Restricted glucose feed for animal cell culture

[0001] This application claims priority to an application filed May 15, 2003, in the United States with serial number 60 / 470,937, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the invention The invention relates to a method of improving protein production by cultured animal cells. More specifically, the invention relates to a method of controlling lactic acid production by cultured animal cells (preferably mammalian cells) at low levels in fed-batch cell culture. In some embodiments, the invention provides methods for maintaining lactate production of cultured cells at low levels by using glucose delivery systems that are not based on sampling the culture at regular intervals. In particular, the invention relates to culturing animal cells under conditions in which the culture is fed with glucose in a restricted manner, e.g. at a rate that is a function of the expected or previously modeled rate of glucose uptake by animal cells when exposed to medium containing high glucose levels. Due to said limited feed, production of cultured lactic acid cells is controlled at low levels throughout the culture. The effect of this is increased production of recombinant protein by cultured cells e.g. to facilitate commercial production.

Background Art Related to the Invention [0003] A large proportion of biotechnology products, commercially available or under development, are protein therapeutic agents. In addition, to produce many forms of protein therapeutic agents (such as glycosylated proteins or monoclonal antibodies produced in hybridoma cells (Mabs)), generally speaking, cell machinery of an animal cell (as opposed to a bacterial cell) is necessary. Consequently, the demand for the production of these proteins in animal cell cultures increases.

[0004] However, compared to bacterial cell cultures, animal cell cultures have lower rates and usually lower production yields. It has been found that maintaining glucose concentrations in cell culture media at low levels (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 at an osmolality of about 400 to 600 mOsm increases the production of recombinant proteins in animal cell cultures, especially after pre-culturing at an osmolality of about 280 to 330 mOsm (US Patent No. 5,856 179; each US patent cited herein is incorporated by reference in its entirety) and when grown in all phases are conducted at a selected concentration of glutamine (preferably between about 0.2 and about 2 mM; U.S. Patent No. 6,180,401).

[0005] An increase in recombinant protein production may in some cases be the result of a reduction in lactate production that occurs when glucose levels in culture media are kept low. Lactate is widely known as a potent inhibitor of cell growth and protein production, and maintaining low glucose levels in culture media may result in low levels of lactate production. (Glacken et al. (1986) Biotechnol. Bioeng. 28: 1376-1389; Kurokawa et al. (1994) Biotechnol. Bioeng. 44: 95-103; U.S. Patent No. 6156570). As a result, depending on other culture conditions, maintaining glucose levels at low levels relative to cell concentration is one factor that contributes to lower lactate production levels, and hence higher cell concentrations and increased production of recombinant proteins in cultures animal cells.

[0006] When cells are exposed to low glucose in the medium, their metabolism changes so that both glucose uptake and lactate production rates are lower compared to cells grown in fed-batch processes in which the media contained high glucose levels at the beginning of the process (US Patent No. 6,156,570). In addition, the duration of the periodic culture with feeding may be extended. As a result, both the rate of cell growth and protein production can be maintained for a longer period of time compared to control fed-batch cultures in which cells are grown in media that promotes high levels of lactate production (e.g., media containing high glucose levels at the beginning of the culture period).

[0007] One way to control lactate production by cultured cells at low levels is invariant, glucose feed in a batch fed process with constant rate (Ljunggren and Haggstrom (1994) Biotechnol. Bioeng. 44: 808-18; Haggstrom et al. ( 1996) Annals NY Acad. Sci. 782: 40-52). Although said invariable, glucose feeding in a batch fed process can help control lactic acid production at low levels, maximum cell concentrations, maximum growth rates and viability, and maximum protein production rates are not achieved under these conditions glucose supply usually results in the formation of glucose craving cells with increasing concentration.

[0008] Another way to control lactate production by cultured cells at low levels is to use glucose delivery systems based on sampling the cultures at regular intervals. Samples are taken from the culture at regular intervals and after determining the glucose concentration in the samples (e.g. by flow injection analysis, as in Male et al. (1997) Biotechnol. Bioeng. 55: 497-504, or in Siegwart et al. (1999 ) Biotechnol. Prog. 15: 608-16; or using high performance liquid chromatography as in Kurokawa et al. (1994) Biotechnol. Bioeng. 44: 95-103), in order to maintain stable, low in relation to cell concentration glucose concentration in media, its measured amounts are added. However, cells can adapt to low glucose levels by, for example, increasing their glucose uptake capacity and thus producing excessive amounts of lactic acid despite low glucose levels.

[0009] WO 2004/048556 A1, a state of the art in accordance with Art. 54 (3) of the European Patent Convention for the present application, discloses a method of controlling low-level lactic acid production in a batch fed glucose feed with limited supply for cell culture.

[0010] Furthermore, when using such feedback control methods based on sampling, there is a significant risk of microbial contamination. It is not surprising then that the use of these methods for the commercial production of recombinant proteins in animal cell cultures has proved impossible. Since the publication of the first article on the subject, feedback control methods based on sampling to maintain low levels of glucose in cell culture media have found limited use only in scientific research. Glacken et al. (Glacken et al., Supra) report that glucose concentrations in culture media were determined on an ongoing basis using an automated analyzer in which the glucose containing sample was mixed with o-toluidine and glucose concentration determined by colorimetry at a wavelength of 660 nm.

[0011] As indicated by the abovementioned indications, there is still a need for alternative methods to control and maintain lactate production by cultured cells at low levels in culture media.

SUMMARY OF THE INVENTION [0012] The present invention relates to an embodiment characterized by the claims. The present invention provides a method for the limited feeding of animal cell cultures with glucose in fed-batch processes. Due to this limited supply, lactate production by cultured cells can be kept controlled in a low way without the need for a constant glucose supply at a constant rate. In some embodiments, low-level lactate production by cultured cells can be controlled without the need for culture samples to be sampled at regular intervals to determine glucose concentration in a feedback control method. In particular, the present invention provides a solution to the long-awaited need for a method of flexibly controlling lactate production by cultured cells at low levels, so as to support increased production of recombinant proteins in animal cell cultures, especially on a commercial scale.

