feeding with glucose (Fig. 6). In turn, glucose consumption rates in control cultures were not limited by a limited rate of glucose delivery. As a result, the rate of glucose consumption in control cultures was maintained at higher levels for a few days than in fed-batch cultures (Table 6). Also lactate concentration values ​​(Fig.

[0066] In the second experiment (i.e. the modeled feed experiment) a gradient program of a syringe pump (KD Scientific, Holliston, MA) was used. In the model power supply experiments, the glucose concentration at high gradients was 0.28 g / ml, and in the low gradient - 0.2 g / ml. In contrast to the expected growth experiment, in which the syringe pump delivered glucose continuously over the entire experiment at a set rate of, e.g., 0.046 g / h. from 20 to hours 20.7 and 0.068 g / hour. from 27.5 to hours 51.5, etc. The gradient program of the syringe pump in modeled power experiments allowed for a gradual increase in the rate of limited glucose feeding to values ​​close to 33% or 45% of the exponential fit curve of the amount of glucose consumed in the control culture. Setting the gradient function allowed the initial and the final limited speed to be programmed for each time period, e.g. 12 hours. During this time period, the pump changed the speed continuously and linearly. Table 2 presents representative data on the addition of glucose for the modeled fertilizer experiment.

[0067]

Table 2. Modeled power supply experiment: glucose replenishment

<td colspan="2">Supplementation of glucose up to 11 control cultures (initial glucose concentration: 8.4 g / l)</td><td></td>

<td>Time point (hours)</td><td>Quantity (g)</td><td></td>

<td>-</td><td>-</td><td></td>

<td>-</td><td>-</td><td></td>

<td>-</td><td>-</td><td></td>

<td>67.5</td><td>2.2</td><td></td>

<td>94.75</td><td>2.2</td><td></td>

<td>119.75</td><td>2.2</td><td></td>

<td>143.75</td><td>2.2</td><td></td>

<td>167.75</td><td>2.2</td><td></td>

<td>191</td><td>2.2</td><td></td>

(continuation)

<td colspan="4">Arranged power supply</td>

<td colspan="2"></td><td colspan="2">Speeds of glucose replenishing fed-batch cultures with limited feeding (initial glucose concentration: 0.88 q / l) of a ml / qd syringe.</td>

<td>Time periods (hours)</td><td>Syringe speed ml / hour</td><td>Low gradient (power supply 0,2 g / ml) g / hour. added to 1 I</td><td>High gradient (power supply 0.28 g / ml) g / hour. added to 1 liter</td>

<td>0-19.75</td><td>0</td><td>0</td><td>0</td>

<td>^^. ^ 775-4 3.75</td><td>0.160-0.184</td><td>0.0320-0.0368</td><td>0.0448-0.0515</td>

<td>43.75 - 67.75</td><td>0.184-0.224</td><td>0.0368 -00448</td><td>0.0515- 0.0627</td>

<td>67.75 - 91.75</td><td>0.224-0.264</td><td>0.0448 -00528</td><td>0.0627- 0.0739</td>

<td>91.75-115.75</td><td>0.264-0,304</td><td>0.0528 - 0.0608</td><td>0.0739 -0.0851</td>

<td>115.75- 139.75</td><td>0.304-0.344</td><td>0.0608 -.06868</td><td>0.0851 - 0.0963</td>

<td>139.75-140.25</td><td>0.344 - 0.346</td><td>0.0688 -00692</td><td>0.0963 - 0.0969</td>

<td>140.25- 1 &amp; 4.25</td><td>0.400 -0.450</td><td>0.0800 -00900</td><td>0.1120- 0.1260</td>

<td>164.25-191</td><td>0450</td><td>.0900</td><td>.1260</td>

[0068] Fig. 2 shows the increasing amount of glucose delivered during the course of the expected power increase experiment, and Fig. 3 shows the increasing amount of glucose delivered during the experiments in which the power was modeled. Both Figures 2 and 3 take into account the initial amounts of glucose delivered to the bioreactors of control and restricted feeding (and not the amount of glucose provided by the syringe pump). For both experiments in the control trial, the initial glucose concentration was high, and the daily addition of glucose (2.2 g) began after about 72 hours. For restricted-feed cultures, the concentration of glucose in the bioreactor on day 0 was 1 g / l, and glucose delivery started after about 20 hours.

