EA045030B1 - METABOLICALLY OPTIMIZED CELL CULTURE - Google Patents

Description

This application claims priority under 35 USC § 119(e) to US Provisional Patent Application No. 61/889,815, filed October 11, 2013, which is incorporated herein by reference in its entirety.

Field of technology to which the present invention relates

The present invention relates to cells that exhibit a metabolic shift to lactate consumption in cell culture. The transition to a metabolic profile of lactate consumption in the culture in a seed fermenter system is characterized by beneficial effects on the production crop. When inoculated into a production reactor, cells exhibit more efficient lactate metabolism with low rates of lactate production, low maximum lactate contents, early transition to lactate consumption, and hence increased productivity in fed-batch mammalian cell culture. Thus, an improved method for large-scale production of proteins and/or polypeptides in cell culture is provided.

BACKGROUND OF THE INVENTION

Biological agents, in particular proteins and polypeptides, are increasingly being developed as new pharmaceutical products. Engineered cells that produce atypically high levels of a particular protein of interest become critical for the successful commercial production of these pharmaceuticals. Control and optimization of cell culture conditions are subject to change and have a significant impact on the content and quality of the therapeutic protein produced in the culture.

It is common practice to produce proteins by cell culture in a batch or fed-batch culture method. The early stages of inoculum growth after the vial is thawed involve culturing the cells in a seed culture. Typically, cells are grown at an exponential growth rate, such as in seed-seeded bioreactors, to ensure continuous expansion of the size and/or volume of the cell population. After a proportional increase in cell mass as a result of passing through several stages in the bioreactor, the cells are transferred to the production bioreactor while the cells are still in the exponential growth phase (logarithmic growth phase) (Gambhir, A. et al., 2003, J Bioscience Bioeng 95(4 ):317-327). It is generally considered undesirable to allow cells in a batch culture, such as a seed culture, to enter stationary phase without going through a logarithmic growth phase. Recommendations are that cultures should be subcultured while they are in logarithmic growth phase, before cells such as adherent cells become confluent due to contact inhibition, or waste accumulation inhibits cell growth, among other reasons (Cell Culture Basics, Gibco/Invitrogen Online Handbook , www.invitrogen.com; ATCC® Animal Cell Culture Guide, www.atcc.org).

After transfer to fed-batch culture, the cells are cultured for a specified period of time while the composition of the medium is monitored and controlled to allow production of the protein or polypeptide of interest. Once a certain product yield or cell viability level has been reached, waste accumulation or nutrient depletion determines the decision to stop culture and the protein or polypeptide produced is isolated. Over the past decade, numerous significant advances have been made to improve recombinant protein yields, which currently reach titers in the order of grams per liter. Advances in protein production methods, as well as in the engineering of cell lines and the development of cell culture media and feeding systems are contributing to increased protein yields.

Fed-batch production involves adding small volumes of feed to replenish the nutrients in the bioreactor as cells grow and product is produced. It should be understood that mammalian cells generally have a tendency to continuously metabolize carbohydrates, resulting in the accumulation of lactate, thus requiring the addition of a base to neutralize lactic acid. The addition of base increases the osmolarity in the cellular environment, which in turn severely limits the overall achievable cell viability and/or productivity in the bioreactor. Accumulation of lactate in the medium negatively affects cell growth and is one of the common factors limiting the maximum productivity that can be achieved in batch culture. In a typical batch cell culture, growth and productivity are inhibited once the lactate concentration in the culture reaches approximately 30-50 mM and/or the ammonia concentration reaches 3-5 mM (Ozturk, S.S., Riley, M.R., and Palsson, V.O. 1992. Biotechnol. and Bioeng. 39: 418-431). Currently, commonly used regimens include nutrient replenishment and the development of serum-free media with established chemistry to support continued cell growth and optimal product secretion.

Attempts related in particular to reducing the output of metabolic waste products, such as lactate accumulation, in cell culture have improved the overall number of final protein titers. These efforts have focused on methods using fed-batch culture with controlled glucose or limited nutrient content (see, for example, WO2004104186; US8192951B2), improved cell culture media (for example, US7390660; Zagari, et al ., 2013, New Biotechnol., 30(2):238-45) or cell engineering, including directing enzymes into the glycolytic pathway (e.g., Kim, S.H. and Lee, G.M., 2007, Appl. Microbiol. Biotechnol. 74, 152-159; Kim, S. H. and Lee, G. M., 2007, Appl. Microbiol. Biotechnol. 76, 659-665; Wlaschin, K. F. and Hu, W. S., 2007, J. Biotechnol. 131, 168-176).

Controlled cell feeding is used in an attempt to achieve a more efficient metabolic phenotype (Europa, A. F., et al., 2000, Biotechnol. Bioeng. 67:25-34; Cruz et al., 1999, Biotechnol Bioeng, 66(2): 104-113 ; Zhou et al., 1997, Cytotechnology 24, 99-108; Xie and Wang, 1994, Biotechnol Bioeng, 43:1174-89). However, this is complicated by the fact that nutrient deficiencies, as well as rapid changes such as ammonia concentrations observed in fed-batch culture at high cell densities, can induce apoptosis (programmed cell death) (Newland et al., 1994 , Biotechnol Bioeng 43(5):434-8). Therefore, a common approach to optimization is to grow cells to moderately high densities in fed-batch culture and then deliberately induce a prolonged, productive stationary phase, for example by changing temperature or pH (Quek et al., 2010, Metab Eng 12 (2): 16171. doi: 10.1016/j.ymben.2009.09.002. Epub 2009 Oct 13).

The optimization techniques discussed above have focused on fed-batch cell culture, and this nutrient-dependent method must be tailored to each host cell engineered to produce the polypeptide of interest. Methods for adapting cells to lactate consumers in culture are essential in the production of biological therapeutics. Optimization of a cell line with a metabolic phenotype characterized by lactate consumption would be useful for the commercial production of polypeptides.

Brief Disclosure of the Present Invention

The present invention provides cells and methods for culturing cells that have undergone a metabolic shift to consume lactate. Metabolically adapted cells are optimal for large-scale protein production.

According to one aspect of the present invention, there is provided a method of culturing cells comprising transferring cells from a first cell culture to a second cell culture after the first culture has undergone a metabolic shift to consume lactate in the cells.

According to another aspect of the present invention, there is provided a method of culturing cells comprising culturing cells in a first cell culture, determining that a metabolic shift to lactate consumption in the cells has occurred in the first cell culture, and transferring the cells to a second cell culture after the metabolic shift to lactate consumption has occurred in the first cell culture. lactate consumption in the cells, the concentration of lactate in the second cell culture indicating the net consumption of lactate during the cultivation of the second culture. In one embodiment, the method further comprises reducing lactate accumulation in the second cell culture compared to that determined in an otherwise identical cell culture under otherwise identical conditions, except that transfer of cells to the second cell culture occurs before a metabolic shift occurred in the first cell culture.

According to a second aspect of the present invention, there is provided a method for producing protein, comprising transferring cells from a first cell culture to a second cell culture after a metabolic shift to lactate consumption has occurred in the cells, and maintaining the second cell culture for a period of time so that the protein accumulates in cell culture. In a related aspect, the present invention provides a method for producing a protein comprising culturing cells in a first cell culture, determining that a metabolic shift to lactate consumption in the cells has occurred in the first cell culture, transferring the cells to a second cell culture after the metabolic shift to lactate consumption has occurred in the first cell culture. consuming lactate in the cells, and maintaining a second cell culture for a period of time so that the protein accumulates in the cell culture. In one embodiment, the method further comprises increasing the productivity in the second cell culture compared to said parameter determined in an otherwise identical cell culture under otherwise identical conditions except that the transfer of cells into the second cell culture occurs before the first cell culture experienced a metabolic shift.