[0013] The present invention relates to a method of culturing animal cells under conditions in which cell cultures are fed with glucose in a restricted manner (so-called restricted feeding), whereby the levels of lactate produced by cultured cells are low. This type of limited or slow feed is obtained by continuous or intermittent (periodic) glucose feeding of cell cultures at a rate that is less than (i.e. at a rate that is a function of) the expected or previously modeled glucose consumption rate of animal cells exposed to high glucose medium. In particular, the invention relates to a method of increasing protein production in animal cell cultures by controlling lactate production at low levels by limited glucose feed.

[0014] Although some embodiments of the present invention may utilize sampling-based feedback control, other embodiments of the present invention do not require sampling-based feedback control. For example, the estimated or expected capacity for glucose uptake by cultured animal cells can be increased by measuring cell concentration, which in some embodiments is performed without sampling (e.g., photometrically). Based on cell concentration measurements, the glucose delivery rates of cell cultures (in real time if needed) can be calculated to feed glucose cell cultures in a limited manner, i.e. at a rate of not more than 45% of the expected or previously modeled glucose consumption rate in animal cells in an analogous culture with very similar culture conditions, but in which the glucose concentration, rather than limited, is such that any increase in this concentration does not change the glucose consumption rate through the cells. As shown in embodiments of the present invention, the limited glucose supply allows the lactic acid to be controlled at low levels by cultured cells.

[0015] pH monitoring is included as an additional method for estimating lactate consumption and preventing glucose starvation in cultured cells. PH monitoring uses the fact that in the absence of glucose, the cultured cells consume lactate from the culture. When cells consume lactate, the culture pH increases. Therefore, an increase in pH signals a lack of glucose in cell culture (i.e., signals the state of glucose starvation of cells). Thus, in some embodiments, a feed strategy that provides glucose feed in a single dose of glucose feed and / or increases the rate of restricted glucose feed for cell culture following an increase in pH can protect cells from glucose craving. In some embodiments, pH measurements are performed without sampling (i.e. pH measurements are carried out using an in-situ pH sensor to which cell-containing portions are not taken from the culture for pH measurement).

[0016] In particular, the invention provides a method of culturing cells for controlling lactate production at low levels in a fed-batch culture, which includes: mixing animal cells with the culture medium to form a cell culture; glucose supply of cell culture in a limited manner. Limited glucose supply occurs when glucose is delivered at a rate that is a function of the expected glucose consumption rate of animal cells when exposed to medium containing high glucose. This function is a multiplication by a percentage value not exceeding 45%, and includes, without limitation, percentages such as at least 33% of the expected speed. In other related embodiments, the restricted glucose supply of the cell culture is achieved without using a feedback control based on sampling during the culture. [0017] In some embodiments of the present invention, a cell concentration sensor is used to monitor the cell concentration in cell culture, and the cell concentration measurement based on this sensor is additionally used to calculate the rate at which the cell culture will be restricted in glucose feed. A pH sensor is used to monitor the pH of the cell culture, and if the pH rises above a predetermined value (e.g. about 7) glucose is added to the cell culture (e.g., a single portion of glucose feed and / or at a new glucose feed rate in a limited manner that is faster than the glucose feed immediately preceding it). In some embodiments, the new rate may be 15%, or not more than 50%, greater than the speed immediately preceding it, assuming that such new rate does not reach or exceed 45% of the expected glucose consumption rate of control cells exposed to high levels glucose). In other embodiments, a cell concentration sensor system along with a pH sensor system can be used to determine the glucose feed rate of cell cultures in a limited manner. Both the cell-based sensor system and the pH-based system, and both systems can be used simultaneously without "sampling" from the cell culture.

[0018] According to a further embodiment of the method of the invention, there is provided a method of culturing cells for controlling lactic acid production at low levels in a fed-batch cell culture, which comprises: (a) mixing animal cells with a high glucose containing culture medium to form a first culture cell; (b) determining the glucose consumption rate (i.e. previously modeled rate) for animal cells cultured in the first cell culture; (c) mixing animal cells and culture medium to form a second cell culture; and (d) feeding the second cell culture with glucose in a restricted manner at a rate that is a function of the specific glucose consumption rate of step (b) (i.e. a function of previously modeled glucose consumption rate ). This function is a multiplication by a percentage value not exceeding 45%, such as at least 33%, of the specific glucose consumption rate (i.e. the previously modeled rate). In other related embodiments, the restricted glucose feed of the second cell culture is carried out without controlled sampling with the second cell culture.

[0019] In some embodiments of the present invention, the cell concentration sensor is used to monitor the cell concentration in the second cell culture, where the measurement based on the cell concentration sensor is additionally used to calculate the glucose feed rate in a limited manner of the second cell culture. The pH sensor is used to monitor the pH of the second cell culture and in response to an increase in pH above a predetermined value (e.g. about 7) glucose is added to the second culture (e.g., a single portion of the feed and / or at a new feed rate in a limited manner that is greater than the immediately preceding glucose addition rate. In some embodiments, the new rate may be 15% or not more than 50%, greater than the speed immediately preceding it. In other embodiments, the cell concentration sensor system together with the pH sensor system can be used to determine the glucose feed rate of the second cell culture in a limited manner. Both the cell based sensor system and the pH sensor based system, as well as both systems can be used simultaneously without the need for cell culture sampling.

[0020] In the method of the present invention, the cells are adapted to grow under culture conditions in which glucose is added to the test cell cultures at limited rates compared to the glucose consumption rate under control culture conditions (e.g., wherein the glucose concentration is such that any 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 rates at which the cultures are restricted-fed glucose) produced lactate at lower levels than the control cell cultures. They also showed different rates of growth and production of recombinant protein. Cultures fed in a limited way of the "low gradient" type "Low-ramp") showed lower levels of lactate production than restricted high-yielding cultures

"High-ramp"). Cultures fed with a "low gradient" type of feed also showed higher growth rates and higher recombinant protein production rates than high gradient feeds with a "low gradient" type. In general, both the low gradient restricted feed cultures and the high gradient restricted feed cultures exhibited lower lactate production rates, higher growth rates, and higher recombinant protein production rates than control cultures.

[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 glucose supply compared to restricted glucose supply: best fit curve.

[0023] Fig. 2. Increasing glucose: control feed compared to the expected feed increase.

[0024] Fig. 3. Increasing glucose: control feed compared to previously modeled feed.

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

[0026] Fig. 5 BMP-2 titer (normalized): control power supply compared to the expected power increase.

[0027] Fig. 6 Glucose and lactate concentrations: control feed compared to the expected increase in feed.