[0069] The experiment regarding the expected feed increase showed that the cell growth in the restricted feed was initially slower compared to the control culture. Nevertheless, on day 6, cell growth in restricted culture reached a higher final concentration than in control (Figure 4). In the control culture, the cell concentration reached its maximum much earlier and the viability began to decrease rapidly after Day 4 (Fig. 4). In contrast to the control culture, the rate of cell growth in cultures with restricted feed remained positive up to day 6 inclusive (Table 3). [0070] Although the restricted feeding culture compared to the control culture reached a higher final cell concentration by day 6, the day cultures had similar concentrations (Figure 5). These data show that the reduced cell viability in the control culture compared to the restricted feed culture was not dependent on the cells reaching the maximum capacity of the bioreactor. In contrast, the data presented in Fig. 4 in combination with the data of Fig. 6, which shows a low level of lactate in restricted culture compared to control cultures, suggests that the increase in cell viability in restricted feed cultures was a function of the low lactate level achieved in the cultures. result of feeding the glucose in a limited way.

[0071]

Table 3. Experiment of expected power increase: cell growth rate (Cedex μ / hr value, Cedex value = cell concentration in units of 10⁵/ Ml).

<td>Day</td><td>Growth rates in the control culture (μι x (hours ¹))</td><td>Growth rates in culture with limited supply (μ x (hours ~¹))</td>

<td>1</td><td>0.027</td><td>0.027</td>

<td>2</td><td>0.032</td><td>0.026</td>

<td>4</td><td>0.016</td><td>0.017</td>

<td>5</td><td>0.001</td><td>0.007</td>

<td>e</td><td>-0,014</td><td>0.005</td>

[0072] Fig. 5 presents graphs of values ​​of the normalized BMP-2 titre. The BMP-2 titer values ​​were normalized as part of the BMP-2 titer value on day 6 in the expected power increase experiment. Table 4 shows the values ​​corresponding to the production rates of BMP-2. The BMP-2 production rates are normalized as part of the BMP-2 production rate in the control culture on day 1. After day 4, BMP-2 titer levels for the control culture stabilized, while in the restricted feed cultures, the titers increased further (Fig. 5 ). In contrast to the control culture, the production rates of BMP-2 in culture with restricted feed remained positive up to day 6 (Table 4). It should be noted that a slight reduction in BMP-2 titers in control cultures, contrary to what is suggested in Fig. 5,

[0073]

Table 4. Experiment of expected power increase: BMP-2 production rate (normalized).

<td>Day</td><td>Production rates in control cultures</td><td>Production speed in culture with limited power supply</td>

<td>1</td><td>1.00</td><td>0.84</td>

<td>2</td><td>0.71</td><td>0.77</td>

<td>4</td><td>0.40</td><td>0.65</td>

<td>5</td><td>-0.07</td><td>0.21</td>

<td>6</td><td>0.08</td><td>0.40</td>

[0074] Maintaining a low level of lactate using a restricted glucose feeding strategy has intensified cell growth and protein production. In the restricted end-fed culture, BMP-2 was about 70% higher (Figure 5) and the rate of BMP-2 production did not reach a negative value, as was the case for the control culture (Table 4).

[0075] Fig. 6 shows the glucose (g / l) and lactate (g / l) profiles in the experiment of the expected power increase, and Table 5 presents the corresponding representative glucose (g / l) and lactate (g / l) data. ). Table 6 shows the values ​​for the corresponding glucose consumption rates, and Table 7 shows the values ​​corresponding to the lactate production rate.

[0076]

Table 5. Experiment of expected power increase: glucose and lactate concentration

<td></td><td colspan="2">Glucose concentration (g / l)</td>

<td>Hours</td><td>Control</td><td>Limited power supply</td>

<td>0</td><td>10.38</td><td>1.10</td>

<td>20.75</td><td>8.96</td><td>0.22</td>

<td>51.25</td><td>5.20</td><td>0.06</td>

<td>75.5</td><td>7.40</td><td>-</td>

<td>92.5</td><td>0.78</td><td>0.07</td>

<td>99.5</td><td>2.98</td><td>-</td>

<td>115.25</td><td>0.10</td><td>0.08</td>

<td>123.5</td><td>2.30</td><td>-</td>

<td>142.5</td><td>0.10</td><td>0.07</td>

<td>0</td><td>0.12</td><td>0.16</td>

<td>20.75</td><td>1.40</td><td>1.14</td>

<td>51.25</td><td>3.40</td><td>2.26</td>

<td>75.5</td><td>-</td><td>-</td>

<td>92.5</td><td>5.78</td><td>3.82</td>

(continuation)