According to a third aspect of the present invention, there is provided an improved method of culturing cells, wherein the cells contain a gene encoding a polypeptide of interest, comprising the following steps: culturing the cells in a first cell culture, maintaining the first cell culture under conditions that allow cell mass to increase, transferring the cells to a second cell culture. cell culture after a metabolic shift to lactate consumption has occurred in the cells, maintaining the second cell culture under conditions that allow expression of the polypeptide of interest, and collecting the polypeptide of interest from the second cell culture. In one embodiment, the method further comprises determining that the first cell culture has undergone a metabolic shift to lactate consumption in the cells.

According to a fourth aspect of the present invention, there is provided an improved method for producing a polypeptide in cell culture, comprising the following steps: transfecting cells with DNA encoding the polypeptide of interest, culturing the cells in a first cell culture, transferring the cells to a second cell culture after a metabolic shift to lactate consumption has occurred in cells, wherein the polypeptide of interest is expressed under the conditions of the second cell culture, and maintaining the second cell culture for a period of time so that the polypeptide accumulates in the cell culture. In one embodiment, the method further comprises determining that the first cell culture has undergone a metabolic shift to lactate consumption in the cells.

According to a fifth aspect of the present invention, there is provided a method for producing a metabolically shifted cell line, comprising the following steps: maintaining a cell population in a first cell culture under conditions that allow cell mass to increase, determining when a metabolic shift to lactate consumption has occurred in the cells, transferring a fraction of the cell population from the first cell culture to the second cell culture after a metabolic shift to lactate consumption has occurred in the cells, maintaining the cell population in the second cell culture for a period of time, and optionally harvesting the cells, thereby obtaining a metabolically shifted cell line.

According to another aspect of the present invention, there is provided a metabolically shifted cell line obtained by any of the methods disclosed herein according to the present invention.

In some embodiments, the metabolically shifted cell comprises a nucleic acid sequence stably integrated into the cell's genome, wherein the nucleic acid sequence encodes a polypeptide or protein of interest. In other embodiments, the metabolic shift cell comprises an expression vector encoding a polypeptide or protein of interest.

In one embodiment, the metabolic shift to lactate consumption is detected by measuring the pH, lactate or base content of the first cell culture. In other embodiments, the cells are transferred to a second cell culture when lactate consumption is detected. In other embodiments, a metabolic shift to lactate consumption is detected after increasing the pH of the first cell culture medium without adding base. In other embodiments, a metabolic shift to lactate consumption is detected when lactate levels in the first cell culture plateau. In other embodiments, the method further comprises determining a metabolic shift by: measuring the pH of the first cell culture, adding a base to maintain the pH above a predetermined lower limit, determining that the pH is above the predetermined lower limit for successive time intervals, and stopping addition of base, thereby determining that the first cell culture has undergone a metabolic shift to lactate consumption.

In other embodiments, the metabolic shift to lactate consumption is detected using indicators or products of cellular metabolism, including but not limited to oxygen consumption and metabolites such as glycine, tryptophan, phenylalanine, adenine, palmitic acid, glutamic acid, methionine, and asparagine. In another embodiment, the metabolic shift to lactate consumption is detected using metabolomic analysis or proteomic analysis.

In one embodiment, a metabolic shift occurs when cells exit logarithmic (ie, exponential) growth phase in the first cell culture. In another embodiment, the cells are transferred after the cells exit logarithmic growth phase in the first cell culture.

In another embodiment, the metabolic shift occurs when the cells have reached the stationary growth phase in the first cell culture. In another embodiment, the cells are transferred after the cells have reached the stationary growth phase in the first cell culture.

In one embodiment, the metabolic shift occurs in the first cell culture on day 3 or after 3 days of cell growth in the first cell culture. In another embodiment, the metabolic shift occurs in the first cell culture at day 3.5 or after 3.5 days of cell growth in the first cell culture.

- 3 045030

In some embodiments, the first cell culture is a seed culture. In some embodiments, the second cell culture is a fed-batch culture. In other embodiments, the second cell culture is a production culture. In other embodiments, cultivation of the second cell culture is carried out in a production bioreactor.

In other embodiments, the cells are transferred to a second cell culture at an initial cell density of greater than or equal to about 0.5 x 10 ⁶ cells/ml. In some embodiments, the cells are transferred to a second cell culture at an initial cell density of approximately 0.5-3.0 x 10 ⁶ cells/ml.

In some embodiments, the lactate concentration in the second cell culture indicates net lactate consumption, e.g., net lactate consumption is achieved on day 2, 3, 4, or 5, or after 2 days, 3 days, 4 days, or 5 days of cell growth in the second cell culture. In other embodiments, the reduction in lactate accumulation is a reduction in the maximum lactate concentration in the second cell culture. In other embodiments, the decrease in maximum lactate concentration occurs in the second cell culture on day 5 or after 5 days of cell growth in the second cell culture. In other embodiments, the maximum lactate concentration in the second cell culture is less than about 6 g/L, 5 g/L, 4 g/L, 3 g/L, 2 g/L, or less than about 1 g/L.

In some embodiments of the present invention, the cell or cells are selected from the group consisting of the following: CHO cell, COS, retinal cell, Vero cell, CV1, HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21, HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL60, Jurkat, Daudi, A431, CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT, PER.C6, mouse lymphoid cells and mouse hybridomas.

Brief description of graphic materials

Fig. 1: A seed vessel containing the fusion protein-producing cell line CHO was used to inoculate production bioreactors in parallel at (Figure 1A) three different metabolic states (on-line pH and off-line lactate) and viable cell counts (VCCs). . Base consumption normalized to 1 for the seed vessel is also shown. The parameters (time, pH, lactate content, VCC and base content) for each cell culture (condition #1, #2 and #3) for which cells were transferred to production bioreactors are indicated by white rectangles (dashed line). All production bioreactors operated under the same operating conditions. The impact of each seed fermenter system and its metabolic state on protein (Figure 1B) and lactate (Figure 1C) titers in the production bioreactor is shown. Lines representing the main trend characteristic of the production bioreactor represent the average of the bioreactors in two parallels, with error bars representing ± one standard deviation.

Fig. 2: A seed vessel containing the antibody-producing cell line CHO was used to inoculate production bioreactors in parallel in a chemically established manner under (FIG. 2A) four different metabolic states (off-line pH and lactate measurements) and viable cell counts. The parameters (time, pH, lactate and VCC) for each cell culture (condition #1, #2, #3 and #4) for which cells were transferred to production bioreactors are indicated by white rectangles (dashed lines). All production bioreactors operated under the same operating conditions. Condition No. 1 was lost after one week. The impact of each seed fermenter system and its metabolic state on protein titer (Figure 2B) and lactate accumulation (Figure 2C) in the production bioreactor is also shown. Lines representing the main trend characteristic of the production bioreactor represent the average of bioreactors in two parallels, with error bars representing ± one standard deviation.

Detailed Disclosure of the Present Invention

It should be understood that the present invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used herein is presented solely for the purpose of describing specific embodiments and is not intended to be limiting as the scope of the present invention is defined by the claims.

As used in this specification and the accompanying claims, the singular forms include references to the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to a method includes one or more methods and/or steps of the type described herein and/or that will become apparent to those skilled in the art upon reading the present disclosure.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as would normally be understood by one skilled in the art.

- 4 045030 technology to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice of the present invention, specific methods and materials are described below. All publications referred to herein are incorporated herein by reference in their entirety.

Cell culture.

As used herein, the phrase batch culture or batch mode is a phrase that refers to a setup (eg, a culture vessel) that is filled with cells and characterized by an initial full working volume of media that is never replaced. In such a batch culture, all cell culture components are supplied to the culture vessel at the beginning of the culture process. Cultivation typically occurs until nutrients are depleted or waste reaches toxic levels and the cells stop growing.