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

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

[0030] Fig. 9. Glucose and lactate concentrations: control feed compared to previously modeled feed.

DETAILED DESCRIPTION OF THE INVENTION [0031] Definitions: The expression "animal cells" includes invertebrate cells, non-mammalian vertebrate cells (e.g., birds, amphibians and reptiles) and mammalian cells. Non-limiting examples of invertebrate cells include the following insect cells: Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori (silkworm larva / silkworm moth). Preferred mammalian cells include baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO) cells, human kidney cells (293), FRhL-2 (normal fetal rhesus diploid) cells and mouse melanoma cells (e.g., SP2 / 0 and NS0).

[0032] The expression "basal inoculum medium" means a solution or substance containing nutrients, except glucose, in which cell culture is initiated. "Basic feed medium" contains the same nutrients as the basic inoculum medium, but is the solution or substance that the cell culture is fed after initiating the culture.

[0033] "Batch culture" means a cell culture in which the cells receive a basic glucose-containing inoculation medium at the time of initiation of the culture, and in which the cell culture provides a product, e.g. a recombinant protein, only at the end of the culture. Similarly, "batch fed" cell culture provides the product only at the end of the culture. However, cells in fed-batch culture receive a basal glucose containing inoculation medium at the time of initiation of the culture, and basal glucose containing feed medium once or several times after initiation of the culture but before it ends.

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

[0035] "Glucose Consumption Rate" reflects the glucose consumption of animal cells in culture at a given time point. Glucose consumption rates can be represented graphically (like the upper best fit curve in Fig. 1) or by means of a mathematical function (as in the legend of Fig. 1).

[0036] The terms "restricted glucose supply" and / or "restricted glucose supply" and / or "restricted glucose delivery" and / or similar terms mean providing a limited amount of glucose to the culture, such that the limited amount supplied determined or calculated by function and is not more than 45% of the expected or specified quantity to be used by the control culture. "Control culture" means the culture of the same animal cells under similar culture conditions (e.g., culture of the same cells in similar basic inoculum and feed media, at the same temperature, starting from the same initial cell concentration, etc.) except for this that the glucose level in this culture is high. Thus, the function by which the limited amount of glucose delivered can be determined or calculated, can be a function of the expected glucose consumption rate, or a function of the specific glucose consumption rate of the cells in the control culture. The glucose supply in a limited manner can take place in such a way that glucose is delivered in a certain concentration or in concentrations over a certain period of time, i.e. at a certain speed or rates and / or in such a way that glucose is delivered in one or more bolus doses. [0037] The expressions "function of the expected glucose utilization rate" or "function of the specified glucose utilization rate" (where the specified utilization rate is a previously modeled rate) may include many mathematical relationships between the expected or previously modeled glucose utilization rate and the rate of restricted glucose supply (or reduced rate glucose addition), including relationships where glucose feed rate is the result (e.g. the result of multiplication) (1) the expected or previously modeled glucose consumption rate at any point in time during the cell culture and (2) a percentage of not more than 45%. The invention also includes many other mathematical relationships, such as quadratic, cubic and exponential functions. However, the functions that can be used within the scope of the invention do not include those in which the rate of restricted glucose supply is constant and constant throughout the cell culture.

[0038] "Low lactic acid" (or "low lactate") in cell culture means a concentration of lactic acid (or lactate) that is lower than the concentration of lactic acid (or lactate) in cells grown at high glucose levels.

[0039] "Sampling" includes collecting 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 measuring the concentration of cells in which samples or non-cell portions are taken from the culture for measuring the concentration of the cells. For example, estimation based on photometric measurements of cell concentration can be carried out without "sampling" from a culture in a transparent or translucent container. In addition, sampling does not include the use of an in situ pH sensor to measure the pH of the medium in which animal cells are grown, and no samples or aliquots containing cells are taken from the culture for pH measurement. The use of a probe to measure the pH of a cell culture medium is not "sampling" as used herein if samples or aliquots containing no cells are taken or extracted from the culture.

[0040] According to an established convention, the singular terms used in this patent application, including claims, preceded in English by the terms "a" and "an" mean "one or more". Although the invention has been described to some extent in detail, it is known that in the light of the disclosure to a person skilled in the art, certain alternatives, modifications and changes will be apparent. Accordingly, it is assumed that all those alternatives, modifications, and changes that fall within the scope of the invention and are in accordance with its spirit are encompassed by the claims.

[0041] The present invention relates to a method of growing animal cells, such that low levels of lactic acid are maintained in these cultures, which increases the viability of the cells for a longer period of time, and the production of recombinant protein occurs at increased levels of efficiency. One of ordinary skill in the art will know that the method disclosed herein can be used to culture many well-known animal cells routinely used and grown in the art, i.e. the method disclosed herein is not limited to use only with the cells mentioned in this document in the definition of animal cells.

[0042] The method of the invention relates to feeding glucose into animal cell cultures in a limited manner. As further detailed in Example 2, glucose feed may be restricted at a glucose delivery rate, which is a function of the expected or determined glucose utilization rate (e.g., previously modeled glucose utilization rate) by animal cells in control culture, i.e. cells cultured at high glucose. The restricted glucose supply may also include providing glucose in one or more portions.

[0043] Control culture conditions can be determined by a person skilled in the art without undue experimentation. For example, one skilled in the art understands that animal cells are usually cultured in "medium", which generally means a solution containing nutrients, including glucose. Accordingly, one of ordinary skill in the art will know that glucose should be added to the basal inoculum and feed media prior to inoculation and feeding of animal cells, respectively. It will be understood that the amount of glucose added to the basal inoculation medium and the basal feed medium may vary. Furthermore, one of skill in the art will know which medium is suitable for growing the animal cells concerned (e.g. CHO cells), and will also know the amount of glucose this medium should contain to obtain high glucose animal cell cultures (see, e.g., Mather, JP, et al. (1999) "Culture media, animal cells, large scale production. "Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation. Vol. 2: 777-785). In other words, a person skilled in the art will understand what is the glucose concentration at which a cell must be cultured so that any increase in glucose concentration in the culture does not affect the rate of glucose consumption by the cell. High glucose in cell culture should be distinguished from high glucose added to basal inoculation and feed media. In the latter case, glucose levels (e.g. 44, 200 or 280 g / L) is usually reduced by dilution upon addition to the cell culture.