<td></td><td colspan="2">Concentration of lactate (g / l)</td>

<td>Hours</td><td>Control</td><td>Limited power supply</td>

<td>99.5</td><td>-</td><td>-</td>

<td>115.25</td><td>6.50</td><td>4.20</td>

<td>123.5</td><td>-</td><td>-</td>

<td>142.5</td><td>6.96</td><td>4.60</td>

[0077]

Table 6. Experiment of expected power increase: rate of glucose consumption

<td>Day</td><td>Qgi control_ATgoat (mg / 10⁶ cells / day)</td><td>Power limited Qglucose (mg / 10⁶ cells / day) j</td>

<td>1</td><td>1.90</td><td>1.32</td>

<td>2</td><td>1.54</td><td>1.02</td>

<td>4</td><td>0.90</td><td>0.66</td>

<td>5</td><td>0.51</td><td>0.53</td>

<td>6</td><td>0.39</td><td>0.48</td>

[0078]

Table 7. Experiment of expected power increase: rate of lactate production

<td>Day</td><td>Q check_gand_ATgoat (mg / 10⁶ cells / day)</td><td>Power limited Qglucose (mg / 10⁶ cells / day)</td>

<td>1</td><td>1.71</td><td>1.41</td>

<td>2</td><td>0.82</td><td>0.55</td>

<td>4</td><td>0.32</td><td>0.26</td>

<td>5</td><td>0.13</td><td>0.08</td>

<td>6</td><td>0.08</td><td>0.06</td>

[0079] In the experiment of the expected feed increase, from day 1 to day 3, a lower rate of glucose consumption was observed in the fed-batch fed-batch culture than in the control culture (Table 6). During the periodic batch culture also lactate production rates were lower compared to the lactate production rate in the control culture (Table 7). This allowed a lower lactate concentration to be achieved during the periodic batch culture (Fig. 6).

[0080] The osmolality profiles and the amount of titrant (mixture of sodium carbonate and sodium bicarbonate) used daily for each bioreactor were also measured. Table 8 presents osmolality profiles, and Table 9 - the amount of titrant consumed per day (per 1l working volume) in both cultivation conditions.

[0081]

Table 8. The experiment of the expected power increase: osmolality

<td>Day</td><td>Osmolality in control culture (mOsm / l)</td><td>Osmolality in culture with limited glucose feeding (mOsm / l)</td>

<td>0</td><td>286</td><td>289</td>

<td>1</td><td>295</td><td>312</td>

<td>2</td><td>340</td><td>324</td>

<td>4</td><td>382</td><td>371</td>

<td>5</td><td>394</td><td>362</td>

<td>6</td><td>413</td><td>375</td>

[0082]

Table 9. Experiment of expected power increase: titrant consumption

<td>Day</td><td>Titrant consumption in control culture (ml / day)</td><td>Titrant consumption in culture with limited feeding (ml / day)</td>

<td>0-1</td><td>3</td><td>1</td>

<td>1-2</td><td>16</td><td>3</td>

<td>2-4</td><td>25</td><td>20</td>

<td>4-5</td><td>10</td><td>1</td>

<td>5-6</td><td>6</td><td>9</td>

[0083] An average lower osmolality level (Table 8) and lower titrant consumption (Table 9) in restricted feed cultures (compared to the control culture) is associated with a lower amount of lactate produced (requiring the use of less titrant for neutralization).

[0084] In experiments with modeled feeds, one standard fermenter was run in one bioreactor with power supply as a control. One bioreactor was intended for low-gradient culture of limited glucose supply, and the other for high-gradient culture of limited glucose supply. For each test bioreactor, one syringe pump was used, which continuously increased the speed of limited feeding with glucose. The concentration of the glucose solution fed to the low gradient bioreactor was 0.2 g / ml. The concentration of glucose solution fed to the high gradient bioreactor was 0.28 g / ml.