As used herein, the phrase seed culture or seed fermenter system (also called an inoculation system) includes a source of inoculation of a cell population that is intended to be propagated in a batch culture, or series of batch cultures, until they are ready for commercial scale. . The cell expansion process in a seed fermenter system represents the initial cell growth phase, or inoculum growth phase, after thawing frozen cells. The interval between thawing of cells and accumulation of sufficient cell mass to inoculate the production bioreactor constitutes the expansion phase of the seed fermenter system. The cell mass can be proportionally increased through several stages in the bioreactor in a seed culture, and the cells are grown in a cell culture medium under conditions favorable for the survival, growth and viability of the cell culture. It should be understood that the seed fermenter system is designed to maximize the exponential growth phase, or to achieve the maximum growth rate for the particular cell type to be cultured. Therefore, passaging cells from one bioreactor or vessel to another may represent one way to achieve maximum growth rates. The exact conditions will vary depending on the cell type, the organism from which the cells originate, and the nature and character of the polypeptide or protein expressed. A shift to metabolism characterized by lactate consumption may occur or be detected in any of the vessels in the cell expansion system of seed fermenters.

As used herein, the phrase fed-batch cell culture or fed-batch culture refers to a batch culture characterized by animal cells and culture medium being supplied to the culture vessel initially and additional culture nutrients being added slowly, continuously or in discrete increments. in incremental steps, into culture during cultivation, with or without periodic collection of cells and/or product before cessation of culture. Fed-batch culture includes semi-continuous fed-batch culture, which is characterized in that periodically the entire culture (which may include cells and media) is removed and replaced with fresh media. Fed-batch culture differs from simple batch culture because in batch culture, all cell culture components (including the animal cells and all culture nutrients) are supplied to the culture vessel at the beginning of the culture process. Fed-batch culture may further differ from perfusion culture in that the supernatant is not removed from the culture vessel during the process, whereas in perfusion culture cells are maintained in culture, for example by filtration, and culture medium is continuously or periodically introduced and removed from culture vessel. However, provision is made for sample removal for research purposes during fed-batch cell culture. The fed-batch culture process continues until it is determined that maximum operating volume and/or protein production has been achieved.

As used herein, the phrase continuous cell culture refers to a technique used to grow cells continuously, typically at a specific growth phase. For example, if a constant supply of cells is required or a specific polypeptide or protein of interest is required, it may be necessary for the cell culture to be maintained at a specific growth phase. Thus, conditions must be continuously monitored and adjusted accordingly to maintain cells in this particular phase.

As used herein, the phrase logarithmic growth phase refers to a period of cell growth typically characterized by a doubling of the number of cells. The phrases exponential growth phase or exponential phase are used interchangeably with logarithmic growth phase. In the logarithmic growth phase, the number of new cells appearing per unit time is proportional to the existing cell population, therefore, the graph of the natural logarithm of the number of cells versus time is a straight line. If growth is not limited,

- 5 045030 doubling will continue at a constant rate, therefore both the number of cells and the rate of population growth doubles for each subsequent period of time.

As used herein, the phrase stationary phase refers to the point where the rate of cell growth equals the rate of cell death. When plotting a graph, the stationary phase is represented as a plateau, or smoothed, horizontal linear part of the curve.

As used herein, the term cell includes any cell that is suitable for expressing a recombinant nucleic acid sequence. Cells include eukaryotic cells, such as non-human animal cells, mammalian cells, human cells, or fusions of cells, such as, for example, hybridomas or quadromas. In certain embodiments, the cell is a human, non-human monkey, ape, hamster, rat, or mouse cell. In other embodiments, the cell is selected from the following cells: CHO cells (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero cells, CV1 cells, kidney cells (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal cells), CV-1, U937, 3T3 , L cell, C127 cell, SP2/0, NS-0, MMT cell, tumor cell and a cell line derived from the above cell. In some embodiments, the cell contains one or more viral genes, for example, a retinal cell that expresses a viral gene (eg, a PER.C6® cell). In some embodiments, the cell is a CHO cell. In other embodiments, the cell is a CHO K1 cell.

As used herein, the term cell line refers to a cell or cells obtained from a particular lineage through serial passaging or subculturing of cells. The term cells is used interchangeably with the term cell population.

Taking into account modern feeding strategies in line with the latest advances, CHO cells reach cell counts of 11x106 cells/ml (on day 8) and titers of, for example, 2.3 g/l human IgG (collected on day 14), amounts that represent typical industrial values for fed-batch cultures of CHO cells (Kim, BJ, et al. Biotechnol Bioeng. 2012 Jan;109(1): 137-45. doi: 10.1002/bit.23289. Epub 2011 Oct 3). Antibody production of even more than 10 g/L has been reported from CHO cells, which have become widely recognized as an important industrial mammalian cell line (Omasa et al., Current Pharmaceutical Biotechnology, 2010, 11:233-240).

The terms cell culture medium and culture medium refer to the nutrient solution used to grow mammalian cells, which typically provides essential nutrients to enhance cell growth, such as carbohydrate energy source, essential amino acids, trace elements, vitamins, etc. The cell culture medium may contain extracts, such as serum or peptones (hydrolysates), that provide raw materials that support cell growth. Media may contain yeast or soy extracts instead of animal extracts. Chemically defined medium refers to a cell culture medium in which all chemical components are known. The chemically defined media is absolutely free of animal components such as whey peptones or animal peptones.

One aspect of the present invention relates to a growth phase in which cell culture conditions are modified to enhance the growth of recombinant eukaryotic cells. During the growth phase, the main culture medium and cells are fed into the culture vessel periodically.

The culture vessel is inoculated with cells. Suitable seeding density for the initial phase of cell growth varies depending on the starting cell line, for example, in the range of 0.2-3x106 cells/ml. Culture vessels include, but are not limited to, well plates, T-flasks, shake flasks, stirrer flasks, roller flasks, hollow fiber bioreactors, air-lift bioreactors, and the like. A suitable cell culture vessel is a bioreactor. Bioreactor refers to any culture vessel that is manufactured or designed to manipulate or control environmental conditions. Such culture vessels are well known in the art.

Bioreactor processes and systems have been designed to optimize gas exchange, to supply sufficient oxygen to support cell growth and productivity, and to remove CO ₂ . Maintaining efficient gas exchange is an important criterion to ensure successful proportional expansion of cell culture and protein production. Such systems are well known to one skilled in the art.

The exponential growth phase or seed culture (ie, the first cell culture) is typically followed by a separate second culture known as the polypeptide production phase. In one embodiment, cells undergoing a metabolic shift to consume lactate in the first cell culture are transferred to a second cell culture. According to one embodiment, the cultivation of the second cell culture is carried out in a different culture vessel than the cell growth phase or seed culture. In some embodiments, cultivation of the second cell culture occurs in a production bioreactor. In this context, cell transfer refers to extracting a fraction of a cell population from a first cell culture vessel and placing a fraction of the cell population into a second cell culture vessel to establish a second cell culture.

In other aspects, cell transfer may refer to a volume of cells containing cells of a first cell culture that is transferred to another vessel, and the volume of inoculum is a fraction of the final volume of the second cell culture, for example, about 20%, 30%, 40%, or 50% , or 60%, or 70%, or 80% of the final volume. In other aspects, cell transfer may refer to the volume of cells containing cells of the first cell culture that remains in the original vessel, and the medium is added such that the original volume (the first cell culture) is a fraction of the final volume of the second cell culture. In this context, the first cell culture is diluted, thereby effecting the transfer of cells into the second cell culture.

As used herein, the phrase exits or exits refers to a changeover from one phase to another phase, or events related to a changeover from one phase to another phase. Exit from a particular phase, such as the growth phase, involves a period of time when measurements indicate that the first phase is slowing down or almost completed, and the next phase begins. Exit from the logarithmic growth phase, for example, indicates that the cells are ending the logarithmic growth phase and/or have begun or reached stationary phase. Growth phases are typically measured by the concentration of viable cells.