[0044] The skilled person will also know that the optimal concentration of other nutrients (e.g. glutamine, iron, trace elements D) or factors intended to control other culture variables (e.g. foam formation and osmolality) will vary depending on from a given animal cell. The mere adjustment of the concentrations of such nutrients or agents in primary inoculum or supply media is routine in this field. In addition, one skilled in the art will know at what temperature and at what concentration to grow 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 to calculate the rate of restricted glucose feed. Methods for measuring cell concentration and / or pH of the culture are well known in the art. Such methods include, but are not limited to, measuring cell concentration using a Cedex measuring instrument (Innovatis GmbH, Bielefeld, Germany) and / or CASY (Scharfe GmbH system, Reutlingen, Germany) and / or measuring using a pH sensor. Particularly useful for measuring pH in the claimed invention are methods for determining cell concentration and pH, which do not require sampling, i.e. taking samples containing cells from animal cell cultures, including, but not limited to, methods using a capacitive probe, an optical density probe and / or a turbidity probe and / or a potentiometric probe or pH sensitive dyes.

[0046] In some embodiments of the present invention, the measurement of the cell concentration and / or the measurements from the pH sensor indicate that the glucose feed should be restricted in a limited manner at a new rate that is faster than the one immediately preceding it. The new rate at which glucose is administered in a restricted manner is not greater than 45% of the expected or specified glucose consumption rate. In other embodiments, the new speed increases by 1-15% compared to the speed immediately preceding it. In some embodiments of the present invention, the new speed increases by at least 15% over the immediately preceding speed. In other embodiments of the present invention, the new speed increases by no more than 50% compared to the speed immediately preceding it.

EXAMPLES

EXAMPLE 1

medium

Example 1.1: Inoculum medium [0047] Basic inoculum medium was prepared to contain the same ingredients as DMEM / F12 medium as well as additional ingredients: 200 mg / L dextran sulfate (US Patent 5,318,898 describes the use of dextran sulfate in culture media) , 10 mg / L Nucellin (human insulin analogue based on recombinant DNA; Eli Lilly (Indianapolis, IN)) and 2.4 g / L polyvinyl alcohol (PVA). The basic inoculum medium prepared for the purposes of these experiments did not contain glucose. To make a control inoculation medium before inoculation, approximately 10 g / L glucose was added to the basal inoculation medium. To make the inoculum medium used in glucose restricted cultures, approximately 0.8 g / L glucose and 1.3 g / L NaCl were added to the basal inoculation medium. NaCl was added so that the initial osmolality of the inoculum medium used in the glucose restricted cultures was similar to the initial osmolality of the control inoculum medium.

Example 1.2 Feed media [0048] The primary feed medium was prepared to contain the same ingredients as the basic inoculum medium based on DMEM / F12 medium. The primary feed medium prepared for these experiments did not contain glucose. To make a control feed medium, approximately 44 g / L glucose was added to the basal feed medium.

EXAMPLE 2

Determining the rate of glucose addition [0049] One approach to determining the rate of glucose addition to a restricted feed culture involves testing the glucose consumption rate of CHO cells during a typical control fed-batch culture. The concentration of glucose in a typical control culture starts at high values ​​(i.e. about 10 g / L) and then gradually decreases during normal growth in the exponential phase. Glucose is added after 3 days. Glucose supplementation in the above control cultures is needed to prevent its depletion (see glucose concentration profile for the control culture in Fig. 9).

[0050] Glucose concentrations for control cultures were determined using sampling-based methods at which different times are taken after inoculation and the glucose concentration of the samples is determined. Samples were taken daily and analyzed on a Bioprofile 100 Analyzer (Nova Biomedical Corp., Waltham, MA), which measures glucose, lactate, glutamine, glutamate and ammonium. Glucose concentrations were also determined using the Glucose HK kit (Sigma-Aldrich Co., St. Louis, MO; Cat No. GAHK-20).

[0051] In this sampling approach, the glucose consumption rate during the exponential growth phase in control culture media is plotted as a function of time (hours and days elapsed).

The exponential best fit curve (i.e. y = sq^bx) was generated using these data points (for the best fit curve in Fig. 1 for example a = 2.058 and b = 0.0064). Glucose consumption rates (g / L / hr) were extrapolated from this curve to obtain amounts for limited glucose feed with high and low gradient at any time point. For low feed cultures at low gradient to estimate the rate at which glucose was to be added to low feed cultures at low gradient, the values ​​for the best fit control curve plotted were multiplied by 33% (see filled "low gradient" triangles on Fig. 1). Similarly, to estimate the rate at which glucose was to be added to the restricted feed cultures at high gradient, the values ​​in the graph for the best fit control curve were multiplied by 45% (see the filled "high gradient" squares in Fig. 1). Multipliers 33% and 45% were chosen arbitrarily. All multipliers with a percentage less than 100%, or functions of the dependence of the rate of limited glucose supply on the expected or previously modeled glucose consumption rate, in which the calculated rate of limited glucose supply is less than the expected or previously modeled glucose consumption rate, are within the scope of the invention (except that acceptable functions do not include such dependencies, in which the resulting glucose addition rate is constant and constant throughout the cell culture).

[0052] Another approach to feeding glucose in a restricted manner is the use of a system responsive to changes in pH in the programmed system for limited glucose supply. When the culture of mammalian cells (such as CHO cells) is devoid of glucose, the cultured cells begin to consume lactate as an alternative source of energy from carbohydrates. A decrease in lactate concentration in cell culture causes an increase in pH (to which the pH control system responds).

[0053] In practicing this approach, the syringe pump delivering glucose solution has been programmed to deliver glucose at a limited rate (i.e. 0.032 g / l / hour; see the initial rate of addition at a low gradient in Table 2), except when the cell culture pH increased by 0.02 pH units above a predetermined value of 7.00, then the delivery of a single glucose feed (0.05 to 0.2 g glucose supplied from the feed medium per 1 liter of culture), and additionally the limited delivery rate using the syringe pump was then increased to 15% to 50% higher than the previous limited delivery rate. For example, supplying a single 0.25 to 1.0 mL aliquot of 0.2 g / mL glucose feed medium provides a 1 L cell culture of approximately 0.05 to 0.2 g glucose.