[0085] Fig. 7 graphically depicts the cell concentration (solid line) and their viability (dashed line) during the course of the culture in the control and test bioreactors. Table 10 presents cell growth rate values ​​for the control bioreactor and test bioreactors.

[0086]

Table 10. Modeled feeding experiments: cell growth rate (Cedex μ / hour value, Cedex value = cell concentration in 10 units)⁵ cells / ml).

<td>Day</td><td>Control speeds (ux (hr '))</td><td>Speeds in a low gradient (gx (hr ')¹))</td><td>High gradient speeds (gx (hr ')¹)))</td>

<td>1</td><td>0.030</td><td>0.026</td><td>0.024</td>

<td>2</td><td>0.035</td><td>0.029</td><td>0.030</td>

<td>3</td><td>0.029</td><td>0.026</td><td>0.025</td>

<td>4</td><td>0.009</td><td>0.016</td><td>0.017</td>

<td>5</td><td>0.005</td><td>0011</td><td>0.010</td>

<td>6</td><td>-0,004</td><td>0012</td><td>0.009</td>

<td>7</td><td>-0,013</td><td>0.006</td><td>0.005</td>

<td>8</td><td>-0,012</td><td>0.002</td><td>0.003</td>

[0087] In both cultures with restricted feed, the cell concentration was continuously increased until day 8 inclusive (192 h). Furthermore, the cell viability remained high throughout this period (Figure 7). In contrast to the test culture, in the control culture, the cell concentration reached a maximum of 5 days (120 h), and then drastically decreased, which was accompanied by an even stronger decrease in cell viability (Fig. 7). The cell concentration in the low graded culture exceeds day 8, 12x10^scells / ml, and cell viability remained above 90% (Figure 7).

[0088] BMP-2 titer levels observed in restricted feed cultures confirm the uselessness of the methods of the invention to increase protein production in animal cell cultures (particularly for low gradient cultures). Fig. 8 shows BMP-2 titer levels for control and test bioreactors normalized as part of the maximum BMP-2 titer (day 5) for the control culture. Table 11 shows the rate of BMP-2 production for control and test bioreactors normalized as part of the BMP-2 production rate in control culture on day 1 (also normalized in Table 4).

[0089]

Table 11 Modeled power experiments: BMP-2 production rate (normalized)

<td>Day</td><td>Control rate of production</td><td>Production speed at low gradients</td><td>Production speed with a high gradient</td>

<td>1</td><td>1.00</td><td>1.19</td><td>1.17</td>

<td>2</td><td>1.01</td><td>0.75</td><td>0.77</td>

<td>3</td><td>0.94</td><td>0.94</td><td>1.02</td>

<td>4</td><td>0.67</td><td>0.86</td><td>1.02</td>

<td>5</td><td>0.16</td><td>1.06</td><td>0.56</td>

<td>6</td><td>-0.19</td><td>1.15</td><td>0.59</td>

<td>7</td><td>-0.39</td><td>0.52</td><td>-0.02</td>

<td>8</td><td>-0.02</td><td>0.43</td><td>-0.03</td>

[0090] The highest final titer was achieved in a batch culture with low-gradient feed. The level of this titer is more than three times higher than the maximum BMP-2 titer achieved in the control culture (Figure 8). In low-gradient culture, the rate of BMP-2 production remained high for six days (Table 11). The rate of production of BMP-2 in batch culture with high gradient feed has dropped earlier than the rate of production of BMP-2 in batch culture with fed in a low gradient table (Table 11). This decrease is most likely caused by the presence of inhibitors, such as lactate, which in high-gradient cultures occur at a higher level than in low-gradient cultures. [0091] FIG. 9 presents concentration profiles (g / l) of glucose (solid lines) and lactate (dashed lines) in the modeled sludge experiment for control and test bioreactors, and Table 12 presents the corresponding representative data on glucose and lactate concentration (g / l) for this experiment . Table 13 shows the rate of glucose consumption, and Table 14 - lactate production rates for the control and test bioreactors.