The phrase cell density refers to the number of cells per sample volume, for example as the number of total (viable and dead) cells per ml. The number of cells can be counted manually or automatically, for example using a flow cytometer. Automated cell counters have been adapted to count the number of viable or dead cells, or both viable and dead cells, using, for example, the standard trypan blue uptake technique. The phrase viable cell density or viable cell concentration refers to the number of viable cells per sample volume (also called viable cell count). Any number of well-known manual or automated techniques can be used to determine cell density. Real-time measurements of crop biomass can be made, where capacity or optical density is correlated with the number of cells per unit volume.

The final cell density in the first cell culture, such as that in the seed fermenter system, varies depending on the starting cell line, for example, in the range of approximately 1.0-10 x 106 cells/ml. In some embodiments, the final density in the seed fermenter system reaches 1.0-10 x 106 cells/ml before the cells are transferred to a second cell culture. In other embodiments, the final density in the seed fermenter system is reached to 5.0-10 x 106 cells/ml by transferring the cells into a second cell culture.

In some embodiments, a fraction of the cell population in the first cell culture is transferred to a second cell culture. In other embodiments, the cell population in the first cell culture is transferred to a second cell culture such that the first cell culture is a fraction of the second cell culture. The initial cell density of the second culture can be selected by one skilled in the art. In some embodiments, the initial cell density in the second cell culture is approximately 0.5x106 cells/mL to approximately 3.0x106 cells/mL. In other embodiments, the initial cell density in the second cell culture is approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1 ,4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6 , 2.7, 2.8, 2.9 or 3.0x106 cells/ml.

In certain embodiments, the cell supernatant or cell lysate is collected after the product formation phase. In other embodiments, the polypeptide or protein of interest is recovered from the culture medium or cell lysate using techniques well known in the art.

The properties of the cells and the location of the product being produced dictate the method used for culture and production and therefore determine the selection of the appropriate type of bioreactor or culture vessel. (Bleckwenn, N.A. and Shiloach, J. 2004 Large-scale cell culture Curr Protoc Immunol. 59: Appendix 1U.1-Appendix 1U.44.)

Metabolic shift.

As used herein, the phrase metabolic shift refers to a change in cellular metabolism, or utilization of carbon nutrient sources, from lactate production to net lactate consumption. Without wishing to be bound by any theory, the most common carbon sources of nutrients in serum-free media are glucose and

- 7 045030 glutamine, which support rapid cell growth. Glucose can be completely oxidized to CO2 and

H2O, or based on oxygen availability, can be converted to lactate, for example in aerobic glycolysis. Fast-growing cells rapidly consume glucose and glutamine, resulting in incomplete oxidative metabolism and therefore excess lactate production. Carbohydrate metabolism may switch to lactate consumption and thus reduce lactate accumulation.

As used herein, the phrase lactate consumption refers to the use of lactate as a carbon source in cellular metabolism.

As used herein, the phrase net lactate consumption refers to lactate consumption in which cells simultaneously consume lactate and produce lactate as a byproduct of cellular metabolism, and the total rate of consumption is greater than or equal to the rate of lactate production. If net lactate consumption increases, the total accumulation of lactate in the cell culture medium decreases.

When establishing a fed-batch culture, the accumulation of lactate and possibly ammonia causes a rapid decrease in cell viability. It was reported that in fed-batch cultures that were not characterized by a metabolic shift, it was not possible to achieve above 90% viability when the cell concentration reached its maximum. (Xie and Wang, 1994, Biotechnol. Bioeng. 43(11): 1175-1189). This metabolic shift, although desirable for optimal process performance, is not typical and is not easy to control (Zagari, et al., 2013, New Biotechnol. 30(2):238-245). The present inventors have discovered that the timing and conditions of transferring cells from a first batch culture (eg, a seed culture) to a second batch culture (eg, a fed batch or production culture) have a significant impact on the final protein titer. Surprisingly, it was determined that cells cultured for a longer period of time in a first batch culture would switch to lactate consumption and provide a metabolic preference, or metabolic phenotype, characterized by lactate consumption. The purpose of the present invention is to create cells in a state of constant metabolic shift, and therefore cells with metabolic memory for lactate consumption. The method of the present invention is well suited for preconditioning cells into a state of metabolic shift so that the cells can be used in any second or subsequent cell culture where lactate consumption is preferred.

In one embodiment, overall lactate accumulation is reduced in the second cell culture. In some embodiments, net lactate consumption is achieved during the second cell culture, for example, net lactate consumption is achieved on day 2, 3, 4, or 5, or after 2 days, 3 days, 4 days, or 5 days of cell growth in the second cell culture. In other embodiments, the reduction in lactate accumulation is a reduction in the maximum lactate concentration in the second cell culture. In other embodiments, the decrease in maximum lactate concentration occurs in the second cell culture on day 5 or after 5 days of cell growth in the second cell culture. In other embodiments, the maximum lactate concentration in the second cell culture is less than about 6 g/L, 5 g/L, 4 g/L, 3 g/L, 2 g/L, or less than about 1 g/L.

In some embodiments, the metabolically shifted cells produce at least 2-fold, or 3-fold, or 4-fold, or 5-fold, or up to 10-fold lower lactate concentrations in the second cell culture. In some further embodiments, the reduced lactate concentration in the second cell culture or the overall reduced lactate accumulation in the second cell culture is compared to that determined in an otherwise identical cell culture under otherwise identical conditions except that the cells are transferred to a second cell culture. cell culture occurs before a metabolic shift has occurred in the first cell culture. In other embodiments, total lactate accumulation is reduced in the second cell culture on day 5 or after 5 days of cell growth in the second cell culture.

In another embodiment, the total titer of the product is increased in the second cell culture. In other embodiments, metabolically shifted cells produce at least 2-fold, or 2.5-fold, 3-fold, or 4-fold, or 5-fold, or up to 10-fold higher titer of product in the second cell culture. In other embodiments, the elevated protein titer values in the second cell culture are compared to that determined in an otherwise identical cell culture under otherwise identical conditions except that the transfer of cells into the second cell culture occurs before the first cell culture culture has undergone a metabolic shift.

There are many benefits to optimizing the metabolic control of cells in culture prior to the fed-batch or product generation stage. The metabolic shift to lactate consumption in the first culture can be determined by numerous parameters. Determination of metabolic shift involves a number of methods known to one skilled in the art.

- 8 045030 techniques for determining the metabolic status of growing cells.

Measurement of lactate concentration values in the first cell culture can be accomplished using a variety of bioanalytical systems and kits well known to one skilled in the art, such as electrochemical analyzers (e.g., Bioprofile® Flex, Nova Biomedical, Waltham, MA), or Raman spectroscopy scattering and can be used for off-line or real-time monitoring of lactate accumulation in cell culture.

It should be understood that the accumulation of lactate has an adverse effect on the cell culture and, therefore, has a negative effect on the yield of the protein product.

In one embodiment, a metabolic shift is determined in the first cell culture when the net accumulation of lactate slows down or stops.

In one embodiment, the metabolic shift to lactate consumption is detected using lactate measurements in a first cell culture. In some embodiments, a metabolic shift is determined in a first cell culture by detecting a plateau or substantially horizontal line on a graph representing the measurement of successive lactate concentration values in the culture. In other embodiments, the lactate concentration value remains below the upper acceptable limit for successive measurements. In other embodiments, the upper permissible limit for the lactate concentration does not exceed 4 g/L. It should be understood that lactate levels reach a plateau when cells experience net lactate consumption.