[0054] In another approach, to assist in calculating the rate of limited glucose supply, the cell concentration in the cell culture is measured without sampling. In the initial tests of this approach, glucose was supplied to the culture in such a way that its concentration remained at the level considered appropriate for the cell concentration in the culture. A Wedgewood spectrophotometer (653 Absorbance Controller 653 and Model BT65 Series Insertion Sensor, Wedgewood Technology Inc., San Carlos, CA) was used to determine the cell concentration in real-time cultures. Alternatively, a turbidity laser probe can be used to determine cell concentration (e.g. Model LA-300LT, ASR Co., Ltd., Tokyo). The laser probe for turbidity measurements emits a laser beam that passes from the probe's light source through cell cultures. A calibration curve is used to convert the optical density value to a cell concentration value. Although the light absorbance properties of the cells are not constant and the cell size distribution changes over time, Zhou and Hu ((1994) Biotechnol. Bioeng. 44: 170-77) determined the total cell concentration of the mouse-mouse hybridoma line,

MAK, correlated linearly with the signal from a laser probe measuring turbidity below 3.0x10⁹ cells / liter. Consequently, spectrophotometric measurements of cell concentration can be performed without sampling to facilitate calculation of the glucose restricted feed rate.

[0055] Another approach combines a pH sensor based response system and a system based on measuring cell concentration using a power gradient increasing program (set to approximate the expected glucose demand during the cell culture). Both the pH sensor based response system and the cell concentration based system, or both, can be used without cell culture sampling.

[0056] The use of a probe with a glucose sensor for direct glucose measurement (and not indirectly by measuring pH or cell concentration) in real time (and in a way that does not require sampling from the culture) is not required to implement the invention, in particular because the invention provides limited rate glucose delivery to animal cell cultures rather than simply maintaining low glucose levels in the culture, to avoid cell adaptation to low glucose levels. However, the use of a glucose sensor when practicing the disclosed methods may fall within the scope of the present invention.

EXAMPLE 3

Production of BMP-2 in CHO cells [0057] The purpose of these experiments was to implement a limited glucose supply strategy to control low-level lactate production in fed-batch cultures (more specifically in one-liter (1 L) cultures) CHO cells (more specifically EMCG5 cells ) for the production of recombinant bone morphogenetic protein (BMP-2) (US Patent No. 5,318,898; U.S. Patent No. 5,618,924 and U.S. Patent No. 5,631,142 provide more data on BMP-2 proteins and their production). During the experiment, the effects of limited glucose supply strategies on cell concentration and cell viability, lactate production, protein production efficiency, and extended periodic culture were monitored.

[0058] The sterile glucose solution was fed into the bioreactor in a restricted manner using a syringe pump programmed to increase the amount of glucose administered during the fed batch culture. In one series of tests, glucose was added to the culture as a function of dependence (e.g. as a percentage) of a predetermined rate of glucose uptake by animal cells when exposed to high glucose levels (e.g. as a function of previously modeled speed).

[0059] Glucose cravings were monitored using a pH sensor (Bradley-James Corp.), the use of which did not require sampling. Accordingly, an increase in pH of 0.02 units over a predetermined value of 7.00 was to be a signal that the glucose supply in the culture media was completely depleted and the cells began to consume lactic acid. A decrease in lactic acid levels in cultures causes an increase in the pH of the culture media. This relationship allows the prediction of craving glucose moments using a system based on a pH sensor.

[0060] To evaluate the effectiveness of a pH sensor-based system in preventing craving moments in glucose restricted experiments, the syringe pump was programmed to deliver a single dose of glucose supply to the bioreactor if the pH increased above a predetermined value of 7.00. An increase in pH of 0.02 units results in some tests giving a portion of glucose supply and an increase in limited continuous glucose by 15%, and in other tests by 50%. [0061] In these experiments, Applikon® 2 L bioreactors were used with a working capacity of 1 L ((Applikon Biotechnology, Foster City, CA). If necessary, aeration was provided to maintain dissolved oxygen at 23% air saturation, while emulsion C was used to combat foam for medical purposes ((Dow Coming Corporation, Midland, MI). The temperature was maintained at 37 ° C throughout the feeding period. . To deliver glucose to control or restricted culture cultures in bioreactors, Becton Dickinson® syringes (Becton, Dickinson and Company, Franklin Lakes, NJ) were filled with aseptically control or experimental glucose solution.

[0062] In the first experiment (i.e., the experiment with the expected increase in feed), a representative control culture used increasing amounts of glucose each day, and an exponential best fit curve was used to estimate the amount of glucose consumed each day by the control culture. Using the best fit curve as a basis, the syringe pump (Yale Apparatus, Wantagh, NY) was set to feed glucose into the experimental culture with a limited feed rate, i.e. about 50-70% of the amount consumed by the control culture. Every day, the limited feed rate was changed taking into account the increasing cell density. 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. 75.5, 99.5 and 123.5 hours of culture) (Table 1, Fig. 2 and Fig. 6). The initial glucose concentration in the restricted-feed culture (1 L) was 1.1 g / L. The rate of continuous limited glucose supply of restricted culture was increased four times (27.5, 51.5, 75.5 and 99.5 hours each). culture) starting from an initial continuous limited feed rate of 0.046 g / L / h. (which was maintained during the culture from 20 to 27.5 hours) (Table 1).

[0064] Table 1. Experiment with the expected increase in feed: glucose addition

<td colspan="2">Addition of glucose to 1 l culture control (initial concentration glucose: 10.38 g / l)</td><td colspan="2">Addition rates for restricted culture power supply (initial glucose concentration: 1.1 g / l)</td>

<td>Time point (hours)</td><td>Quantity (g)</td><td>Time period (hours)</td><td>Speed ​​(g / l / h)</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 glucose consumption rate of the fed batch culture was close to the limited glucose supply rate throughout the entire period of continuous limited glucose feed in the restricted feed culture. This is demonstrated by the glucose concentration in the restricted-feed culture, which remained close to zero when after 20 hours. continuous restricted glucose feed was started (Fig. 6). In turn, glucose consumption rates in control cultures were not limited by the limited glucose delivery rate. As a result, glucose consumption rates in control cultures for several days remained at higher levels than in fed batch cultures (Table 6). Also lactate concentrations (Fig. 6) and production rates (Table 7) throughout the experiment with the expected increase in feed remained lower in cultures with restricted feed than in control cultures.