[0092]

Table 12. Experiments of modeled power supply; glucose and lactate

<td></td><td colspan="3">Concentration of lactate (g / l)</td>

<td></td><td></td><td colspan="2">Limited power supply</td>

<td>Hours</td><td>Control</td><td>Low gradient</td><td>High gradient</td>

<td>0</td><td>10.46</td><td>1.09</td><td>1.09</td>

<td>18.75</td><td>8.68</td><td>0.01</td><td>0.00</td>

<td>42.75</td><td>6.38</td><td>0.12</td><td>0.08</td>

<td>66.25</td><td>3.07</td><td>0.05</td><td>0.05</td>

<td>67.75</td><td>5.82</td><td>-</td><td>-</td>

<td>92.25</td><td>1.61</td><td>0.16</td><td>0.11</td>

<td>94.75</td><td>4.36</td><td>-</td><td>-</td>

<td>115.75</td><td>0.44</td><td>0.06</td><td>0.05</td>

<td>119.75</td><td>3.19</td><td>-</td><td>-</td>

<td>139.75</td><td>0</td><td>0</td><td>0</td>

<td>143.75</td><td>2.75</td><td>-</td><td>-</td>

<td>164.5</td><td>0.54</td><td>0.07</td><td>0.22</td>

<td>167.75</td><td>3.29</td><td>-</td><td>-</td>

<td>187.25</td><td>0.87</td><td>0.06</td><td>0.39</td>

<td>191</td><td>3.62</td><td>-</td><td>-</td>

<td colspan="4"></td>

<td></td><td colspan="3">Concentration of lactate (g / l)</td>

<td rowspan="2">Hours</td><td rowspan="2">Control</td><td colspan="2">Limited power supply</td>

<td>Low gradient</td><td>High gradient</td>

<td>0</td><td>0.01</td><td>0.02</td><td>0.01</td>

<td>18.75</td><td>1.20</td><td>1.06</td><td>1.08</td>

<td>42.75</td><td>2.90</td><td>1.58</td><td>1.88</td>

<td>66.25</td><td>4.68</td><td>2.06</td><td>2.77</td>

<td>67.75</td><td>-</td><td>-</td><td>-</td>

<td>92.25</td><td>5.92</td><td>2.20</td><td>3.43</td>

<td>94.75</td><td>-</td><td>-</td><td>-</td>

<td>115.75</td><td>7.76</td><td>1.86</td><td>3.68</td>

<td>119.75</td><td>-</td><td>-</td><td>-</td>

<td>139.75</td><td>8.04</td><td>1.24</td><td>4.08</td>

<td>143.75</td><td>-</td><td>-</td><td>-</td>

<td>164.5</td><td>7.60</td><td>1.18</td><td>4.36</td>

<td>167.75</td><td>-</td><td>-</td><td>-</td>

<td>187.25</td><td>7.72</td><td>1.09</td><td>4.76</td>

<td>191</td><td>-</td><td>-</td><td>-</td>

[0093]

Table 13. Modeled power supply experiment: speed of glucose consumption

<td>Day</td><td>Qgiukoza (mg / 10^b cells / day) in the control culture</td><td>Qgiukoza (mg / 10^b cell / day) in culture at low gradient</td><td>Qgiukoza (mg / 10^b cells / day) in culture at high gradient</td>

<td>1</td><td>2.90</td><td>1.67</td><td>1.69</td>

<td>2</td><td>1.43</td><td>0.45</td><td>0.68</td>

<td>3</td><td>0.99</td><td>0.36</td><td>0.48</td>

<td>4</td><td>0.73</td><td>0.22</td><td>0.33</td>

<td>5</td><td>0.63</td><td>0.22</td><td>0.29</td>

<td>6</td><td>0.49</td><td>0.18</td><td>0.26</td>

<td>7</td><td>0.40</td><td>0.18</td><td>0.27</td>

<td>8</td><td>0.65</td><td>0.18</td><td>0.26</td>

[0094]

Table 14. Experiment model recharges: the rate of lactate production

<td>Day</td><td>Qmieczan (mg / 10⁶ cells / day) in the control culture</td><td>Qmieczan (mg / 10⁶ cell / day) in culture at low gradient</td><td>Qmieczana (mg / 10⁶ cells / day) in culture at high gradient</td>