In other embodiments, determining the metabolic shift involves measuring lactate in the first cell culture at intervals, and determining that the lactate content is below a predetermined upper limit for successive time intervals, thereby determining that a metabolic shift to lactate consumption in the cells has occurred.

pH management and control is an important aspect of cell maintenance in a bioreactor culture. Most cell growth is optimal within narrow pH limits. Typically, the cell culture is maintained at a neutral pH of 7.0, within the upper and lower pH range. The established values are determined by one skilled in the art depending on the specific cell line in culture, the composition of the medium and the optimal growth conditions for a given cell. As used herein, the expression neutral pH value means a pH value of about 6.85 to about 7.4. The expression neutral pH value includes pH values of approximately 6.85, 6.9, 6.95, 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7. 3, 7.35 and 7.4.

Monitoring pH and adding base online, or in real time, can be accomplished using any number of methods well known to one of ordinary skill in the art. In an online system, real-time measurements of biological and chemical parameters in cell culture through a direct connection to the analyzer provide feedback for additional actions, such as adding base or adding nutrients to the culture medium. It is also possible to carry out autonomous measurements, in which periodic sampling and non-automated operator manipulations take place. Continuous pH measurement allows monitoring of the cellular environment and addition of base, for example, if the acidity reaches a lower set value outside the acceptable range. If the pH reaches the specified upper tolerance limits (ie becomes too basic), CO ₂ can be added.

Real-time monitoring can be accomplished using a variety of methods. Electrodes, such as flow electrodes, are widely used to measure the pH value or other parameters such as dissolved O ₂ (dO ₂ ) and temperature in cell culture media. These flow electrodes connect directly to any standard strip recorder for continuous recording or can be connected to any standard laboratory pH meter or millivoltmeter. pH can also be measured by optical measurement using a fluorescent sensor zone installed in the bioreactor.

Any such monitoring system will integrate a tolerance limit (or sensor deadband) around specified upper and lower values. The sensor deadband prevents the dosing system from switching on and off too quickly. During pH monitoring, no dosing or titration will occur if the deviation of the pH value from the set value is within acceptable limits. If the measured pH values exceed the lower acceptable limit (acidic), then a liquid base (eg KOH, NaOH, NaHCO ₃ ) or gaseous NH3 will be added. If the measured pH values exceed the upper limit of acceptable values (basic), then acid or CO2 gas will be added. The set pH value and the control strategy, such as the dead band of the sensor, are associated with numerous parameters such as dissolved CO2 content, base consumption to control the pH value, and therefore osmolarity. (See, for example, Li, F., et al., 2010, mAbs 2(5):466-479.)

- 9 045030

In one embodiment, the metabolic shift is determined in the first cell culture when base addition (ie, titration) is stopped. The base content trend includes a real-time trend, and an automated monitoring method can be used to determine the pH value and periodically add base. In the present method, the set pH values may vary, but an increase in the pH value beyond the lower limit of the dead zone indicates a metabolic shift in the first cell culture. Real-time and manual methods for measuring the trend of base content are known in the art, including methods for monitoring the mass of a vessel, or the volumetric flow rate of a pump to detect base addition or stop base addition.

In another embodiment, a metabolic shift is determined in the first cell culture when base addition is no longer required to raise the pH above the lower tolerance limit.

In some embodiments, a metabolic shift is determined in the first culture if the pH value is increased without adding base. In other embodiments, the pH value increases above the lower acceptable limit for sequential measurements.

In other embodiments, determining the metabolic shift involves: (a) adjusting the pH meter to detect the noise level in the first cell culture, (b) continuously measuring the pH of the first cell culture at regular intervals, (c) adding base as needed to maintain the pH. above a predetermined lower limit, (d) determining that the pH value is above a predetermined lower limit for several consecutive time intervals, and (e) stopping the addition of base, thereby determining that a metabolic shift to lactate consumption in the cells has occurred.

In one embodiment, the lower acceptable limit is a pH value of approximately 6.5, 6.55, 6.6, 6.65, 6.7, 6.75, 6.8, 6.85, 6.9 , 6.95, 7.0, 7.05 or approximately 7.1.

In some embodiments, the metabolic shift to lactate consumption is detected using indicators or products of cellular metabolism in the first cell culture. One such indicator of cellular metabolism is oxygen consumption (Zagari, et al., 2013, New Biotechnol. 30(2):238-245). Accurate measurement of the rate of oxygen depletion in cell culture media can be used to determine the presence of viable cells in the culture after inoculation, as well as the rate of cell growth in the culture (see, for example, US Pat. Nos. 6,165,741 and 7,575,890). Measuring oxygen consumption is well known in the art.

Other indicators of cellular metabolism, such as enzymes and metabolites, can be measured using proteomic or metabolomic techniques such as immunochips, nuclear magnetic resonance (NMR), or mass spectrometry. There has been a correlation between the content of metabolites such as glycine, tryptophan, phenylalanine, adenine, palmitic acid, glutamic acid, methionine and asparagine with an increase in cellular biomass (see, for example, Jain, M., et al., Science. 2012 May 25; 336(6084): 1040-1044. doi: 10.1126/science.1218595; and De la Luz-Hdez, K., 2012, Metabolomics and Mammalian Cell Culture, Metabolomics, Dr Ute Roessner (Ed.), ISBN: 978-953 -51-0046-1, InTech, Available from: http://www.intechopen.com/books/metabolomics/ metabolomics-and-mammalian-cell-cultures). Any number of molecular changes that coincide with or directly lead to a metabolic shift in the first cell culture can be used to determine that a metabolic shift has occurred.

Getting protein.

The methods of the present invention involve the production of a protein or polypeptide of interest in a cell culture. To enable protein production in the methods of the present invention, cells are engineered to recombinantly express the polypeptide or protein of interest.

The cells are transferred to a second cell culture, such as a production culture, after a metabolic shift to lactate consumption has occurred in the cells, and will be maintained in the second cell culture for a period of time so that the polypeptide or protein accumulates in the cell culture.

As used herein, a polypeptide is a single unbranched polymer chain of amino acids linked together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The term protein can also be used to describe a large polypeptide, such as a seven transmembrane domain protein, with a particular fold or spatial structure. Thus, the term protein is intended to include quaternary structures, tertiary structures, and other complex macromolecules consisting of at least one polypeptide. When a protein is composed of more than one polypeptide that are physically linked to each other, the term protein as used herein refers to multiple polypeptides that are physically linked and function together as a distinct structural unit. The term protein includes a polypeptide.

Examples of polypeptides and proteins produced by the methods of the present invention include antibodies, fusion proteins, Fc-containing fusion proteins, receptors, receptor-containing Fc fusion proteins and the like.

The term immunoglobulin refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) chains and one pair of heavy (H) chains, all four of which can be interconnected by disulfide bonds. The structure of immunoglobulins has been extensively characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically contains a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (CH). The heavy chain constant region typically contains three domains, _CH1 , CH2 and CH3. The C _H 1 and CH2 domains are connected by a hinge. Each light chain typically contains a light chain variable region (abbreviated herein as V _L or VL) and a light chain constant region. There are two types of light chains in humans and other mammals: kappa (k) chain and lambda (λ) chain. The light chain constant region typically contains one domain (CL). The VH and _VL regions can be further subdivided into hypervariable regions (or hypervariable regions that may be hypervariable in sequence and/or may form structurally definable loops), also called complementarity determining regions (CDRs), interspersed with regions that are more conserved , which are called frame regions (FR). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus (N-terminus) to the carboxy terminus (C-terminus) in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 , FR4 (see also Chothia and Lesk J. MoI. Biol. 196, 901-917 (1987)). As a rule, the numbering of amino acid residues in this region is carried out according to IMGT, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) or by the EU numbering system according to Kabat (also known as EU numbering or EU index), for example, as in Kabat, EA et al. Sequences of Proteins of Immunological interest. 5 ^th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

The term Fc refers to the portion of the heavy chain constant region that contains at least the CH2 and CH3 domains that typically bind to the Fc receptor, e.g. FcyR, namely FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16) or FcRn, i.e. neonatal Fc receptor. It should be understood that an Fc fusion protein may contain all or part of the native Fc domain or contain deletions, substitutions and/or insertions or other modifications that render it unable to bind to any Fc receptor, thereby rendering the domain non-functional or lacking effector functions in regarding its typical biological function, which is achieved through the Fc receptor.