[0066] In the second experiment (i.e. experiment with previously modeled feed) a programmable dual-syringe pump gradient (KD Scientific, Holliston, MA) was used. In experiments with previously modeled feed, the glucose concentration with glucose feed with high gradient was 0.28 g / ml and with low gradient - 0.20 g / ml. In contrast to the experiment with the expected increase in feed, in which the syringe pump supplied glucose continuously and with a pre-set limited feed rate in time, e.g. 0.046 g / h. from 20 to 20.7 and 0.068 g / h from 27.5 to 51.5, etc. The syringe pump gradient program in experiments with previously modeled feed allowed for a gradual increase in the glucose limited feed rate to values ​​close to 33% or 45% of the exponential curve of best glucose fit consumed in the control culture each day.

Gradient programming allowed programming of the initial and final limited speed for each period of time e.g. 12 hours. During this period of time, the pump changed speed continuously and linearly. Table 2 shows representative data on glucose addition for this experiment with previously modeled feed.

[0067] Table 2. Experiment with previously modeled feed: glucose addition

<td colspan="3">Addition of glucose to 1 L of control culture (initial glucose concentration: 8.4 g / L)</td>

<td>Time point (hours)</td><td>Quantity (g)</td><td rowspan="9"></td>

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

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

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

<td> 67,5</td><td> 2,2</td>

<td> 94,75</td><td> 2,2</td>

<td> 119,75</td><td> 2,2</td>

<td> 143,75</td><td> 2,2</td>

<td> 167,75</td><td> 2,2</td>

<td colspan="2"> 191</td><td> 2,2</td><td></td>

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

<td colspan="2"></td><td colspan="2">Addition rates to restricted-feed cultures (initial glucose concentration: 0.88 g /,)</td>

<td>Period of time (Hrs.)</td><td>Speed syringes ml / hr.</td><td>Low gradient (0.2 power supply) g / ml) g / h added to 1 l</td><td>High gradient (power supply 0.28 g / ml) g / h added to 1 l</td>

<td> 0-19,75</td><td> 0</td><td> 0</td><td> 0</td>

<td> 19,75 - 43,75</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 - 0,0448</td><td> 0,0515 - 0,0627</td>

<td> 67,75 - 91,75</td><td> 0,224 - 0,264</td><td> 0,0448 - 0,0528</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 - 0,0688</td><td> 0,0851 - 0,0963</td>

<td> 139,75 140,25</td><td> 0,344 - 0,346</td><td> 0,0688 - 0,0692</td><td> 0,0963 - 0,0969</td>

<td> 140.25 164.25</td><td> 0,400 - 0,450</td><td> 0,0800 - 0,0900</td><td> 0,1120 - 0,1260</td>

<td> 164,25-191</td><td> 0,450</td><td> 0,0900</td><td> 0,1260</td>

[0068] Fig. 2 shows the increasing amount of glucose delivered during the experiment with the expected increase in feed, and Fig. 3 shows the increasing amount of glucose delivered during the experiment with previously modeled with asilane. Both Figures 2 and 3 take into account the initial amounts of glucose delivered to control culture bioreactors and fed in a limited manner (and not only total glucose delivered by syringe pump). For both experiments in the control sample, the initial glucose concentration was high and the daily glucose addition (2.2 g) started after about 72 hours. For the restricted-feed culture, the glucose concentration in the bioreactor on day 0 was 1 g / L, and glucose delivery by the syringe started after about 20 hours.

[0069] The experiment with the expected increase in feed showed that the cell growth in the restricted feed culture was initially slower compared to the cell growth in the control culture, but on day 6 the cell growth in the restricted supply culture reached a higher final concentration than in the control (Fig. 4). In the control culture, the cell concentration reached its maximum much earlier, and viability began to decline rapidly after day 4 (Fig. 4). In contrast to the control culture, the rate of cell growth in restricted feeding cultures remained positive up to and including Day 6 (Table 3).

[0070] Although the restricted feed culture reached a higher final cell concentration by day 6 compared to the control culture, cultures had similar concentrations on day 5 (Fig. 5). These data show that the reduced cell viability in the control culture compared to the restricted supply culture was not a function of the fact that the cells reached maximum bioreactor capacity. In contrast, the data presented in Fig. 4 in combination with the data from Fig. 6, which presents low lactate levels in restricted cultures compared to lactate levels in control cultures, suggests that the increase in viability of cells cultured in restricted cultures was a function of the low lactate level achieved as a result of restricted glucose supply.

[0071] Table 3. Experiment with expected feed increase: cell growth rate (Cedex μ / h value; Cedex μ value = cell concentration in units of 10)⁵/ Ml).

<td>Day</td><td>Control growth rates ^ • (hr.^-1))</td><td>Growth rates with limited power ^ • (hr.^-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>Day</td><td>Control growth rates (μ / h^-1))</td><td>Growth rates with limited power (Μ / hr.^-1))</td>

<td> 4</td><td> 0,016</td><td> 0,017</td>

<td> 5</td><td> 0,001</td><td> 0,007</td>

<td> 6</td><td> -0,014</td><td> 0,005</td>

[0072] Fig. 5 shows graphs of the values ​​of normalized BMP-2 titers. BMP-2 titers were normalized as part of the BMP-2 titer value on day 6 in the experiment with the expected increase in feed. Table 4 shows the values ​​corresponding to the BMP-2 production rates. BMP-2 production rates are normalized as part of the BMP-2 production rate in control culture on day 1. After day 4, BMP-2 titer levels for the control culture stabilized, while in restricted cultures these titers continued to increase (Fig. 5). In contrast to the control culture, the rates of BMP-2 production in the restricted supply culture remained positive until day 6 (Table 4). It should be emphasized that a slight decrease in BMP-2 titer in control cultures, contrary to what is suggested in Fig. 5, can only be a reflection of the variability between experiments, and not a real decrease in BMP-2 titer in culture.

[0073] Table 4. Experiment with the expected increase in feed: BMP-2 production rate (normalized).

<td>Day</td><td>Control speed preparation</td><td>Production speed with limited power</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] Keeping lactic acid low using a glucose feed strategy limited the cell growth and protein production efficiency. In a restricted fed culture, the final BMP-2 titer was about 70% higher (Fig. 5), and the BMP-2 production rate did not reach a negative value, as was the case with the control culture (Table 4).