<td>1</td><td>1.94</td><td>1.61</td><td>1.66</td>

<td>2</td><td>1.06</td><td>0.35</td><td>0.53</td>

<td>3</td><td>0.53</td><td>0.17</td><td>0.31</td>

<td>Day</td><td>Qmieczan (mg / 106 cells / day) in the control culture</td><td>Qmieczan (mg / 106 cells / day) in culture at low gradient</td><td>Odmieczana (mg / 106 cells / day) in culture at a high gradient</td>

<td>4</td><td>0.22</td><td>0.03</td><td>0.13</td>

<td>5</td><td>0.30</td><td>-0.05</td><td>0.04</td>

<td>6</td><td>0.04</td><td>-0.07</td><td>0.05</td>

<td>7</td><td>-0.08</td><td>-0.01</td><td>0.03</td>

<td>8</td><td>0.03</td><td>-0.01</td><td>0.04</td>

[0095] The profiles (dashed lines) in Fig. 9 underline the differences between the three cultures in the amount of lactate produced. The comparison of the profiles in Fig. 9 shows that the lowest levels of lactate were obtained under conditions where the limited glucose feed was done in low gradient mode. The very low rate of lactate production in low-gradient fed culture (Fig. 9 and Table 14) is most likely the reason why these cells maintain high production yields. The rate of glucose consumption stabilized at the level of 0.2 mg / 10^scells / day in low-gradient culture (Table 13).

[0096] Table 15 shows the osmolality profiles for restricted control and culture, and Table 16 shows titrant consumption in these cultures (as above 1 1 of working volume).

[0097]

Table 15. Experiments of modeled feedings: osmolality

<td>Day</td><td>Kontrol. Osmium. (MOsm / l)</td><td>Low Gr.-Osm. (mOsm / l) and</td><td>High Gr.-Osm. (MOsm / l)</td>

<td>0</td><td>290</td><td>288</td><td>271</td>

<td>1</td><td>299</td><td>290</td><td>293</td>

<td>2</td><td>320</td><td>298</td><td>304</td>

<td>3</td><td>349</td><td>306</td><td>324</td>

<td>4</td><td>on</td><td>on</td><td>on</td>

<td>5</td><td>408</td><td>296</td><td>334</td>

<td>6</td><td>427</td><td>287</td><td>353</td>

<td>7</td><td>437</td><td>221</td><td>308</td>

<td>8</td><td>413</td><td>237</td><td>366</td>

[0098]

Table 16. Model power experiments: titrant consumption

<td>Day</td><td>Control - titrant (ml / day)</td><td>Low hail. - titrant (ml / day)</td><td>High hail. - titrant (ml / day)</td>

<td>1</td><td>1</td><td>0</td><td>5</td>

<td>2</td><td>8</td><td>2</td><td>3</td>

<td>3</td><td>10</td><td>2</td><td>4</td>

<td>4</td><td>13</td><td>2</td><td>6</td>

<td>5</td><td>10</td><td>1</td><td>4</td>

<td>6</td><td>8</td><td>3</td><td>8</td>

<td>7</td><td>5</td><td>1</td><td>2</td>

<td>8</td><td>5</td><td>0</td><td>7</td>

[0099] In a batch culture with low-gradient feed, the osmolality decreased slightly from the initial value of 288 mOsm / L to 306 mOsm / L on day 3, and then its level stabilized at 237 mOsm / L on day 8 (Table 15) . Cultivation conducted in a low gradient from day 1 to day 8 inclusive also required the use of a relatively small amount of titrant (Table 16). In the control culture, however, the osmolality increased by almost 50% up to and including day 7 (Table 15), and the titrant consumption in the control culture was always higher than the titrant consumption in the batch culture with high gradient feed (Table 16). Similarly, with the exception of days 1 and 8, the titrant consumption in the control culture exceeded titrant consumption in the batch culture with the high gradient fed feed (Table 16).

[0100] Feeding the cell culture with glucose in a restricted manner (and thus keeping lactate production low in the media) induced several positive effects in this experiment (particularly related to protein production, Fig. 5 and Table 4, Fig. 8 and Table 11 ). Positive effects were obtained by planning delivery of glucose to increase during the periodic fed-batch farms. The goal was to anticipate the glucose demand set for the expected or modeled increase in glucose demand (e.g. as a result of increasing cell concentration) when fed in a limited manner.