As used herein, the term antibody (Ab) refers to an immunoglobulin molecule or derivative thereof that has the ability to specifically bind an antigen. The variable regions of the heavy and light chains of an immunoglobulin molecule contain a binding domain that interacts with an antigen, as described above when describing the term immunoglobulin. The antibody may also be a bispecific antibody, diabody, or similar molecule (see, for example, Holliger, et al., 1993, PNAS USA 90(14), 6444-8, for a description of diabodies). In addition, it has been shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody, i.e. antigen binding fragments or antigen binding proteins. Like full-length antibody molecules, antigen-binding proteins can be monospecific or multispecific (eg, bispecific). Examples of binding molecules or fragments contemplated within the term antibody include, but are not limited to, the following: (i) a Fab' or Fab fragment, a monovalent fragment consisting of the V _L , V _H , C _L and _CH 1 domains, or a monovalent antibody , described in international patent publication No. WO2007059782; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting essentially of the VH and CH1 domains; (iv) an Fv fragment consisting essentially of the VL and VH domains, (v) a dAb fragment (Ward et al., 1989, Nature 341, 544-546), which consists essentially of a Vh domain and is also called domain antibodies ( Holt et al., 2003, Trends Biotechnol. 21(11):484-90); (vi) camel antibodies or nanobodies (Revets et al., 2005, Expert Opin Biol Ther. 5(1): 111-24) and (vii) a dedicated complementarity determining region (CDR).

As used herein, the terms monoclonal antibody or monoclonal antibody composition refer to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits uniform specificity and binding affinity for a particular epitope.

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The term human antibody, as used herein, is intended to include antibodies containing variable and constant regions derived from human immunoglobulin germline sequences. Human antibodies may include amino acid residues not encoded by human immunoglobulin germline sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or during gene rearrangement or by somatic mutation in vivo). The term mouse monoclonal antibody, or mouse monoclonal antibody, refers to antibodies exhibiting a single binding specificity that contain variable and constant regions derived from murine immunoglobulin germline sequences.

As used herein, the term fusion protein includes an Fc-containing fusion protein and a receptor-containing Fc fusion protein. A fusion protein can be any polypeptide formed by expressing a chimeric gene formed by combining more than one DNA sequence of different origins, typically by cloning one gene into an expression vector in frame with a second gene so that the two genes encode one contiguous polypeptide .

According to one aspect of the present invention, there is provided a method described herein for producing a recombinant polypeptide or protein of interest. In some embodiments, the recombinant polypeptide or protein of interest is selected from the group consisting of an antibody, an antigen binding protein, a fusion protein containing an Fc fusion protein, and a receptor containing Fc fusion protein.

Cell expression systems.

The use of a cell expression system is a necessary prerequisite for large-scale production of such polypeptides or proteins in cell culture.

The product of the present invention is a polypeptide or protein that is expressed in cells and collected from a culture system, i.e. cells and/or cellular environment. It may be any polypeptide or protein of interest (formerly).

Expression vectors typically use strong gene promoters to drive transcription and product RNA. In a further aspect, the present invention provides an expression vector encoding a polypeptide of interest, such as an antibody, antigen binding protein, or fusion protein. Such expression vectors can be used in the methods of the present invention to recombinantly produce polypeptides or proteins of interest by cell culture.

An expression vector in the context of the methods of the present invention can be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (a nucleic acid sequence containing a suitable set of expression control elements). Examples of such vectors include SV40 derivatives, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. Such nucleic acid vectors and their use are well known in the art (see, for example, US Pat. Nos. 5,589,466 and 5,973,972).

A vector containing a nucleic acid molecule encoding a polypeptide or protein of interest is delivered to a host cell, the nucleic acid molecule being operably linked to an expression control sequence suitable for expression in a mammalian host cell.

Expression control sequences are designed to control and direct the transcription of polypeptide-encoding genes of interest and subsequent expression of polypeptides or proteins in various cellular systems. Plasmids combine an expressible gene of interest with expression control sequences (ie, expression cassettes) that contain necessary elements such as, for example, promoters, enhancers, selectable markers, operators, etc. In an expression vector, the nucleic acid molecules may contain or be associated with any suitable promoter, enhancer, selectable marker, operator, repressor protein, polyadenylation termination sequences and other expression facilitating elements.

As used herein, the term promoter refers to a DNA sequence sufficient to direct transcription of the DNA sequence to which it is operably linked, i.e. bound in such a way as to control the transcription of a nucleotide sequence. Expression of the nucleotide sequence can be placed under the control of any promoter or enhancer element known in the art. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter/enhancer or CMV IE major promoter (CMV-MIE), as well as RSV, SV40 late promoter, SL3-3, MMTV, ubiquitin (Ubi) promoters C (UbC) and HIV LTR).

In some embodiments, the vector contains a promoter selected from the group

- 12 045030 consisting of SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi, UbC and HIV LTR.

The nucleic acid molecules encoding the polypeptide or protein of interest may also be operably linked to an effective polyadenylation termination sequence, an origin of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and/or a suitable cloning site (eg , polylinker). Nucleic acids may also contain a regulated inducible promoter (inducible, repressible, regulated by environmental conditions) as opposed to a constitutive promoter such as CMV IE (one skilled in the art will appreciate that such terms are actually descriptors of the degree of gene expression under certain conditions) .

Selectable markers are elements well known in the art. Under selective conditions, only cells expressing the appropriate selectable marker can survive. Typically, selectable marker genes express proteins, usually enzymes, that provide resistance to various antibiotics in cell culture. In other selective conditions, cells that express a fluorescent protein marker are visualized and thus selected. Embodiments include the following: beta-lactamase (bla) (beta-lactam antibiotic resistance or ampicillin resistance gene, or ampR), bls (acetyltransferase gene conferring blasticidin resistance), bsd (deaminase gene conferring blasticidin resistance -S), bsr (blasticidin-S resistance gene), Sh ble (Zeocin® resistance gene), hygromycin phosphotransferase (hpt) (hygromycin resistance gene), tetM (tetracycline resistance gene, or tetR), neomycin phosphotransferase II (npt ) (neomycin resistance gene, or neoR), kanR (kanamycin resistance gene), and ras (puromycin resistance gene). Selectable (or selectable) markers are typically used in the development of stable cell lines.

In certain embodiments, the vector contains one or more selectable marker genes selected from the group consisting of bla, bls, BSD, bsr, Sh ble, hpt, tetR, tetM, npt, kanR, and races. In other embodiments, the vector contains one or more selectable marker genes encoding green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), blue fluorescent protein (CFP), enhanced blue fluorescent protein (eCFP), yellow fluorescent protein (YFP) or similar.

For purposes of the present invention, gene expression in eukaryotic cells can be precisely regulated using a strong promoter that is controlled by an operator, which in turn is regulated by a regulatory fusion protein (RFP). RFP consists essentially of a transcription blocking domain and a ligand binding domain that regulates its activity. Examples of such expression systems are described in US20090162901A1, which is incorporated herein by reference in its entirety.

As used herein, the term operator refers to a DNA sequence that is introduced into or adjacent to a gene of interest such that the gene can be regulated by binding of RFP to the operator and thereby prevent or permit transcription of the gene of interest. A number of operators in prokaryotic cells and bacteriophages have been well characterized (Neidhardt, ed. Escherichia coli and Salmonella; Cellular and Molecular Biology 2d. Vol 2 ASM Press, Washington D.C. 1996). These include, but are not limited to, the operator region of the E. coli LexA gene, which binds the LexA peptide, and the lactose and tryptophan operators, which bind the repressor proteins encoded by the E. coli LacI and trpR genes. They also include bacteriophage operators from lambda PR and the ant/mnt genes of phage P22, which bind repressor proteins encoded by lambda cI and P22 arc. In some embodiments, in which the transcription blocking domain of the RFP is a restriction enzyme such as NotI, the operator is a recognition sequence for that enzyme. One skilled in the art will appreciate that the operator must be located adjacent to, or 3' to, the promoter so that it is capable of controlling transcription by the promoter. For example, US Pat. No. 5,972,650, which is incorporated herein by reference, discloses that tetO sequences are within a specific distance of the TATA box.