[0075] Figure 6 shows glucose (g / L) and lactate (g / L) profiles in an experiment with the expected increase in feed, and Table 5 shows the corresponding representative data on glucose (g / L) and lactate (g / L) l) in this experiment. Table 6 presents values ​​for corresponding glucose consumption rates, and Table 7 presents values ​​for corresponding lactate production rates.

[0076] Table 5. Experiment with the expected increase in feed: glucose and lactate

<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 colspan="3"></td>

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

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

<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 with the expected increase in feed: glucose consumption rate

<td>Day</td><td>Qglucose control (mg / 10⁶cells / day)</td><td>Limited Qglucose (mg / 10⁶cells / day)</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 with the expected increase in feed: lactate production rate

<td>Day</td><td>Qml Lactate Control (mg / 10⁶cells / day)</td><td>Limited supply of Qml lactate (mg / 10⁶cells / 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 with the expected increase in feed, from day 1 to day 3 a slower glucose consumption rate was observed in the fed batch culture than in the control culture (Table 6). During the fed-batch culture, also lactate production rates were lower compared to the lactate production rate of the control culture (Table 7). This allowed lower lactate concentrations to be achieved during fed-batch culture (Fig. 6).

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

[0081] Table 8. Experiment with the expected increase in feed: osmolality

<td>Day</td><td>Control osmolality (MOsm / l)</td><td>Osmolality with limited glucose (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>Day</td><td>Control osmolality (MOsm / l)</td><td>Osmolality with limited glucose (MOsm / l)</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 with expected increase in feed: titrant consumption:

<td>Day</td><td>Control titrant consumption (Ml / day)</td><td>Titrant consumption with limited glucose (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] Generally, a lower level of osmolality (Table 8) and a lower level of titrant consumption (Table 9) in a restricted-feed culture (compared to a control culture) are associated with less lactate produced (which requires the use of less titrant for neutralization).

[0084] In experiments with previously modeled feed, a standard fed batch culture was carried out in one bioreactor as a control. In addition, two limited supply bioreactors were installed, one bioreactor for limited glucose delivery at low gradient and the other for limited glucose delivery at high gradient. One syringe pump was used for each test bioreactor to continuously increase the rate of restricted glucose supply. The concentration of glucose solution fed into the bioreactor with a low gradient was 0.2 g / ml. The concentration of glucose solution fed into the high gradient bioreactor was 0.28 g / ml.

[0085] Fig. 7 graphically presents the cell concentration (solid line) and their viability (broken line) during the culture in the control and test bioreactors. Table 10 presents cell growth rate data for control bioreactors and test bioreactors.

[0086] Table 10 Experiment with previously modeled feed: (values

Cedex μ / hr .; Cedex values ​​μ = cell concentration in units of 10⁵/ Ml).

<td>Day</td><td>Check rates (μ / h^-1))</td><td>Rates at low gradient (μ / h "¹))</td><td>High gradient rates (μ / h^-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> 0,011</td><td> 0,010</td>

<td> 6</td><td> -0,004</td><td> 0,012</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 restricted feed cultures, cell concentration continued to increase up to and including day 8 (192 hours). Furthermore, cell viability remained high throughout this period (Fig. 7). In contrast, in the control culture, the cell concentration reached a maximum of day 5 (120 hours), and then the cell concentration dropped dramatically, accompanied by an even greater decrease in cell viability (Fig. 7). Cell concentration in the low gradient culture reached 12x10 on day 8⁶cells / ml, and cell viability remained higher than 90% (Fig. 7).

[0088] BMP-2 titer levels observed in restricted feed cultures confirm the utility of the methods of the invention to increase protein production in cultured animal cells (especially in low gradient culture). Fig. 8 shows BMP-2 titer levels for control and test bioreactors normalized as part of the maximum BMP-2 titer (day 5) for control culture. Table 11 presents BMP-2 production rates 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. Experiment with previously modeled feed: BMP-2 production rate (normalized)

<td>Day</td><td>Control speed preparation</td><td>Production speed at low gradient</td><td>Production speed at 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 feed fed batch culture with a low gradient. The level of this titer is more than three times higher than the maximum BMP-2 titer achieved in the control culture (Fig. 8). In low gradient culture, the production rate of BMP-2 remained high for six days (Table 11). The rate of BMP-2 production in the high gradient fed batch culture decreased earlier than the rate of BMP-2 production in the low gradient fed batch culture (Table 11). This rapid decrease in BMP-2 production rate is most likely due to the presence of higher levels of inhibitors, such as lactate, in high gradient culture than in low gradient culture.

[0091] Fig. 9 shows glucose (solid lines) and lactate (dashed lines) concentration (g / l) profiles in a previously modeled feed experiment for control and test bioreactors, and Table 12 shows the corresponding representative data for glucose and lactate ( g / l) for this experiment. Table 13 presents data on glucose consumption rate, and Table 14 - data on lactate production rate for control and test bioreactors.

[0092] Table 12. Experiment with previously modeled feed: glucose and lactate concentrations

<td rowspan="2"></td><td colspan="3">Glucose concentration (g / l)</td>

<td colspan="3">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 rowspan="2"></td><td colspan="3">Lactate concentration (g / l)</td>

<td colspan="3">Limited power supply</td>

<td>Hours</td><td>Control</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. Experiment with previously modeled feed: glucose consumption rate

<td>Day</td><td>Qglucose control (mg / 10⁶cells / day)</td><td>Low gradient Qglucose (mg / 10⁶ cells / day)</td><td>High gradient Qglucose (mg / 10⁶ cells / day)</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 with previously modeled feed: lactate production rate

<td>Day</td><td>Qml Lactate Control (mg / 10⁶cells / day)</td><td>Low Qml lactate gradient (Mg / 10⁶ cells / day)</td><td>High gradient Qlactate (mg / 10⁶ cells / day)</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> 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 highlight the differences between the three said cultures in the amount of lactate produced. Comparison of these profiles in Fig. 9 shows that the lowest lactate levels were obtained under conditions when the limited glucose feed was in low gradient mode. Very low lactate production rate in a limited feed culture with a low gradient (Fig. 9 and Table 14) is probably the reason why these cells are able to maintain such high production efficiency. Glucose consumption rate stabilized at 0.2 mg / 10⁶ cells / day in a low gradient culture (Table 13).

[0096] Table 15 presents the osmolality profiles for control and fed cultures in a limited manner, and Table 16 presents the titrant consumption in these cultures (as above per 1 liter working volume).