[0101] This limited feeding strategy has resulted in a significant reduction in the lactate production rate (for the duration of the expected power increase experiment - Table 7, see also Fig. 6 and during the set of power supply experiments modeled - Table 14, see also Fig. 9) in comparison for control cultures in which the culture medium initially contained a high concentration of glucose (e.g., about 10 g / l). The cell concentration (see Tables 3 and 10) and the level of protein production (see Table 4 and 11) in the restricted feed cultures increased steadily, although the concentration of cells in the control cultures reached a maximum.

[0102] Given the achieved normalized BMP-2 titer level (Fig. 8), it seems that the main advantage of the limited feed to control the production of lactic acid and to keep it low is to increase the efficiency of the process (especially taking into account the rate of protein production ). The limited glucose feeding, used for controlled production of lactic acid at a low level, can also facilitate achieving efficient cell growth (Fig. 7).

[0103] It is important that the benefits of the invention have been achieved by using limited glucose delivery to test cultures rather than by maintaining low glucose concentrations in these cultures. For example, glucose concentration profiles in modeled feed cultures performed at both low and high gradients remained significantly low, while the lactate production profile at low gradient was significantly lower compared to the high gradient cultivation profile (Fig. 9 and Table 12). Thus, favorable metabolic profiles have been achieved by adapting animal cell cultures to growth under conditions in which glucose availability is limited by limited supply to the culture, in particular, when this limited power is based on the expected or modeled rates of possible glucose consumption by the cultured animal cells. When glucose is only supplied to the culture in a limited manner, it is not important how many glucose transporters express cells because they are able to take enough glucose to produce a small amount of lactic acid.

EXAMPLE 4

System based on cell concentration sensors [0104] To facilitate the delivery of glucose at a limited rate in real time, a sensor sensitive to the concentration of cells can be used, the use of which does not require sampling. A computer monitoring system that allows cell concentration to be determined without sampling (e.g., by using a system in which the cell concentration of the culture is estimated by photometric or turbidity measurement) is programmed to record the concentration of cells every 5 min. and transfer this data to a computer system connected to it that controls the delivery of glucose to animal cell culture. The aforementioned connected computer system is in turn programmed like this, to calculate the rate of limited glucose delivery and to deliver glucose for cell culture at this rate. Said limited glucose delivery rate is a function of the expected or modeled rate of glucose consumption for the cells at their fixed concentration.

[0105] The glucose delivery system based on the use of a syringe is arranged to deliver glucose to the culture as in the previous example in a restricted manner and at a low gradient (i.e., using a 0.2 g / ml feed solution). For a cell concentration of 1.4x10^s cells / ml and 1.6x10^s cells / ml in 1 l cell culture, the limited rate of glucose delivery to the culture is (as a function of the expected or modeled rate of glucose consumption) 8.4 mg glucose / hour, whereas for cell concentration 1.9x106 cells / ml and 2.1x106 cells / ml in a 1 liter culture system, the limited rate of glucose delivery to the culture is (again as a function of the expected or modeled rate of glucose consumption) 11 mg glucose / hour. Thus, when the cell concentration measured by the computer monitoring system is about 1.5x106 cells / ml connected to this system, the glucose delivery system for the culture determines in real time the rate of glucose delivery to the culture in such a way that the syringe adds 0.042 ml glucose solution with a concentration of 0, 2 g / ml per hour (glucose is delivered to the cell culture at a rate of 8.4 mg / hour). When the concentration of cells, measured by a computer monitoring system, reaches about 2.0x10^s cells / ml, the combined glucose delivery system determines in real time the rate of glucose delivery to the culture that the syringe replenishes glucose in a 0.055 ml / hr dose. a 0.2 g / ml glucose feeding solution (glucose is delivered to the cell culture system at a rate of 11 mg / hour).

[0106] The above described invention of limited glucose feeding of cell cultures provides a practical way to improve the quality of animal cell cultures. Said practical method provides direct opportunities to improve the quality of cell culture on an industrial scale.

[0107] The above detailed description and examples are given only for the purpose of explaining and allowing an understanding of the invention. The present invention is not limited to the particular details presented in the specification, since the variant verifiable by the person skilled in the art will be within the scope of the invention as defined by the claims.