In certain embodiments, the operator is selected from the group consisting of the following: a tet operator (tetO), a NotI recognition sequence, a LexA operator, a lactose operator, a tryptophan operator, and an Arc operator (AO). In some embodiments, the repressor protein is selected from the group consisting of TetR, LexA, LacI, TrpR, Arc, LambdaC1, and GAL4. In other embodiments, the transcription blocking domain is derived from a eukaryotic repressor protein, for example, a GAL4-derived repressor domain.

In an exemplary cell expression system, cells are engineered to express a tetracycline repressor protein (TetR) and the polypeptide of interest is placed under the transcriptional control of a promoter whose activity is regulated by TetR. Two tandem TetR operators (tetO) are located immediately downstream of the CMV-MIE promoter/enhancer in the vector. Trance

- 13 045030 transcription of a gene encoding a protein of interest, directed by the CMV-MIE promoter, in such a vector can be blocked by TetR in the absence of tetracycline or other specific suitable inducer (eg, doxycycline). In the presence of an inducer, the TetR protein is unable to bind to tetO, hence transcription and thus translation (expression) of the polypeptide of interest occurs. (See, for example, US Pat. No. 7,435,553, which is incorporated herein by reference in its entirety.)

Such cell expression systems can be used to trigger production of the polypeptide of interest only during cultivation of the production culture. Thus, antibiotics, such as tetracycline or other suitable inducers, can be added to the bioreactor to the first cell culture.

Another exemplary cell expression system includes regulatory fusion proteins, such as a TetR-ER _LBD T2 fusion protein, in which the transcription blocking domain of the fusion protein is TetR, and the ligand binding domain is an estrogen receptor ligand binding domain (ER _LBD ) with T2 mutations ( ER _LBD T2; Feil et al., 1997, Biochem. Biophys. Res. Commun. 237:752-757). If tetO sequences are located downstream and adjacent to the strong CMV-MTE promoter, transcription of the nucleotide sequence of interest from the CMV-MIE/tetO promoter was blocked in the presence of tamoxifen, and this blocking was reversed when tamoxifen was removed. In another example, the use of an Arc2-ER _LBD T2 fusion protein, a fusion protein consisting of a single chain dimer consisting of two Arc proteins connected by a 15 amino acid residue linker and the ER _LBD T2 (formerly), involves an Arc operator (AO), more specifically two tandem operators a, immediately downstream of the CMV-MIE promoter/enhancer. Cell lines can be regulated by Arc2-ER _LBD T2, with cells expressing the protein of interest driven by the CMV-MIE/ArcO2 promoter and induced by tamoxifen removal. (See, for example, US 20090162901A1, which is incorporated herein by reference.) In some embodiments, the vector comprises a CMV-MIE/TetO or CMV-MIE/AO2 fusion promoter.

Suitable vectors used in the methods of the present invention may also utilize Cre-lox recombination technology tools to facilitate replication of the gene of interest. The Cre-lox strategy requires the presence of at least two components: 1) Cre recombinase, an enzyme that catalyzes recombination between two loxP sites; and 2) loxP sites (eg, a specific 34-bp sequence consisting of an 8-bp core sequence where recombination occurs and two flanking 13-bp inverted repeats) or mutant lox sites. (See, for example, Araki et al., 1995, PNAS 92:160-4; Nagy, A. et al., 2000, Genesis 26:99-109; Araki et al., 2002, Nuc Acids Res 30(19) :e103; and US20100291626A1, all of which are incorporated herein by reference). In another recombination strategy, the yeast FLP recombinase can be used with the FRT consensus sequence (see also, eg, Dymecki, S., 1996, PNAS 93(12): 6191-6196).

In another aspect, a gene (i.e., a nucleotide sequence encoding a recombinant polypeptide of interest) is introduced into an expression enhancing sequence of an expression cassette and is optionally operably linked to a promoter, wherein the promoter-associated gene is flanked by a 5' first recombinase recognition site and a 3' second recognition site recombinase. Such recombinase recognition sites enable Cre-mediated recombination in the host cell of the expression system. In some cases, a second promoter-bound gene is located downstream (3') of the first gene and is flanked 3' by a second recombinase recognition site. In other cases, the second promoter-bound gene is flanked 5' by a second recombinase recognition site and flanked 3' by a third second recombinase recognition site. In some embodiments, the recombinase recognition sites are selected from a loxP site, a lox511 site, a lox2272 site, and an FRT site. In other embodiments, the recombinase recognition sites are different. In a further embodiment, the host cell contains a gene capable of expressing Cre recombinase.

In one embodiment, the vector contains a first gene encoding an antibody light chain or an antibody heavy chain of interest and a second gene encoding an antibody light chain or an antibody heavy chain of interest.

It should be understood that one or more vectors carrying one or more nucleic acid sequences encoding and expressing a protein of interest can be used in such an expression system.

Cells of the present invention can also be engineered to increase product expression by co-expressing proteins such as chaperones, apoptosis inhibitors, protein degradation inhibitors, or other protein that can enhance product expression or stability.

In some embodiments, the vector further comprises an X-box binding protein 1 (mXBP1) and/or EDEM2 gene capable of enhancing protein production/secretion by controlling the expression of genes involved in protein folding in the endoplasmic reticulum (ER). . (See, for example, Ron D, and Walter P., 2007, Nat Rev Mol Cell Biol.8:519-529; Olivari et al.,

2005, J. Biol. Chem. 280(4): 2424-2428, Vembar and Brodsky, Nat. Rev. Mol. Cell. Biol. 9(12): 944-957, 2008).

The use of transiently transfected cells that quickly produce significant quantities of product can also be implemented to optimize the cell culture process, although stable transfection is typically used for large-scale industrial production.

In the context of the present invention, a metabolically shifted cell may contain any or all of the elements of the cell expression system described herein necessary for efficient recombinant production of the protein of interest.

In a further aspect, the present invention relates to a metabolically shifted recombinant eukaryotic host cell that produces a protein of interest. Examples of host cells include mammalian cells such as CHO, PER.C6, murine lymphoid and murine hybridoma cell lines (formerly). For example, in one embodiment, the present invention provides a metabolically shifted cell comprising a nucleic acid sequence stably integrated into the cellular genome that contains a sequence encoding a protein of interest. In another embodiment, the present invention provides a metabolically shifted cell containing a non-integrated (ie, episomal) nucleic acid sequence, such as a plasmid, cosmid, phagemid, or linear expression element, that contains a sequence encoding a protein of interest.

Harvesting or collection of cells occurs at the end of the production run during the accumulation of cell mass. Cells are separated from the medium using a number of methods, such as filtration, cell encapsulation, cell adherence to microarrays, cell sedimentation, or cell centrifugation. Protein purification occurs in additional steps to isolate the protein product. Polypeptides or proteins can be collected either from cells or from cell culture media.

Protein purification strategies are well known in the art. Soluble forms of the polypeptide, such as antibodies, antibody binding fragments and Fc-containing proteins, can be subjected to commercially available concentration filters and subsequently can be affinity purified using well known methods such as affinity resins, ion exchange resins, chromatography columns and the like. Membrane-bound forms of the polypeptide can be purified by obtaining a total membrane fraction from the expression cell and extracting the membranes with a nonionic detergent such as TRITON® X-100 (EMD Biosciences, San Diego, CA, USA). Cytosolic or nuclear proteins can be obtained by lysing host cells (through mechanical action, sonication, detergent, etc.), removing the cell membrane fraction by centrifugation, and storing the supernatant.