[0097] Table 15. Experiment with previously modeled feed: osmolality

<td>Day</td><td>Osmo Control. (MOsm / l)</td><td>Osmo. at low power (MOsm / l)</td><td>Osmo. at high power supply (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. Experiment with previously modeled feed: Titrant consumption

<td>Day</td><td>Control titrant (Ml / day)</td><td>Titrant at low gradient (ml / day)</td><td>Titrant at high gradient (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 fed-batch, low gradient culture, osmolality increased slightly from an initial value of 288 mOsm / L to 306 mOsm / L on Day 3, and then stabilized at 237 mOsm / L on Day 8 (Table 15) . Breeding with a low gradient from day 1 to day 8 also required the use of a relatively small amount of titrant (Table 16). In contrast, osmolality increased by almost 50% in control culture up to day 7 inclusive (Table 15), and titrant consumption in control culture was always greater than titrant consumption in fed-batch culture with low gradient (Table 16). Similarly, except on days 1 and 8, the titrant consumption in the control culture exceeded the titrant consumption in the fed batch culture at high gradient (Table 16). As in the experiment with the expected increase in feed, in experiments with previously modeled feed a lower level of osmolality (Table 15) and generally lower titrant consumption (Table 9) in cultures with limited feed (both conducted at high and low gradient) compared to the culture control results from the production of smaller amounts of lactate (which requires less titrant to neutralize).

[0100] Feeding glucose cell culture media in a restricted manner (and thus keeping lactate production in the media low) had several positive effects (particularly associated with protein production) in this experiment; Fig. 5 and Table 4, Fig. 8 and Table 11). These positive effects were obtained by programming glucose delivery to increase during these fed-batch cultures to predict the glucose requirement established for the expected or previously modeled increase in glucose demand (e.g., as a result of increased cell concentration) with restricted feeding .

[0101] This limited feed strategy resulted in significant reductions in lactate production rate (throughout the duration of the experiment with the expected increase in feed - Table 7, see also Fig. 6 - and during the set of experiments with previously modeled feed - Table 14, see also Fig. 9 ) compared to control cultures in which the culture medium initially contained high glucose (e.g. about 10 g / L). Cell concentration (see Tables 3 and 10) and protein production levels (see Tables 4 and 11) in the restricted feed test cultures continued to increase, although the cell concentration in the control cultures reached a maximum. Particularly in the previously modeled feed experiment, the restricted feed culture at a low gradient achieved a final recombinant protein concentration three times greater than the maximum titre in the control culture (Fig. 8).

[0102] Given the achieved normalized BMP-2 titer levels (Fig. 8), it seems that the main advantage of the limited feed in controlling lactic acid production and keeping its production at low levels is to increase the process production efficiency (in particular measured as production rate protein). Feeding glucose in a manner limited to controlled production of lactic acid at low levels may also facilitate achieving process production efficiency when the latter is measured as cell growth (Fig. 7).

[0103] It is important that the benefits of the invention have been achieved due to the limited glucose delivery rate to the test cultures, and not simply by maintaining low glucose concentrations in these cultures. For example, the glucose concentration profiles in previously modeled feed cultures at both low and high gradient remained similarly low, but the lactate production profile of the low gradient culture remained significantly lower compared to the high gradient culture profile (Fig. 9 and Table 12). Thus, favorable metabolic profiles have been achieved as a result of adaptation of cultured animal cells to growth under culture conditions in which glucose availability is limited by limited glucose supply to the culture, in particular when limited delivery is based on expected or previously modeled rates of glucose utilization by cultured animal cells. When glucose is supplied to the culture only in a restricted manner, it is not important how many glucose transporters the cells express because they are able to take up enough glucose to produce only a small amount of lactic acid.

EXAMPLE 4

Sensor based system and cell concentration [0104] To facilitate the delivery of glucose at a limited rate in real time, a cell concentration sensor that does not require sampling can be used. A computerized monitoring system by which cell concentrations can be determined without sampling (e.g. by using a system in which cell concentration in animal cell culture is estimated on the basis of photometric measurements or turbidity of the culture) is programmed in such a way as to record cell concentrations every 5 min. and send this data to a computer system connected thereto which controls the delivery of glucose to the culture of animal cells. Said connected computer system is in turn programmed to calculate the rate of limited glucose delivery and to supply glucose to the animal cell culture at that speed. Said limited glucose delivery rate is a function of the expected or previously modeled glucose consumption rate for cells at their fixed concentration.

[0105] The syringe-based glucose delivery system is set up as for the glucose restricted culture with the low gradient from the previous example (i.e. using 0.2 g / ml glucose feed solution). For cell concentrations between 1.4x10⁶ cells / ml a 1.6x10⁶ cells / ml in a 1 L animal cell culture system, the specified limited glucose supply rate for culture is (as a function of the expected or previously modeled glucose consumption rate) 8.4 mg glucose / hour, while for cell concentrations between 1.9x10⁶ cells / ml a 2.1x10⁶ cells / ml in a 1 L animal cell culture system, the limited rate of glucose delivery to the culture is (again as a function of the expected or previously modeled glucose consumption rate) 11 mg glucose / hour. Thus, when the cell concentration measured by the computerized monitoring system is about 1.5x10⁶ cell / ml, the culture glucose supply control system connected to this system determines in real time the rate of glucose supply to the culture in such a way that the syringe delivers 0.042 ml / h. 0.2 g / ml glucose feed solution (i.e. glucose is supplied to the cell culture at a rate of 8.4 mg / h). Later, when the cell concentration, measured by the computer monitoring system, reaches about 2.0x10⁶ cells / ml, the combined system controlling glucose delivery for animal cell culture sets in real time such a glucose delivery rate for the culture that the syringe delivers glucose at 0.055 ml / h. 0.2 g / ml glucose feed solution (i.e. glucose is delivered to the cell culture system at a rate of 11 mg / h).

[0106] The above described invention for the limited supply of glucose to cell cultures provides a practical method for improving the efficiency of animal cell cultures. This practical method provides a direct opportunity to improve cell culture on an industrial scale.

[0107] The above detailed description and examples are provided solely to explain and enable understanding of the invention. It should not be understood as unnecessary restrictions. The present invention is not limited to the particular details presented and described herein, since a verifiable variant for a person skilled in the art will be within the scope of the invention as defined by the claims.

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