According to a further aspect, the present invention provides a method for producing an antibody or antigen binding protein or fusion protein of interest, the method comprising the following steps: a) culturing cells according to the method described hereinabove, b) harvesting the cells, and c) purifying the polypeptide or a protein, such as an antibody, or antigen binding protein, or fusion protein, from cells or cell culture media.

The following examples are provided to describe to those skilled in the art how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider their invention to be. Attempts have been made to ensure the accuracy of the numerical expressions used (eg amounts, concentrations, temperatures, etc.), but some experimental errors and deviations should be taken into account.

Examples

Example 1 Determination of Metabolic Shift Parameters: Fusion Protein Producing Cell Line.

CHO cells were transfected with DNA expressing the fusion protein. The fusion protein-producing cell line CHO was incubated in seed flask culture in proprietary media containing soy, and parameters such as real-time pH, lactate content, and off-line viable cell counts were measured and recorded to determine metabolic status (see No. 1, No. 2 or No. 3 in Fig. 1A). Base utilization was also monitored and normalized to 1 for the indicated cell line (also see Fig. 1A).

Cells from condition #1 and condition #2 were used to inoculate production bioreactors in parallel when the pH value was controlled at the lower limit of the control range and the lactate and VCC contents were increased. Cells under condition No. 3 were inoculated when the pH value began to increase above the lower limit of the control range, i.e. base use was discontinued, indicating lactate remetabolization (i.e., consumption). Cell growth under condition No. 3 exited the phase following the exponential growth phase. All production bioreactors

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Claims (14)

operated under the same operating conditions. Product titer (see Fig. 1B) and lactate profiles (see Fig. 1C) were measured in each production bioreactor using known methods to determine the impact of metabolic status No. 1, No. 2 or No. 3 in the seed fermenter system. Lines representing the main trend of the production bioreactor represented the mean of the bioreactors in two parallels, with error bars representing ± one standard deviation. Cells under condition No. 3 had the most significant effect on productivity and lactate accumulation in the second cell culture, resulting in a more than 2-fold increase in product titer (compared to conditions No. 1 and No. 2) in the production bioreactor (see FIG. 1B). Cells under condition #3 also resulted in a reduced lactate concentration after transfer to a second cell culture (compared to conditions #1 and #2 - see Fig. 1C). Cells in condition #3 had a lactate profile indicative of net lactate consumption (see Fig. 1C at days 8-12 of cell culture). Cells transferred from the first culture under condition #1 (i.e., before the metabolic shift in the first culture) do not achieve net lactate consumption in the production bioreactor. Example 2 Determination of Metabolic Shift Parameters: Antibody Producing Cell Line. A vessel for seeding the antibody-producing cell line CHO was used to inoculate production bioreactors in parallel similar to example 1, but in a medium with an established chemical composition. Four different metabolic statuses were measured (off-line monitoring of pH, lactate and viable cell counts - see #1, #2, #3 and #4 in Figure 2A). The VCC value continued to increase throughout the incubation in the seed vessel when production bioreactors were inoculated. Condition #1 was inoculated very early in the seed fermenter system when the pH remained at the high end of the control range and when the lactate content was low but increasing. Condition No. 2 was inoculated when the pH value began to decrease and the lactate content increased and reached maximum levels. Condition #3 was inoculated when the pH value was near the lower limit of the control range and the lactate content reached a plateau. Condition #4 was inoculated when the pH value began to decrease below the lower limit of the control range and during lactate remetabolization (ie, lactate consumption). All production bioreactors operated under the same operating conditions. Condition No. 1 was lost after one week. The impact of metabolic status in the seed fermenter system on production bioreactor titer (Figure 2B) and lactate profiles (Figure 2C) was determined. Lines representing the main trend of the production bioreactor represented the mean of the bioreactors in two parallels, with error bars representing ± one standard deviation. Cells under conditions No. 3 and No. 4 had the most significant effect on productivity in the second cell culture. Cells under condition #3 and #4 also resulted in a reduced lactate concentration in the production bioreactor (compared to conditions #1 and #2), indicating a metabolic phenotype directed toward lactate consumption (see Figures 2B and 2C). Similar to Example 2, cells transferred from the first culture under condition No. 1 do not achieve net lactate consumption during the product formation phase. Conditions #2, #3 and #4 achieve net lactate consumption during the production phase, however, condition #4 is the most optimal because net lactate consumption occurs earlier than other conditions and the maximum lactate content is the smallest. CLAIM 1. A method for culturing cells, comprising: (a) culturing the cells in a first cell culture, (b) measuring the lactate concentration in the first cell culture, (c) determining that the first cell culture has undergone a metabolic shift to consume lactate, and (d) transferring the cells of the first cell culture to a second cell culture after a metabolic shift to lactate consumption has occurred in the first cell culture, wherein the metabolic shift occurs when lactate levels plateau in the first cell culture; and wherein the cultivation of the second cell culture is carried out in a different culture vessel than the first cell culture. 2. The method of claim 1, wherein the cells are transfected with DNA encoding the polypeptide of interest before culturing the cells in a first cell culture, and further comprising maintaining the second cell culture under conditions that allow expression of the polypeptide of interest, and collecting the polypeptide of interest from second glue - 16 045030 precision culture. 3. The method according to claim 1 or claim 2, wherein the metabolic shift to lactate consumption is detected by measuring the pH value, lactate content or base in the first cell culture. 4. The method according to any one of claims 1 to 3, wherein the metabolic shift to lactate consumption is detected by an increase in the pH value in the first cell culture medium without adding a base. 5. The method according to any one of claims 1 to 3, wherein the metabolic shift is determined in the first cell culture when the net accumulation of lactate slows down or stops. 6. The method according to any one of claims 1 to 5, wherein the metabolic shift occurs when the cells exit the logarithmic growth phase or reach the stationary phase in the first cell culture. 7. Method according to any one of claims 1-6, in which: (i) a metabolic shift occurs in the first cell culture on day 3 or after 3 days of cell growth in the first cell culture; (ii) the transferred cells have an inoculation cell density of from about 0.5x106 cells/ml to about 3.0x106 cells/ml in the second cell culture; and/or (iii) the metabolic shift determination step involves: a) measuring the pH in the first cell culture, b) adding a base to maintain the pH above a predetermined lower limit, c) detecting that the pH value exceeds a predetermined lower limit for successive time intervals, and d) stopping the addition of base, thereby determining that a metabolic shift to lactate consumption has occurred in the first cell culture. 8. Method according to any one of claims 1 to 7, wherein the first cell culture is a culture in a seed fermenter system. 9. Method according to any one of claims 1 to 8, wherein the second cell culture is a production culture. 10. The method according to any one of claims 1 to 9, wherein the transfer of cells into the second cell culture involves transferring the cells into a production bioreactor. 11. The method according to any one of claims 1 to 10, wherein the population of cells from the first cell culture is transferred to a second cell culture so that the first cell culture is a fraction of the second cell culture. 12. Method according to any one of claims 1-11, in which: (i) one or more nucleic acid sequences are stably integrated into the cellular genome of the cells, and wherein the nucleic acid sequences encode a polypeptide of interest; or (ii) the cells contain one or more expression vectors encoding the polypeptide of interest. 13. The method according to any one of claims 2 to 12, wherein the polypeptide of interest is selected from the group consisting of an antibody, an antigen binding protein and a fusion protein. 14. Method according to any one of claims 1 to 13, wherein the cells are selected from the group consisting of CHO cells, COS cells, retinal cells, Vera cells, CV1, HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21, HeLa, HepG2 , WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431, CV-1, U937, 3T3, L cells, C127 cells, SP2/0 cells, NS-0, MMT, PER.C6, lymphoid and mouse hybridoma cells. -